Annual Progress Report on Malting Barley Research March, 2002

<strong>Annual</strong> <strong>Progress</strong> <strong>Report</strong>
on
Malting Barley Research
March, 2002
Information presented in this report is
confidential and not for publication,
citation or reproduction in any form.
American Malting Barley Association, Inc.
740 N. Plankinton Ave., #830; Milwaukee, Wisconsin 53203; (414) 272-4640; http://www.AMBAinc.org

OFFICERS AND STAFF
Chairman of the Board ............... HEINRICH K. HEISSINGER, Anheuser-Busch, Inc.
Vice Chairman ........................... RICHARD D. MENSING, Minnesota Malting Co.
Secretary/Treasurer .................... DAVID S. RYDER, Miller Brewing Co.
President ..................................... MICHAEL P. DAVIS, AMBA
VP & Technical Director ........... SCOTT E. HEISEL, AMBA
Administrative Assistant ............ JOANN M. WALLDREN, AMBA
BOARD OF DIRECTORS
HEINRICH K. HEISSINGER (Chairman), Anheuser-Busch, Inc.
DOUG EDEN, Cargill Malt
KEN GROSSMAN, Sierra Nevada Brewing Co.
PHILIP HALL, Latrobe Brewing Co.
RICHARD D. MENSING, Minnesota Malting Co.
ROBERT MICHELETTI, Rahr Malting Co.
CHRIS MULDER, Froedtert Malt
DAVID S. RYDER, Miller Brewing Co.
CARL SIEBERT, Briess Malting Co.
GORDON TILLEY, Great Western Malting Co.
TECHNICAL COMMITTEE
MICHAEL P. DAVIS (Chairman), American Malting Barley Association, Inc.
TONY CHADWICK, Great Western Malting Co.
SHERMAN H. CHAN, Rahr Malting Co.
MARVIN DAVELOOSE, Minnesota Malting Co.
DICK DUNCOMBE, Miller Brewing Co.
KEN GROSSMAN, Sierra Nevada Brewing Co.
JOSEPH J. GRUSS, Latrobe Brewing Co.
THOMAS H. HARTZELL, Briess Malting Co.
BERNE L. JONES, USDA/ARS Cereal Crops Research Unit, WI
WILLIAM J. LADISH, Cargill Malt
MARY-JANE MAURICE, Froedtert Malt
LESLIE J. WRIGHT, Anheuser-Busch, Inc.

-i-
INTRODUCTION
The March 2002 <strong>Annual</strong> <strong>Progress</strong> <strong>Report</strong> on Malting Barley Research was
compiled from reports submitted by barley researchers. The reports include research
supported in whole or in part by the American Malting Barley Association and other
barley research conducted during the 2001 crop season and the winter of 2001/2002.
The American Malting Barley Association made grants totaling $652,940 to state
and federal research institutions for the support of malting barley research programs in
ten states during the 2001/2002 fiscal year. This substantial industry support augments
state and federal funds allocated for barley research.
The overall objective of the American Malting Barley Association research
program is the development of barley varieties that combine superior malting and
brewing qualities with superior agronomic characteristics. Attainment of this goal
involves both basic and applied research in breeding, cytogenetics, genetics,
biochemistry, molecular biology, plant physiology, plant pathology, and production. We
gratefully acknowledge the cooperation and contributions of both state and federal
researchers and administrators in helping to achieve this objective.

ANNUAL PROGRESS REPORT ON MALTING BARLEY RESEARCH
March, 2002
TABLE OF CONTENTS
Page
INTRODUCTION ............................................................................................................... i
CALIFORNIA
UNIVERSITY OF CALIFORNIA – DAVIS
2000/2001 Winter Nursery: Barley Stripe Rust Screening
L.F. Jackson ............................................................................................................. 1
Two-Rowed Barley Germplasm Improvement
L.W. Gallagher........................................................................................................... 4
COLORADO
COLORADO STATE UNIVERSITY
Barley stripe Rust International Germplasm Screening
W.M. Brown, Jr., J.P. Hill and V.R. Velasco .......................................................... 6
IDAHO
USDA-ARS NATIONAL SMALL GRAINS GERMPLASM RESEARCH FACILITY
Breeding, Germplasm Development, and Genetics of Improved Spring and Winter
Malting Barley
C.A. Erickson, D.E. Burrup and D.M. Wesenberg .................................................. 10
National Small Grains Collection Barley Germplasm Evaluations
H.E. Bockelman, C.A. Erickson and D.M. Wesenberg ........................................... 27
Development of Six-rowed Malting Barley Germplasm for the Western US Using
Conventional and Marker-assisted Selection Techniques
D.L. Hoffman and A. Hang ....................................................................................... 31
MINNESOTA
UNIVERSITY OF MINNESOTA
Minnesota Barley Improvement Project
K.P. Smith ................................................................................................................ 35
Management and Epidemiology of Diseases in Barley
R. Dill-Macky, C.K. Evans and B. Salas ................................................................. 42
Identification of Novel Disease Resistance Geenes for Barley
B.J. Steffenson ........................................................................................................... 47

Barley Transformation
G.J. Muehlbauer, L. Smith and N. Al Saady ........................................................... 51
MONTANA
MONTANA STATE UNIVERSITY
Developing Improved Malting Barley Varieties for Montana
T.K. Blake ................................................................................................................ 58
Epidemiology and Control of Barley Leaf Diseases Caused by Fungal Pathogens
M.R. Johnston .......................................................................................................... 66
NORTH DAKOTA
NORTH DAKOTA STATE UNIVERSITY
Two-rowed Barley Improvement Project
J.D. Franckowiak ..................................................................................................... 71
Six-rowed Barley Improvement Project
R.D. Horsley ............................................................................................................ 77
Studies on Barley Diseases and Their Control
S. Neate .................................................................................................................... 85
Malting and Brewing Quality of Barley
P.B. Schwarz ............................................................................................................ 89
USDA-ARS NORTHERN CROP SCIENCE LABORATORY
Attempts to Transform Newer Cultivars with Genes Affecting Trichothecene
Toxin and FHB Levels
L.S. Dahleen and M. Manoharan .............................................................................. 96
Developing Barley Tissue Culture Systems to Improve Plant Regeneration and Reduce
Somaclonal Variation
L.S. Dahleen and P. Bregitzer.................................................................................... 99
Barley Virus Diseases
M.C. Edwards .......................................................................................................... 104
OKLAHOMA
USDA-ARS PLANT SCIENCE RESEARCH LABORATORY
Germplasm Enhancement for RWA Resistance
D.W. Mornhinweg, D.R. Porter and J.A. Webster .................................................. 106
OREGON STATE UNIVERSITY
OREGON
The Oregon Barley Improvement Program
P.M. Hayes................................................................................................................. 109

WASHINGTON
WASHINGTON STATE UNIVERSITY
Molecular Marker Assisted Modification of Traditional High Quality Malting Barleys
A. Kleinhofs, D. von Wettstein and S.E. Ullrich ..................................................... 123
Barley Improvement in Washington
S.E. Ullrich, V.A. Jitkov, J.A. Clancy, M.C. Dugger, A. Kleinhofs
and D. v.Wettstein .................................................................................................... 126
WISCONSIN
USDA-ARS CEREAL CROPS RESEARCH UNIT
A Study of the Malting Quality of New Barley Selections
A.D. Budde, B.L. Jones, E. Goplin and D.M. Peterson .......................................... 137
Characterization of the Protein Hydrolyzing Systems of Mashes and Malts
B.L. Jones, D. Fontanini and L.A. Marinac............................................................... 143
Studies on the Proteinases That Are Produced When Fusarium Grows on Cereal
Proteins and Barley Proteins That Inhibit Them
A.I. Pekkarinen and B.L. Jones.................................................................................. 147
Studies of Starch Degradation and the Production of Fermentable Sugars in
Malting Barley
C.A. Henson .............................................................................................................. 132
Directed Expression of Antifungal Genes in Barley and Influences of Native
Antifungal Seed Proteins on Malting Quality
R.W. Skadsen ........................................................................................................... 149

1
2000/2001 WINTER NURSERY: BARLEY STRIPE RUST SCREENING
Lee Jackson, Extension Agronomist/Pathologist
Department of Agronomy & Range Science
University of California, Davis
Objectives
Barley stripe rust (BSR) continues to threaten barley production throughout the western
United States. Since its first occurrence in 1991, it has caused significant yield losses
throughout the region. Public and private breeding programs have developed lines and
varieties that are resistant to stripe rust, but continued screening for new sources of
resistance is needed since new races of the BSR fungus continue to appear. California,
where most barley acreage (primarily spring feed barley) is fall-sown, has experienced
very heavy stripe rust pressure each year since 1996. UC Davis, located in the northcentral
portion of the state, provides an ideal environment for screening. A key
advantage of the UC Davis site is that both winter and spring germplasm can be grown
successfully with fall-sowing (fall-sowing produces sufficient vernalization conditions
for winter barley while winter conditions are mild enough that spring barley has high –
essentially 100% - winter survival).
The main objective of the project is to identify sources of resistance to BSR among
barley accessions (spring and winter) from the National Small Grains Collection (NSGC)
and cooperator lines (breeding lines and newly developed varieties from public and
private breeding programs).
Methodology
About 2500 lines (primarily assembled by Darrell Wesenberg, USDA-ARS, Aberdeen
from the NSGC and public and private breeders; additional lines were received directly
from breeders) were screened in 2000/2001. Germplasm included cooperator lines (from
Hayes, Hensleigh, Clark, Wesenberg, An Hang, McProud, Treat, Roche, Ullrich, Wright,
Carleton, and Pickering (New Zealand)), 1000 lines from the NSGC, and about 100
Hordeum spontaneum accessions from Steffenson. The varieties Russell and Bancroft
were repeated checks throughout the nursery, while spreader rows of the California
susceptible check variety, Max, also were sown throughout the nursery. Single 8-ft rows
of each entry were sown on November 26-27, 2000. Time of initial infection was noted
and disease increase during the season was recorded. Disease severity ratings were made
on April 16-18 and on May 7-8 for the cooperator lines and on April 25-27 and May 9-10
for the winter barley accessions from the NSGC. Assessments of incidence and severity
of other diseases (primarily BYD, scald, and net blotch) also were made.
Results
Stripe rust was first detected in mid-to-late March. By the time of the final disease
severity rating, about 35% of the cooperator lines and 58% of the winter barley
accessions from the NSGC showed a 100S reaction. Many lines (22% of the cooperator

2
lines and 7% of the winter barley accessions from the NSGC) remained BSR-free. Stripe
rust collections were sent to Xianming Chen, USDA pathologist at Washington State
University in Pullman, WA for race identification. Of other diseases observed, both
BYD and scald occurred in high frequency and from low to high severity, depending on
the line. Low levels of net blotch and leaf rust were observed on some lines. Lines that
expressed moderate to high severity of BYD and/or scald were noted, along with their
stripe rust reactions. Disease severity ratings of evaluated lines are given in the attached
files.
Personnel:
Lee Jackson, Extension Agronomist/Pathologist
Diane Prato-Mayo, Lab Assistant

3
2001 Barley Stripe Rust Screening Nursery, UC Davis
Group II: Winter Barleys, National Small Grains Collection
Summary of Ratings
Disease rating 5/9-5/10, 2001 (% severity)
Source # Lines 0 5 to 10 >10 to 20 >20 to 40 >40 to 60 >60 to 80 >80 to 100
Afghanistan 1 0 0 0 0 0 0 1
Albania 1 0 0 0 0 0 0 1
Algeria 1 0 0 0 0 1 0 0
Armenia 7 0 1 1 0 0 1 4
Australia 1 1 0 0 0 0 0 0
Austria 17 2 3 1 1 4 2 4
Azerbaijan 33 3 1 2 5 10 2 10
Belgium 6 1 0 1 1 2 0 1
Bosnia 1 0 0 0 0 0 0 1
Bulgaria 5 0 0 0 2 0 0 3
Canada 31 2 2 1 1 6 1 18
China 17 0 0 1 0 1 0 15
Croatia 1 0 0 0 1 0 0 0
Czechoslovakia 4 0 0 0 2 1 0 1
Czech Republic 1 0 0 0 0 0 0 1
Denmark 1 0 0 0 1 0 0 0
Europe 1 0 0 0 1 0 0 0
Ethiopia 4 2 0 0 0 1 0 1
Former Soviet Union 4 1 0 0 1 1 0 1
France 15 2 1 3 1 1 1 6
Georgia 5 0 0 0 1 2 0 2
Germany 64 19 8 5 6 11 5 10
Greece 8 2 0 1 1 0 0 4
Hungary 8 0 0 0 2 0 2 4
India 9 5 1 1 0 0 0 2
Iran 32 0 1 1 0 0 0 30
Japan 35 0 0 0 0 4 0 31
Kyrgyzstan 1 0 0 0 0 0 1 0
Macedonia 14 1 0 0 0 0 3 10
Moldova 2 0 0 0 0 1 0 1
Morocco 1 0 0 0 1 0 0 0
Netherlands 10 5 1 2 0 0 0 2
North Korea 2 0 0 0 0 0 0 2
Norway 1 0 0 0 0 0 0 1
Pakistan 2 0 0 0 0 0 0 2
Poland 27 3 1 2 5 5 3 8
Portugal 1 1 0 0 0 0 0 0
Romania 10 1 0 0 1 3 3 2
Russian Federation 74 2 1 5 2 12 9 43
Slovakia 1 0 0 0 0 1 0 0
South Korea 95 0 0 0 4 5 2 84
Spain 7 0 0 0 4 1 0 2
Sweden 3 2 1 0 0 0 0 0
Turkey 35 2 0 0 1 2 1 29
Turkmenistan 9 0 0 0 1 0 1 7
Ukraine 7 0 0 1 1 1 1 3
United Kingdom 5 2 0 1 2 0 0 0
United States 362 10 4 13 16 53 40 226
Uzbekistan 2 0 0 0 0 0 0 2
Western Asia 1 0 0 0 0 0 0 1
Yugoslavia 15 1 0 5 3 1 2 3
Total 1000 70 26 47 68 130 80 579
% 7 3 5 7 13 8 58

4
TWO-ROWED BARLEY GERMPLASM IMPROVEMENT
Lynn W. Gallagher
Department of Agronomy & Range Science
University of California, Davis 95616
The production of malting barley in California has been sporadic over the last century.
Prior to World War II as much as 30% of the production of the barley variety Coast was
exported to England for brewing. In earlier times six-rowed Coast’s production was 10%
malted and 37% exported. Atlas barley, a selection from Coast, became the genotype
upon which a whole series of improvements was made ending in the barley yellow dwarf
resistant Atlas 68. None of the Atlas series of varieties gained acceptance as a malting
barley because of thickness of hull, bitterness, and other reasons. The two-rowed variety
Hannchen gained minor acceptance when grown in the Paso Robles area of the Central
Coast. In more recent decades the southern part of the Klamath Basin near Tulelake has
been used from time to time to grow malting barley. Malting barley as a breeding
objective was abandoned by UCD even though beer production and consumption in the
state has been growing and demand for malt increasing. The focus of the breeding
program at UCD has traditionally been six-rowed feed barley. Despite increasing grain
yields per unit area and a deficit in feed grains, the area in barley production has declined
from two million to about one hundred thousand acres during the last fifty years. To
provide greater grower choice, the UCD barley breeding program began to explore the
possibility of creating malting barley for California growing conditions by taking
advantage of two-rowed germplasm not previously evaluated or exploited.
For the last decade ICARDA/CIMMYT barleys have been evaluated at Davis and a
number of two-rowed barleys have exhibited good plant types and moderate levels of
productivity given their earliness to head when compared to the commonly grown feed
barleys. More recently two-rowed barleys from Oregon St. University have been
evaluated at Davis. Two lines from Mexico were identified as having good plant types
and multiple disease resistances. The genotypes 22IBON153 (Emir/5/Apizaco/CM67//
Russell/Aleli) and 23IBON61 (Arupo/K8753//Aleli) were evaluated for malting quality
over three years at the USDA malt lab in Wisconsin (Table 1). These lines had both
positive and negative malting qualities. On the positive side were kernel weight,
plumpness, grain protein, wort clarity, alpha-amylase and beta-glucan values. On the
negative side were low values for wort protein, soluble/total protein, DP, and malt
extract. By contrast BCD 47 from Oregon St. U. exhibited good or higher wort protein,
soluble/total protein, DP, and malt extract but is susceptible to BYDV and scald. The
two Mexican genotypes were hybridized to BCD 47 creating an initial gene pool.
Backcrosses of the F1’s were made to BCD 47. Single crosses are now in F3 bulks
which will be harvested in May 2002. Subsequently these bulks will be divided and
evaluated at Tulelake and Davis, two environments in which varieties are generally not
interchangeable.
In the 2001 growing season four more two-rowed genotypes exhibited near malting
quality. Three lines (SalAjo1, BU 27, and OR 2967102) from Pat Hayes’ program at
Oregon State achieved good malt quality scores. An additional promising line is

5
Triumph/Tyra//Arupo, a selection from an ICARDA/CIMMYT F2 sample received years
ago. These short statured lines provide the plant type required for Central Valley
environments because few tall Midwestern malting varieties are productive after a
shattering episode created by fifty mile per hour spring winds. The diseases of scald,
BYDV, and stripe rust are additional impediments to the utilization of Midwestern
malting barley genotypes in the Central Valley of California.
Table 1. Malting values for selected two-rowed barley genotypes grown at Davis,
California.
Entry KWt 6/64 Color Ext. Wrt
Clr
Wrt
Clrty Prot
Sol.
Pro.
mg % % % % %
S/T DP AA Bglu Score
1999
22IB153 46.8 95.5 68 75.3 1.6 1 11.8 4.03 35.6 81 37.4 91 33
23IB61 51.0 93.9 68 74.7 1.9 2 10.1 3.61 36.6 71 42.9 120 35
BCD47 40.2 75.9 73 76.9 1.6 1 13.0 4.93 38.6 163 74.7 159 21
H MaltChk 37.2 81.2 79 79.8 1.6 1 12.0 5.61 47.3 135 77.0 45 29
2000
22IB153 48.7 99.3 57 74.9 1.3 1 11.6 4.22 35.4 80 39.7 154 29
23IB61 48.0 99.5 52 74.6 1.5 1 11.7 4.11 35.0 82 45.2 257 32
BCD47 44.9 95.0 50 79.7 1.9 1 9.9 4.90 49.6 110 81.5 100 42
H MaltChk 36.2 89.8 57 78.6 1.9 1 12.1 5.39 43.5 79 51.9 354 31
2001
22IB153 48.3 91.9 70 78.8 1.3 1 9.8 3.50 36.9 39 41.1 75 31
23IB61 47.5 98.0 69 78.8 1.5 1 10.0 3.94 39.5 57 46.0 166 31
BCD47 45.7 93.2 70 82.2 1.8 1 9.1 4.64 52.1 79 76.2 67 37
H MaltChk 40.1 95.1 75 81.7 1.8 1 11.6 6.17 56.3 139 77.0 46 38
Harrington 38.2 89.1 81 82.5 1.5 1 8.1 4.49 60.6 62 61.7 24 29
SalAjo1 44.7 89.5 71 79.5 1.6 1 10.9 4.76 45.7 96 77.6 124 50
Tri/Ty//Arup 47.1 96.6 64 80.4 1.7 1 8.8 3.83 47.1 106 42.5 51 38
BU 27 44.2 93.5 74 81.0 1.7 1 8.5 4.52 54.6 73 68.3 56 37
OR2967102 47.2 87.3 70 80.3 1.7 1 9.8 5.01 52.9 89 79.3 134 36

6
BARLEY STRIPE RUST INTERNATIONAL GERMPLASM SCREENING
William M. Brown, Jr., Joseph P. Hill and Vidal R. Velasco
Bioagricultural Sciences and Pest Management
Colorado State University
Fort Collins, CO 80523
Background
Barley stripe rust (BSR), caused by Puccinia striiformis f.sp. hordei, is now well
established in the western U.S. It was introduced into South America from Europe in
1975. It then spread north and was found in Uvalde, TX in 1991. Since that time the
disease has been found in all principal barley growing areas of the west at different times.
It is now endemic to the northwest United States (California, Oregon and Washington)
and has caused significant damage in the Central Valley of California over the last
several seasons. In other malt barley producing areas of the U.S. stripe rust has not yet
developed into a significant problem although in some years it has caused considerable
damage. The extent of damage in the Rocky Mountain area is usually dependent upon
inoculum development and spread from northern Mexico.
There still are only a few commercial barley cultivars with resistance to barley stripe rust.
One of the first barley stripe rust resistant cultivars to be released is Bancroft, which was
developed in this program. Bancroft is a joint release from the USDA-ARS at Aberdeen,
ID, Colorado State University, Idaho State University and Oregon State University.
Additionally, numerous sources of resistance have been identified and several are being
incorporated into commercially available malting varieties to provide sufficient levels of
resistance. Levels of resistance being observed in field trials in cooperator lines is very
encouraging.
Methods
All trials were planted in non-replicated 1 meter long rows (5 gms per entry).
Disease severity was recorded as percentage of rust infection on the plants according to a
modified Cobb scale. Response, referring to infection types, was also recorded according
to the pictorial scale developed by Stubbs and Vench and utilized as a standard by the
International Center for Maize and Wheat Improvement (CIMMYT).
Field trials are conducted with endemic natural inoculum levels of the barley stripe rust
pathogen present in the Toluca Valley of Mexico (8,000 ft), the high valleys of Peru
(winter nursery) at Callejon de Hualas (7,000 ft), Huancayo (11,000 ft) and lower levels
of the pathogen in the San Luis Valley (7,000 ft) of south central Colorado. An additional
set of the test nursery was planted in Yuma, Colorado (4,000 ft). This later plot was an
overhead irrigated plot located in eastern Colorado where the disease is not endemic but
an increasing interest is being expressed in growing malting barley because the area is
free of Fusarium scab and other grain discoloring fungi.
The accessions in the Cooperative BSR Trial were obtained from programs in Idaho
(Coors Brewing Company, Plant Breeders 1, and USDA/ARS), Oregon (Oregon State
University), Montana (Western Plant Breeders and Montana State University), and

7
Washington (Fossum Cereals and Washington State University). These entries were
planted in Toluca, the San Luis Valley and Yuma, Colorado.
Results
In Toluca Valley all varieties were exposed to high levels of natural endemic inoculum of
the stripe rust pathogen. A total of 11.4 % of the 902 entries were rated resistant.
Unfortunately in the San Luis Valley and Yuma, Colorado insufficient levels of infection
developed and accurate evaluations for BSR were not possible.
In Huncayo only 5.1 % of the entries were rated resistant. While in Callejon de Huaylas
63.1 % were rated resistant (Table 1) most of the entries rated resistant were in the range
of 0 infection type.
Table 1. Barley Stripe Rust Results in Mexico and Peru, 2000-01
INFECTION TYPE
HUANCAYO
%
PERU
CALLEJO DE HUYLAS
%
MEXICO
TOLUCA VALLEY
%
0 3.5 56.3 0.0
R 1.1 4.4 4.8
MR 0.5 2.4 0.0
MS 2.9 8.6 6.6
S 92.0 28.3 88.6
Different infection types were observed in certain lines in Huancayo and Callejon de
Huaylas in Peru and the Toluca Valley, Mexico. In Callejon de Huaylas some entries
were resistant while in Huancayo and Toluca Valley they were susceptible. The entries
BCD 47 and 00-2R-0064 were resistant in the Toluca Valley and Callejon de Huaylas
while in Huancayo they were susceptible see
Table 2. Comparison of Barley Stripe Rust Infection Types in Selected Lines in
Huancayo, Callejon de Huaylas, Peru and Toluca Valley, Mexico
PEDIGREE
PERU
HUANCAYO CALLEJON DE HUAYLAS
MEXICO
TOLUCA VALLEY
99ID967 90 S T MR 90 S
BCD 47 100 S 0 5 MS/TR
CENTURY 100 S T MS 70 S
6B98 – 9058 100 S T R 100 S
2B98–5421 100 S 5 MR 90 S
00–2R–0064 50 S 0 T R

8
All field observations have been forwarded to the USDA-ARS laboratory at Aberdeen,
Idaho and entered into the GRIN system.
Discussion and Recommendations
In the Toluca Valley of Mexico, the environmental conditions continue to be conducive
to disease development. In 2001 the high level of natural inoculum continued to be
conducive to disease occurrence and provided good pathogen pressure and subsequent
disease development. Susceptible controls showed a very high degree of infection (100%
in many instances) while the newly released Brancroft (being used as a resistant check)
was generally resistant or, at most sustained a trace of rust.
In the Huancayo Valley the environment was also conducive to development of high
inoculum pressure and disease development. In Callejon de Huaylas 56.3% of the entries
were in the infection type range of 0. This could possibly be due to escapes resulting
from heavy rains and high levels of Helminthosporium teres damage.
The differences in infection type in the same entries in Huancayo, Callejon de Hualas in
Peru and the Toluca Valley in Mexico are very likely due to the presence or emergence of
different BSR races.
The percentage of lines with resistance to BSR from all sources has consistently proved
to be higher than prior years. These lines are essentially the result of the successful
identification and selection by the participating breeders in the various programs. Over
the years this rapid field-screening program has quickly identified lines of varying levels
of resistance. Also, the program has consistently proved to be an effective method for
rapid identification of resistance sources.
While significant progress has been made identifying barley stripe rust resistance, there is
still a major problem in developing agronomically acceptable reliable resistant lines for
deployment in the near future. This is especially clear this year with the high levels of
susceptibility shown at the Hyancayo site in lines that exhibit resistance at the other sites.
While there are more commercially available lines with sufficient levels of resistance to
BSR and the essential qualities necessary for a good malting barley, the breakdown of
resistance as typified by Bancroft and other lines at Hyancayo (refer to GRIN data for
specific lines) again demonstrates the diversity of the stripe rust populations.
The winter nurseries were planted in December 2001 at both locations in Peru. As noted
above these sites provide excellent alternative season (winter) locations for further
screening to develop germplasm in an accelerated manner.
Acknowledgements
Dr. Hugo Vivar and Dr. Flavio Capitini (Dr. Vivar’s replacement) CYMMIT, Mexico
for field management of the trials.
Dr. Luz Gomez Pando, Head, Programa de Cereales y Leguminosas, Universidad
Nacional Agraria La Molina, Lima, Peru for field management and cooperation In the
Peruvian winter nursery.
Dr. Darrel Wesenberg and Dr. Harold Bockelman at the USDA/ARS Small Grains
Laboratory, Aberdeen, ID for providing and preparing barley lines used in this program.

9
This project is supported by the USDA/ARS Small Grains Laboratory, Aberdeen, ID and
the American Malting Barley Association (AMBA).
Papers from the project in 2001
Brown, W.M., J. P. Hill and Vidal R. Velasco. 2001. Yellow Rust in North American.
Annu. Rev. Phytopathol. 39:367-84.
Brown, W.M., J. P. Hill and Vidal R. Velasco. 2001. The Role of Resistance in Stripe
Rust Management. Proceedings of the 33 rd Barley Improvement Conference. San
Antonio, TX January 10-12, 2001. pp 34-39.
Brown, W.M, J. P. Hill and V. R. Velasco. 2001. Importance of Identification of Stripe
Rust Resistance in Barley. In Vivar, H.E. and McNab, A. (eds) 2001. Breeding Barley in
the New Millenium: Proceedings of an International Symposium. Mexico D.F. pp18-24.
Symposium abstracts of papers presented in the First Regional Yellow (Stripe) Rust
Conference for Central & West Asia and North Africa. Karaj, Iran May 8-14, 2001.
Proceedings in press.
Brown, W.M., Jr., Hill, J.P., and Velasco, V.R. 2001. Identification of barley yellow rust
resistance I the Americas: a case study of successful international cooperation. p 8
(abstr.)
-------------------------------------------------. 2001. Pathogen variability and the role of
resistance in barley yellow rust management. p. 72 (abstr.)
--------------- and Velasco, V.R. 2001. Chemical suppression of barley yellow rust. p 60
(abstr.)
Capettini, F. , Brown, W.M. and Velasco, V.R. 2001. Occurrence and spread of barley
yellow rust in the Americas. P. 7 (abstr.)

BREEDING, GERMPLASM DEVELOPMENT, AND GENETICS OF IMPROVED
SPRING AND WINTER MALTING BARLEY
C. A. Erickson, D.E. Burrup, and D.M. Wesenberg
Agricultural Research Service - USDA
National Small Grains Germplasm Research Facility
Aberdeen, Idaho
10
Justification
Malting barley is an important crop in Idaho. Improved, high yielding, short strawed,
lodging resistant germplasm and varieties with genetic resistance to important diseases,
insect pests, and other environmental stresses coupled with improved management could
help to achieve the potential of malting barley in Idaho and the West. Plant pathogens
have had minimal impact on barley production in Idaho and most other western states
historically. The Russian wheat aphid has emerged as a significant threat to barley
production in Idaho and other western states and likewise barley stripe rust was widely
reported in Idaho in 1993, 1995, and 1998, adding another significant threat to barley
production and quality in these environments.
Objectives
Development of improved two-rowed and six-rowed spring and winter malting barley
varieties and germplasm lines adapted to irrigated and dryland production in Idaho and
other western states. Primary emphasis of the program is on the improvement of irrigated
spring barley with secondary emphasis on dryland spring barley. The modest winter
program emphasizes the transfer of desirable malting quality characteristics from spring
barley to winter barley germplasm and varieties. Characteristics given prime
consideration include high yield, resistance to lodging and shattering, short straw,
improved malting and brewing quality, and improved disease and insect resistance.
Significant attention is being given to barley stripe rust resistance. Evaluation of
agronomic performance, malting and brewing quality, and pest resistance response of
promising malting varieties and selections will focus on irrigated barley performance
trials at Aberdeen and nonirrigated or dryland trials at Tetonia.
Preliminary Results
ADVANCED TESTING PROGRAM - TWO-ROWED SPRING BARLEY
Over forty two-rowed barley varieties were tested in replicated trials at Aberdeen and/or
Tetonia in 2001 in trials that included nearly 300 two-rowed barley entries. These
barleys included proprietary varieties and selections plus barleys developed by public
programs in Idaho, Montana, North Dakota, Oregon, Washington, and Canada. Named
varieties in these trials included ‘Acuario’, ‘Alexis’, ‘Bancroft’, ‘Baronesse’, ‘BCD 47’,
‘Bear’, ‘CDC Bold’, ‘B1202’, ‘Camas’, ‘Chinook’, ‘Clark’, ‘Condor’, ‘Cooper’, ‘CDC
Copeland’, ‘Crest’, ‘Criton’ (91Ab3148), ‘Crystal’, ‘CDC Dolly’, ‘Galena’, ‘Garnet’,
‘Harrington’, ‘Hector’, ‘CDC Helgason’, ‘Idagold’, ‘Jersey’, ‘CDC Kendall’, ‘Manley’,
‘CDC McGwire’, ‘Merit’, ‘Merlin’, ‘AC Metcalfe’, ‘Moravian 14’, Moravian 37’,
‘Munsing’, ‘Orca’, ‘Samish 23’, ‘Seebe’, ‘CDC Select’, ‘CDC Stratus’, ‘Targhee’,

11
‘Triumph’, ‘Valier’, ‘Waxbar’, and ‘Xena’. In addition to the various replicated trials
grown at Aberdeen and Tetonia, about 140 two-rowed barleys were grown under
irrigation in nonreplicated trials at Aberdeen in 2001 for preliminary evaluations of
agronomic and quality characteristics. Approximately forty two-rowed entries were also
grown in similar nonreplicated trials on dryland at Tetonia in 2001.
Promising two-rowed spring barley selections developed at Aberdeen and currently being
considered in AMBA-sponsored pilot or plant-scale evaluations of malting and brewing
quality or other advanced trials and evaluations include 90Ab241 (A517/Harrington//
Harrington), 94Ab12990 (85Ab2323/ND9147), 95Ab11469 (87Ab9561/ND9870),
98Ab11865 (85Ab2323/Galena), and 98Ab11993 (90Ab241/Baronesse).
Summaries of agronomic data for two-rowed barley varieties and selections in the
Advanced Yield Nurseries (trial A) grown under irrigation at Aberdeen and Tetonia,
Idaho from 1999 through 2001 are shown in Tables 1 and 2, respectively. The selections
95Ab11469 and 98Ab11865 performed especially well under irrigation at Aberdeen, with
95Ab11469 showing good kernel plumpness along with high yield. The selections
90Ab241 and 95Ab11469 showed fair to good yield at Tetonia, with 95Ab11469 again
showing good plump kernel percentage. Table 3 shows the agronomic performance of
two-rowed varieties and selections on dryland at Tetonia from 1999 through 2001. The
selections 94Ab12990 and 95Ab11469 demonstrated very good yield and were
comparable in performance to 85Ab2323 in these tests.
The selection 90Ab241 was submitted for AMBA pilot-scale tests of malting and
brewing quality in 2001 for a third year of testing. The selection 90Ab241 has a good
agronomic record in irrigated and dryland trials in southern Idaho and promising malting
quality characteristics. 90Ab241 was equal in yield to Merit under irrigation at Aberdeen
in seven years of trials (Table 4) and equal to Garnet under irrigation at Tetonia over five
years (Table 5). Its performance on dryland at Tetonia over five years (Table 6) was not
as good and was significantly less than 85Ab2323. The malting quality data for 90Ab241
at Aberdeen and Tetonia are shown in Tables 7-10. It has shown higher plumps and malt
extract than 85Ab2323 with lower barley and wort protein. It’s alpha amylase activity is
also higher than 85Ab2323. A winter two-rowed selection, 94Ab1274
(MT81616/81Ab1702), was also submitted for a third year of AMBA pilot-scale testing
in 2001. (See details below under WINTER BARLEY.)
The selection 98Ab11865 which was highlighted in last year’s report continued to show
high yield potential under irrigation at Aberdeen (Table 1), outperforming all other
varieties and selections except for Xena over the past two years. It was not submitted for
pilot-scale testing in 2001, but will be included in the 2002 AMBA pilot-scale multilocation
testing program coordinated by Pat Hensleigh at Montana State University at
Bozeman. The selection 98Ab11993 is the other two-rowed entry in this testing program.
This selection has good agronomic performance with a yield equal to or better than
85Ab2323 for three years under irrigation at Aberdeen (Table 1) and with good plump
kernel percentage. The three six-rowed entries in the AMBA program will be discussed
later.

12
The testing of the selection 85Ab2323 has been continued. It has performed especially
well in field trials on dryland at various locations in northern and southern Idaho in recent
years. Agronomic data from Tetonia are shown in Tables 2 and 3. This selection may
have potential as a variety in some production areas of Idaho. Although the University of
Idaho Foundation Seed Stocks Committee approved the selection for release in December
2000, a variety name has not yet been assigned.
Table 1. Agronomic data for selected two-rowed varieties and selections grown in trial A under
irrigation at Aberdeen, Idaho, 1999-2001.
Yield (bu/A) Test Plump Heading
Average Weight* Barley* Date* Height* Lodging*
Entry 1999 2000 2001 1999-2001 (lbs/bu) (%) (Julian) (in) (%)
No. Trials 3 1 1 1 1 1
Bancroft 141.2 135.0 143.9 140.0 54.8 91 176 36 90
Baronesse 139.3 174.4 163.5 159.1 54.4 94 176 34 15
B 1202 135.6 130.0 149.9 138.5 55.2 96 176 33 28
Camas 153.8 153.2 160.8 155.9 57.0 91 174 34 0
Crest 147.9 133.3 153.2 144.8 55.3 92 175 34 19
Criton 146.2 126.1 149.2 140.5 55.1 99 176 35 1
Galena 152.3 149.8 148.2 150.1 56.6 93 179 33 3
Garnet 159.4 140.4 154.3 151.3 56.2 98 175 36 1
Harrington 149.8 113.0 143.1 135.3 53.9 84 175 34 76
Merit 144.0 136.8 143.7 141.5 53.7 78 179 36 23
Metcalfe 143.5 120.0 141.0 134.8 55.9 93 176 38 60
Moravian 14 138.9 138.4 133.1 136.8 56.4 94 171 27 0
Moravian 37 146.1 133.2 139.7 139.7 56.6 99 177 29 3
MT910189 ---- 134.2 144.9 ---- 55.2 89 174 34 93
Orca 120.4 139.1 132.3 130.6 55.6 97 167 32 5
CDC Stratus ---- ---- 122.4 ---- 55.8 96 181 31 3
Xena ---- 160.4 168.8 ---- 55.9 98 178 36 15
85Ab2323 158.0 115.9 150.4 141.4 55.0 94 175 35 46
90Ab241 149.6 140.5 151.2 147.1 54.8 97 175 35 21
91Ab6526 166.3 121.0 148.2 145.2 53.9 93 175 37 65
93Ab859 143.4 154.4 150.9 149.6 56.6 99 176 36 30
94Ab12990 ---- ---- 150.7 ---- 56.3 92 174 37 0
95Ab11469 162.8 137.4 155.2 151.8 54.9 97 172 36 23
95M4623 ---- 131.1 144.6 ---- 54.4 96 176 37 63
95SR149C ---- 149.7 152.7 ---- 52.9 83 179 36 78
95SR316A ---- 116.5 152.6 ---- 54.7 90 179 36 53
98Ab11720 151.8 118.7 156.9 142.5 53.4 91 179 33 38
98Ab11771 ---- ---- 143.6 ---- 54.4 92 174 33 58
98Ab11865 ---- 163.2 163.9 ---- 55.3 91 179 31 3
98Ab11993 ---- 134.3 158.9 ---- 53.6 96 176 33 50
98Ab12019 ---- ---- 156.7 ---- 53.6 92 178 30 18
*2001 data only

13
Table 2. Agronomic data for selected two-rowed varieties and selections grown in trial A under
irrigation at Tetonia, Idaho, 1999-2001.
Test Plump Heading
Yield Weight Barley Date Height Protein Oil
Entry (bu/A) (lbs/bu) (%) (Julian) (in) (%) (%)
No. Trials 3 3 3 3 1 1 1
85Ab2323 115.0 53.4 93 194 30 11.8 2.2
90Ab241 110.0 51.6 95 195 27 9.3 2.0
91Ab6526 123.1 53.8 95 193 33 12.6 2.2
93Ab859 112.2 53.7 94 196 28 11.5 2.1
95Ab11469 110.0 53.4 95 192 29 12.0 1.8
B 1202 107.7 51.3 86 194 28 12.6 2.1
Bancroft 108.5 52.5 88 193 29 11.4 2.2
Baronesse 119.7 51.7 83 193 24 11.8 2.2
Camas 121.0 54.3 92 191 28 12.7 2.1
Crest 110.3 52.9 92 193 28 11.9 2.2
Criton 115.2 52.5 97 194 28 10.7 2.3
Galena 104.9 52.4 85 198 27 10.3 2.3
Garnet 112.2 52.0 95 195 31 11.3 2.1
Harrington 100.9 52.1 83 195 28 11.1 2.2
IdaGold II 105.3 52.7 89 197 26 11.0 1.9
Merit 108.8 51.6 84 198 30 11.6 2.2
AC Metcalfe 111.8 52.6 89 194 30 12.0 2.0
Moravian 14 109.2 54.2 90 189 26 11.8 1.9
Moravian 37 115.1 53.7 96 196 26 10.2 1.7
Orca 95.6 53.3 98 188 29 11.5 2.2

14
Table 3. Agronomic data for selected two-rowed varieties and selections grown on dryland at
Tetonia, Idaho, 1999-2001.
Test Plump Heading
Yield Weight Barley Date Height Protein Oil
Entry (bu/A) (lbs/bu) (%) (Julian) (in) (%) (%)
No. Trials 3 3 3 3 1 1 1
85Ab2323 75.9 52.5 95 195 21 13.4 2.3
90Ab241 66.6 50.5 94 195 21 13.8 2.3
94Ab12990 72.8 53.2 92 193 26 13.3 2.1
95Ab11469 72.4 52.4 96 192 25 13.9 2.1
Bancroft 69.2 51.6 88 194 23 12.7 2.0
Baronesse 70.4 51.0 89 194 22 13.1 2.1
Camas 74.8 53.5 91 193 23 13.3 1.9
Chinook 71.8 52.7 84 195 24 14.3 2.1
Clark 64.1 51.0 83 196 23 15.0 2.0
Criton 70.7 51.0 95 195 22 14.8 2.0
Farmington 59.1 52.0 79 195 20 16.3 2.1
Harrington 66.6 51.2 80 195 24 14.3 2.2
Hector 72.2 52.1 83 194 21 14.9 1.9
Merit 69.3 51.0 85 195 23 13.3 2.3
MT910189 66.3 52.4 89 192 25 14.5 2.2
Munsing 65.7 52.1 84 193 20 18.1 1.9
Targhee 70.2 51.6 87 194 23 14.2 2.3
Valier 65.5 52.1 87 196 22 15.9 1.9
Xena 76.5 52.5 95 195 24 13.9 2.3

15
Table 4. Agronomic data for selected two-rowed varieties and selections grown under irrigation
at Aberdeen, Idaho, 1994 & 1996-2001.
Test Plump Heading
Yield Weight Barley Date Height Protein Oil
Entry (bu/A) (lbs/bu) (%) (Julian) (inches) (%) (%)
No. Trials 7 7 7 7 7 3 3
85Ab2323 138.1 55.0 95 175 37 13.2 1.9
90Ab241 144.7 54.6 97 176 36 13.7 2.1
B 1202 139.0 54.0 96 176 34 14.1 1.8
Bancroft 135.2 54.5 93 175 48 13.5 1.9
Baronesse 151.2 54.3 95 176 34 12.9 2.0
Crest 140.2 53.9 94 174 34 12.7 1.8
Galena 144.3 54.8 94 179 32 12.1 2.1
Garnet 139.4 53.9 97 177 37 14.0 2.2
Harrington 134.2 54.2 90 177 35 13.4 1.9
Merit 145.9 53.7 91 179 36 12.1 2.0
Moravian 14 135.8 55.1 94 171 29 12.7 1.9
Steptoe* 156.5 50.9 95 170 36 11.9 1.7
* Six-rowed check
Table 5. Agronomic data for selected two-rowed varieties and selections grown under irrigation
at Tetonia, Idaho, 1997-2001.
Test Plump Heading
Yield Weight Barley Date Height Protein Oil
Entry (bu/A) (lbs/bu) (%) (Julian) (inches) (%) (%)
No. Trials 5 5 5 5 1 4 3
85Ab2323 112.9 52.8 94 194 30 13.0 1.9
90Ab241 106.6 51.2 95 195 27 12.1 1.8
B 1202 107.4 51.0 89 194 28 13.3 1.8
Bancroft 111.1 51.8 89 193 29 12.9 1.9
Baronesse 120.4 51.7 88 193 24 12.9 2.0
Crest 107.7 52.1 93 193 28 12.8 2.0
Criton 114.6 52.2 97 193 28 12.4 2.0
Galena 110.3 52.0 86 198 27 12.3 2.1
Garnet 106.3 51.6 96 195 31 12.6 2.0
Harrington 99.2 51.9 87 194 28 12.8 2.0
Merit 108.0 50.7 86 197 30 12.7 1.9
Moravian 14 109.6 53.5 91 191 26 13.0 1.9
Steptoe* 121.0 50.5 93 188 30 11.6 2.1
* Six-rowed check

16
Table 6. Agronomic data for selected two-rowed varieties and selections grown on dryland at
Tetonia, Idaho, 1997-2001.
Test Plump Heading
Yield Weight Barley Date Height Protein Oil
Entry (bu/A) (lbs/bu) (%) (Julian) (in) (%) (%)
No. Trials 5 5 5 5 1 3 3
85Ab2323 87.2 52.9 95 194 21 13.4 2.3
90Ab241 77.7 50.5 94 195 21 13.8 2.3
94Ab12990 83.7 53.8 93 192 26 13.3 2.1
Bancroft 83.5 51.9 90 194 23 12.7 2.0
Baronesse 82.4 51.5 90 194 22 13.1 2.1
Camas 86.2 53.6 90 193 23 13.3 1.9
Chinook 84.4 52.7 87 194 24 14.3 2.1
Clark 75.3 51.3 86 195 23 15.0 2.0
Criton 83.1 51.4 96 194 22 14.8 2.0
Harrington 77.2 51.4 83 195 24 14.3 2.2
Hector 84.8 52.4 86 194 21 14.9 1.9
Munsing 73.5 52.0 85 191 20 18.1 1.9
Targhee 84.7 51.9 91 194 23 14.2 2.3
Table 7. Malting quality data* for selected two-rowed varieties and selections grown in six
irrigated trials at Aberdeen, Idaho, 1996-2001.
Plump Malt Barley Wort Alpha Beta
Barley Extract Protein Protein S/T DP Amylase Glucan
Entry (%) (%) (%) (%) (%) (ºASBC) (20ºDU) (ppm)
85AB2323 94 80.2 13.6 5.25 39.7 127 48.5 271
90AB241 97 80.6 13.1 5.12 39.9 128 63.8 284
B 1202 95 79.0 13.9 4.92 36.3 116 52.8 184
Galena 94 79.2 12.7 4.19 33.5 104 38.7 321
Garnet 97 80.4 13.4 5.43 41.9 142 58.2 170
Harrington 86 78.7 13.4 4.62 35.4 101 46.3 378
Merit 91 80.9 12.9 5.20 42.0 142 69.5 178
Moravian 14 93 78.9 13.1 4.29 33.7 110 39.3 277
* Malting quality data courtesy of USDA-ARS Cereal Crops Research Unit, Madison
Wisconsin.

17
Table 8. Malting quality data* for selected two-rowed varieties and selections grown in five
irrigated trials at Tetonia, Idaho, 1995-97 and 1999-2000.
Plump Malt Barley Wort Alpha Beta
Barley Extract Protein Protein S/T DP Amylase Glucan
Entry (%) (%) (%) (%) (%) (ºASBC) (20ºDU) (ppm)
85Ab2323 89 79.0 13.5 4.57 34.4 155 46.0 360
90Ab241 91 79.3 12.8 4.40 35.6 158 57.6 242
Bancroft 83 78.1 13.6 4.36 32.9 155 45.3 417
B 1202 83 77.4 13.8 4.43 32.9 129 50.7 392
Crest 90 78.1 13.3 4.82 36.9 128 57.6 365
Crystal 82 77.6 14.6 4.59 32.2 161 55.9 528
Garnet 92 78.7 13.6 4.44 33.7 170 52.8 291
Harrington 78 77.6 12.9 3.86 30.1 108 40.5 537
* Malting quality data courtesy of USDA-ARS Cereal Crops Research Unit, Madison,
Wisconsin.
Table 9. Malting quality data* for selected two-rowed varieties and selections grown in seven
irrigated trials at Aberdeen, Idaho, 1997-2000 and Tetonia, Idaho, 1997 & 1999-2000.
Plump Malt Barley Wort Alpha Beta
Barley Extract Protein Protein S/T DP Amylase Glucan
Entry (%) (%) (%) (%) (%) (ºASBC) (20ºDU) (ppm)
85Ab2323 92 79.8 13.3 5.05 39.2 141 48.7 321
90Ab241 95 80.7 12.3 4.95 41.2 140 64.0 221
93Ab859 95 80.3 12.4 4.88 41.0 149 58.7 295
B 1202 91 78.8 13.3 4.73 36.6 122 54.5 246
Crystal 89 79.1 13.7 4.99 36.8 147 60.6 395
Garnet 95 80.2 12.9 5.06 40.4 151 57.6 221
Harrington 83 78.3 12.7 4.18 33.6 96 42.6 423
* Malting quality data courtesy of USDA-ARS Cereal Crops Research Unit, Madison,
Wisconsin.

18
Table 10. Malting quality data* for selected two-rowed varieties and selections grown in four
irrigated trials at Aberdeen, Idaho, 1999-2001 and Tetonia, Idaho, 2000.
Plump Malt Barley Wort Alpha- Beta-
Variety or Barley Extract Protein Protein S/T DP amylase glucan
Selection (%) (%) (%) (%) (%) (°ASBC) (20°DU) (ppm)
85Ab2323 91 79.1 14.5 5.18 37.3 140 52.7 383
90Ab241 95 80.2 13.6 5.13 38.8 144 69.0 332
94Ab12990 95 77.9 13.7 4.36 33.0 133 43.8 556
95Ab11469 90 78.1 12.9 3.43 33.5 104 37.8 498
98Ab11865 91 78.7 13.7 4.30 32.9 115 55.7 472
98Ab11993 93 79.0 13.7 4.55 34.8 119 63.7 368
B 1202 90 78.1 14.7 4.89 34.5 115 55.1 286
Galena 87 77.5 13.7 4.23 31.0 106 41.4 384
Garnet 95 79.9 14.0 5.25 39.0 150 61.6 267
Harrington 79 77.8 13.7 4.51 33.1 102 50.0 438
Merit 88 80.0 13.8 5.26 39.3 146 72.9 225
Moravian 14 91 78.0 14.0 4.33 32.2 114 41.5 342
Moravian 37 96 78.7 13.6 4.44 33.9 123 41.4 317
Orca 98 78.2 15.1 6.26 41.9 162 68.4 219
* Malting quality data courtesy of USDA-ARS Cereal Crops Research Unit, Madison
Wisconsin.
ADVANCED TESTING PROGRAM - SIX-ROWED SPRING BARLEY
The six-rowed malting barley improvement program at Aberdeen includes as a major
objective the development of a short-strawed, lodging and shattering resistant, six-rowed
malting barley adapted to irrigated production in Idaho and other western states.
Eighteen six-rowed barley varieties were tested in replicated trials at Aberdeen and/or
Tetonia in 2001 in trials that included about 85 six-rowed barley entries. These barleys
included proprietary varieties plus barleys developed by public programs in Idaho,
Minnesota, North Dakota, Oregon, Utah, Washington, and Canada. Named varieties in
these trials included ‘Barbless’, ‘B2601’, ‘Colter’, ‘Drummond’, ‘Foster’, ‘Lacey’,
‘Larker’, ‘Legacy’, ‘Millenium’, ‘MNBrite’, ‘Morex’, ‘Nebula’, ‘Robust’, ‘CDC Sisler’,
‘Sprinter’, ‘Stander’, ‘Stanwax’, and ‘Steptoe’. Cooperative trials involving six-rowed
and two-rowed barleys developed at Oregon State University and Washington State
University were also grown under irrigation at Aberdeen. Approximately 100 six-rowed
barleys were grown under irrigation in nonreplicated trials at Aberdeen in 2001.
Agronomic data for six-rowed barley varieties grown in Trial B under irrigation at
Aberdeen from 1999 through 2001 are shown in Table 11, while performance on dryland
at Tetonia is shown in Table 12. The last three years data for the Mississippi Valley
Barley Nursery are shown in Table 13.

19
Six-rowed barley selections of current interest include 92Ab5180 (83Ab5432/85SR40),
93Ab688 (M44/80Ab4952//79Ab10719), 94Ab13449 (Russell/M64), 96Ab10468
(Colter/M64), 97Ab8333 (86Ab4120/Russell), 98Ab12362 (90Ab852/Excel), and
98Ab12905 (88Y315/82Ab519//M64). The selection 92Ab5180 was submitted for
AMBA pilot-scale tests of malting and brewing quality for the third year in 2001, while
98Ab12362 was submitted for the first time. The selection 93Ab688 is being proposed
for release in Idaho in 2002 as a feed barley under the variety name of ‘Creel’, but the
other indicated selections have malting quality characteristics of interest. All of the
indicated six-rowed selections have exhibited good to very good agronomic performance
in trials to date, e.g., 96Ab10468, 97Ab8333, and 98Ab12362 all had significantly higher
yields than the six-rowed malting barley varieties in trial B at Aberdeen (Table 11), while
94Ab13449 and 98Ab12905 did significantly better in two different irrigated trials at
Aberdeen in 2000 and 2001 (Table 14). Selections 98Ab12362, 94Ab13449 and
98Ab12905 have been entered in the 2002 AMBA pilot-scale multi-state trials.
Table 11. Agronomic data for selected six-rowed varieties and selections grown in trial B under
irrigation at Aberdeen, Idaho, 1999-2001.
Test Plump Heading
Yield (bu./Ac) Weight* Barley* Date* Height* Lodging
*
Entry 1999 2000 2001 Average (lbs/bu) (%) (Julian) (in) (%)
No. Trials 3 1 1 1 1 1
Colter 173.5 201.0 183.4 186.0 51.2 87 168 36 20
Drummond 156.1 146.0 154.5 152.2 51.3 96 169 37 3
Foster 145.4 163.9 159.1 156.1 50.6 96 168 38 33
Legacy 150.7 154.3 159.4 154.8 52.0 94 170 39 70
Millenium 180.8 200.8 188.4 190.0 50.7 87 164 33 0
Morex 143.7 110.3 136.0 130.0 50.5 88 168 38 75
Nebula 159.4 187.2 149.0 165.2 51.9 96 172 27 0
Stander 141.9 154.9 165.3 154.0 52.8 94 169 37 5
Steptoe 166.6 167.2 157.3 163.7 50.0 92 169 37 83
93Ab688 187.7 182.4 175.4 181.8 51.2 87 169 37 60
92Ab5180 ---- 177.5 166.4 ---- 52.8 93 168 33 10
94Ab13449 ---- ---- 157.0 ---- 51.4 92 168 35 23
96Ab10468 ---- 184.5 171.3 ---- 51.2 89 169 38 25
97Ab8333 ---- 179.8 175.4 ---- 53.1 87 164 34 4
98Ab12362 ---- 163.1 163.3 ---- 52.6 94 170 38 18
98Ab12905 ---- ---- 154.7 ---- 52.0 87 168 35 61
*2001 data only.

20
Table 12. Agronomic data for selected six-rowed varieties and selections grown in trial B on
dryland at Tetonia, Idaho, 1999-2001.
Test Plump Heading
Yield (bu/A) Weight* Barley* Date*
Entry 1999 2000 2001 Average (lbs/bu) (%) (Julian)
No. Trials 3 1 1 1
Legacy 78.4 57.7 63.1 66.4 50.2 87 189
Steptoe 81.4 58.6 71.9 70.6 49.0 97 185
UT4087 92.0 71.1 84.2 82.4 51.7 95 188
UT5828 76.8 54.6 71.2 67.5 48.5 86 185
6B95-2482 ---- 57.9 65.3 ---- 50.3 90 190
93Ab688 77.2 59.5 76.7 71.1 50.5 94 186
*2001 data only.
Table 13. Agronomic summary of varieties and selections grown in the Mississippi Valley
Barley Nursery under irrigation at Aberdeen, Idaho, 1999-2001.
Test Plump Heading
Yield Weight Barley Date Height Lodging Protein Oil
Entry (bu/A) (lbs/bu) (%) (Julian) (in) (%) (%) (%)
No. Trials 3 3 3 3 3 3 1 1
6B95-2482 161.0 54.7 96 172 37 9 12.9 2.2
6B96-3733 172.4 54.4 98 173 40 18 12.1 2.3
Barbless 112.4 48.9 80 181 41 58 14.3 2.3
Colter 177.9 51.8 88 171 36 6 9.6 2.2
Drummond 162.4 53.9 96 172 39 12 12.4 2.1
Foster 173.0 52.8 95 172 38 11 12.5 2.1
Lacey 165.6 53.9 95 171 38 20 11.6 2.1
Larker 138.5 52.2 88 172 39 47 12.5 2.2
Legacy 153.7 53.4 90 174 39 43 12.2 2.0
M103 170.9 53.6 97 172 38 25 12.5 2.2
M104 174.4 53.6 95 171 35 11 11.7 2.1
MNBrite 144.4 53.6 94 173 41 34 14.4 2.1
Morex 131.4 51.8 88 171 40 53 13.2 2.2
ND16301 169.1 53.2 98 172 37 2 11.0 2.4
Robust 144.7 53.7 92 173 41 27 13.1 2.1
Stander 158.6 53.6 92 174 39 24 12.7 2.2

21
Table 14. Agronomic data for selected six-rowed varieties and selections grown under
irrigation at Aberdeen, Idaho, 2000-01.
Test Plump Heading
Yield Weight Barley Date Height Lodging
Entry (bu/A) (lbs/bu) (%) (Julian) (in) (%)
No. Trials 2 2 2 2 2 2
93Ab688 178.9 52.9 90 169 37 41
94Ab13449 171.0 54.0 94 167 35 11
98Ab12905 173.4 54.0 92 167 36 35
Colter 192.2 52.8 91 168 36 13
Drummond 150.3 52.8 96 170 38 8
Foster 161.5 52.9 97 168 38 19
Legacy 156.8 53.7 95 170 39 53
Millenium 194.6 51.7 90 165 33 0
Nebula 168.1 53.2 97 173 28 2
Stander 160.1 54.0 95 169 37 9
Steptoe 162.2 51.4 94 168 36 48
Table 15. Malting quality data* for selected six-rowed barley varieties and selections grown in
two irrigated trials at Aberdeen, Idaho, 1999-2000.
Plump Malt Barley Wort
Alpha- Beta
Variety or Barley Extract Protein Protein S/T DP amylase glucan
Selection (%) (%) (%) (%) (%) (°ASBC) (20°DU) (ppm)
B 2601 90 80.1 12.4 4.97 41.7 129 60.5 414
Colter 92 81.0 10.1 5.24 53.5 90 49.6 332
Foster 97 79.1 12.5 4.95 40.7 140 60.3 368
Sisler 87 79.8 12.7 4.98 39.9 163 70.2 333
Stander 94 79.8 12.7 6.11 50.0 140 65.9 343
92Ab5180 90 79.9 11.3 4.58 42.6 127 55.6 268
94Ab13449 95 81.9 10.8 4.72 44.6 102 53.3 163
96Ab10452 97 80.2 13.5 5.40 41.1 127 65.6 376
96Ab10468 91 80.1 10.8 5.01 49.5 109 43.2 400
97Ab8333 93 81.2 11.0 4.50 42.6 109 55.1 269
98Ab12362 97 80.2 12.8 5.17 41.8 156 58.2 242
98Ab12904 92 80.1 12.1 4.58 38.3 108 54.3 333
98Ab12905 95 82.6 11.0 5.31 51.6 76 73.3 425
* Malting quality data courtesy of USDA-ARS Cereal Crops Research Unit, Madison
Wisconsin.

22
WINTER BARLEY
The winter barley breeding program has produced both six-rowed and two-rowed
selections with promising malting quality characteristics that have the potential to
compete with existing spring malting barley varieties. The six-rowed winter selection
88Ab536-B (NE 76129/Morex//Morex) continues to be tested in regional trials and used
extensively as a parent in barley breeding programs designed to develop winter six-rowed
malting barleys. The selection 88Ab536-B has been evaluated in AMBA-sponsored
evaluations of malting and brewing quality and appears to have malting quality
characteristics similar to the recurrent parent Morex. This selection was included in the
2001 AMBA pilot-scale tests, mainly as a check variety for winter malting barley
Agronomic data for 88Ab536-B and other six-rowed winter barley varieties and
selections are shown in Table 17.
Additional winter barley selections of interest include several two-rowed winter barleys
with promising yield and malting quality characteristics. Two-rowed winter barleys
prominent in the program include 94Ab1269 (MT81616/81Ab1702), 94Ab1274
(MT81616/81Ab1702), and 95Ab2299 (ORWM8406/Harrington). Agronomic data for
these selections plus selected winter barley varieties are shown in Table 18, and malting
quality data in Table 19. The selection 94Ab1274 was submitted in 2001 for a third year
of AMBA pilot-scale evaluations. This is believed to be the first two-rowed winter
selection submitted for AMBA pilot-scale evaluations. In addition, Oregon State
University selections STAB 7, STAB 47, and STAB 113 were also planted in this drill
strip series in September 2000 and submitted in 2001 for AMBA pilot-scale evaluations.
The 2000-01 Aberdeen winter barley nursery included 100 entries in replicated yield
trials. Eighty entries were grown in a 2000-01 nonreplicated nursery and over 6500
headrows were evaluated. In addition, the 2000-01 winter barley nursery included two
replicated trials of winter barleys developed at Oregon State University, one replicated
trial of Russian Wheat Aphid resistant materials from Oklahoma plus over 100
observation plots and 600 headrows. The 2000-01 winter barley trials were the most
extensive winter barley trials ever planted at Aberdeen and all of the trials had good
winter survival. Named varieties planted in the current replicated trials include ‘Boyer’,
‘Cyclone’, ‘Eight-Twelve’, ‘Fanfare’, ‘Hesk’, ‘Hundred’, ‘Kamiak’, ‘Kold’, ‘Mathias’,
‘Merian’, ‘Opal’, ‘Petula’, ‘Pipkin’, ‘Plaisant’, ‘Puffin’, ‘P721’, ‘P954’, ‘Rifle’,
‘Schuyler’, ‘Scio’, ‘Sprinter’, ‘Strider’, and ‘Sunstar Pride’. Agronomic data for three
years of testing at Aberdeen are shown in Table 16.

23
Table 16. Agronomic data for winter varieties and selections grown under irrigation in Winter
Barley Nursery A at Aberdeen, Idaho, 1997-98 thru 2000-01. (excludes 1998-99 season)
Test Plump Heading
Variety or Yield Weight Barley Date Height Lodging
Selection (bu/A) (lbs/bu) (%) (Julian) (in) (%)
No. Trials 3 3 1 3 3 1
86Ab474 186.8 52.3 91 148 29 5
88Ab124 190.4 51.7 86 150 33 4
88Ab926 183.4 51.2 92 149 33 5
88Ab977 192.2 52.0 91 150 33 5
91Ab23 213.1 52.3 93 150 31 4
91Ab36 215.5 51.7 84 150 33 4
91Ab603 198.0 50.8 84 149 32 5
92Ab481 201.8 52.2 93 148 33 6
92Ab561 191.3 52.8 91 149 34 6
96Ab46 193.2 51.7 80 148 31 6
96Ab907 198.3 52.0 93 148 28 4
97Ab23 208.1 52.3 79 150 32 4
Boyer 194.9 51.6 86 150 34 5
Cyclone 200.8 51.7 96 149 34 5
Eight-Twelve 190.1 51.6 95 147 35 4
Hesk 188.7 50.8 85 150 35 5
Hundred 179.5 50.5 86 148 34 3
Kamiak 157.5 51.7 89 143 36 5
Kold 186.4 52.2 82 148 34 7
P721 157.3 53.3 84 146 34 5
P954 166.7 51.8 87 149 33 5
Pipkin 156.2 55.6 98 150 34 6
Puffin 160.5 54.1 99 147 34 6
Schuyler 165.5 52.0 83 150 34 3
Scio 184.0 51.6 89 149 33 5
Sprinter 200.6 52.7 96 150 34 5
Strider 131.4 35.0 95 95 23 5

24
Table 17. Agronomic data for selected winter varieties and selections grown under irrigation in
trial B at Aberdeen, Idaho 1997-98 thru 2000-01. (excludes 1998-99)
Test Plump Heading
Variety or Yield Weight Barley Date Height Lodging
Selection (bu/A) (lbs/bu) (%) (Julian) (in) (%)
No. Trials 3 3 2 3 3 1
88Ab536-B 141.8 52.2 89 139 36 2
88Ab539-B 149.3 52.2 90 141 37 4
88Ab953 189.0 51.2 92 149 32 0
92Ab1810 136.5 52.0 90 140 37 5
92Ab1841 148.4 52.2 89 140 36 3
92Ab1843 159.4 52.2 90 143 37 2
94Ab1777 170.3 53.1 76 144 35 1
96Ab273 166.8 53.6 87 145 35 2
96Ab474 167.5 52.3 80 151 34 2
BZ 5W96-38 174.6 50.6 83 156 26 0
Eight Twelve 182.1 51.8 75 146 33 1
Kold 162.6 53.4 93 145 34 0
Plaisant 155.0 52.8 76 144 33 3
Schuyler 111.2 33.6 47 98 26 1
Table 18. Agronomic data for winter barley varieties and selections grown in trial C under
irrigation at Aberdeen, Idaho, 1998-2001
Spike Test Plump Heading Plant
Variety or Row Yield Weight Barley Date Height Lodging
Selection Number (bu/A) (lbs/bu) (%) (Julian) (inches) (%)
No. Trials 3 3 1 3 3 1
88Ab536-B 6 141.8 52.2 88 140 36 2
94Ab1269 2 169.6 53.4 99 146 30 2
94Ab1274 2 167.9 53.6 97 147 32 2
95Ab2299 2 174.1 55.7 95 150 35 2
Boyer 6 201.6 52.4 86 151 35 5
Eight-twelve 6 191.8 52.7 93 147 33 0
Fanfare 2 165.9 55.1 99 149 33 1
Pipkin 2 155.9 56.4 98 153 36 6
Plaisant 6 155.6 53.1 88 139 37 3
Puffin 2 149.0 54.4 99 146 31 6
Schuyler 6 169.4 52.3 65 150 35 3
Sunstar Pride 6 209.4 51.4 71 157 33 4

25
Table 19. Malting quality data* for winter barley varieties and selection grown under irrigation
at Aberdeen, Idaho, 1998-2001.
Spike Plump Malt Barley Wort Alpha- Beta-
Variety or Row Barley Extract Protein Protein S/T DP amylase glucan
Selection Number (%) (%) (%) (%) (%) (°ASBC) (20°DU) (ppm)
No Trials 2 2 2 2 2 2 2 2
Fanfare 2 95.4 77.7 14.0 4.42 31.7 98 37.4 100
Pipkin 2 92.6 77.3 11.4 3.91 34.2 88 41.9 155
Puffin 2 96.7 76.3 12.8 4.25 33.3 103 32.1 309
Plaisant 6 90.9 77.6 11.9 3.71 31.9 106 37.8 428
88Ab536-B 6 78.5 78.6 12.1 4.65 38.7 156 49.8 187
94Ab1269 2 98.7 80.6 12.5 5.43 45.5 124 68.5 143
94Ab1274 2 97.8 81.9 12.0 5.47 47.6 122 66.9 83
95Ab2299 2 93.3 79.0 12.8 5.47 43.8 129 75.9 151
Morex** 6 75.2 79.4 13.0 6.46 52.1 155 70.8 132
* Malting quality data courtesy of USDA-ARS Cereal Crops Research Unit, Madison
Wisconsin.
** Adjacent spring planted.
EARLY GENERATION SELECTIONS
Seventy-two barley crosses were grown at Aberdeen in 2001 as F2 populations in 230
four-row plots, eight feet in length. Over 8,400 spike selections were made in these F2
populations for evaluation in 2002 F3 barley head rows. F3 spaced plants from 2000-01
Arizona F2 populations were grown at Aberdeen in 2001 and over 4,800 spikes were
selected for evaluation in 2002 Aberdeen F4 head rows. Additional F2 populations are
currently being grown in Arizona for evaluation as F3 head rows at Aberdeen in 2002.
Over 12,000 head rows are currently scheduled for planting in 2002, with additions
expected from the 2001-02 Arizona F2 series. Certain crosses scheduled for planting in
the 2002 barley head row series are listed below (* identifies a six-rowed cross and +
identifies cross with barley stripe rust resistance).
Crystal/Merit Colter/B2912*
Colter/M83* 93Ab375/M83
93Ab688/M83* 95Ab11469/Steffi+
96Ab8309/Steffi+ Crystal/95Ab11469
Garnet/95Ab11469 92Ab1368/Foster8
92Ab5189/B2912//Foster* 92Ab5189/M83//Foster*
93Ab375//92Ab5189/M83* 93Ab375/B2912//Foster*
93Ab375/M83//Foster* 92Ab5189/B2912//Foster*
92Ab5189/M83//Foster* 93Ab375//92Ab5189/M83

26
93Ab375/B2912/Foster* 93Ab375/M83//Foster*
93Ab688/Foster* 96Ab10452/Foster*
97Ab7367/Foster* 97Ab7367/UT4198
97Ab8188/Foster* Colter/Foster*
Crystall/95Ab11469
Nearly 700 head rows representing a wide range of crosses that were cut in 2001 will be
evaluated in nonreplicated trials in 2002. All of these new selections will be evaluated at
Aberdeen and possibly on dryland at other Idaho locations. Crosses represented in this
new series of Aberdeen selections entering preliminary nonreplicated trials in 2002
include the following (* identifies a six-rowed cross and + identifies cross with barley
stripe rust resistance):
86Ab599//83Ab6461/Colter* 93Ab688/M81*
86Ab599//92Ab5228/88Y394* 92Ab310/WA9593-87
90Ab321/91Ab3148 94Ab12981/91Ab3148
92Ab4186/Dolly 91Ab6526/Condor
94GH21-3/2B91-4947 94GH21-3/91Ab3148
94GH63-8/Condor 78Ab10274/Acclaim
92Ab3159/BZ594-19 Colter/M83*
Over 2700 winter barleys are currently being evaluated at Aberdeen in a series of winter
barley head rows. These winter barley head rows include F4 and F5 populations of
several two-rowed and six-rowed winter barley crosses. Selected crosses in this series
are listed below (* identifies a six-rowed cross).
85Ab216/Fanfare 88Ab536-B/92Ab1308*
85Ab216/Trasco 88Ab536-B/93Ab375*
93Ab428/Crystal 92Ab1308/B2912
Fanfare/93Ab428 95Ab15017/88Ab536-B*
93Ab428/Orca 95Ab15178/88Ab536-B*
Advanced generation selections from crosses involving barley stripe rust resistant parents
are being evaluated for stripe rust reaction in cooperation with William M. Brown, Jr. and
associates (Colorado State University, Fort Collins, CO); Xianming Chen (ARS,
Pullman, Washington); Patrick M. Hayes (Oregon State University, Corvallis, Oregon);
and Lee F. Jackson (University of California - Davis, Davis, California).
NEW CROSSES
Nine-six new barley crosses were planted as F1 populations in the field at Aberdeen in
2001. Certain of these crosses (* identifies a six-rowed cross and + identifies cross with
barley stripe rust resistance) are listed below.

Garnet/98Ab11865 Garnet/98Ab12895
95M4623/Garnet 96Ab8289/Garnet
98Ab11993/Garnet 98Ab12210/Garnet
Bancroft/96Ab8289+ 98Ab12895/Bancroft+
85Ab2323/96Ab8289 98Ab11993/85Ab2323
94Ab12990/Baronesse 95M4623/94Ab12990
98Ab11993/94GH86-5+ Garnet/Moravian 32
McGwire/99Ab38-5 95Ab17891/Xena
97Ab8093/Lacey* 98Ab12362/Lacey*
Colter/6B95-2089* 93Ab688/Millenium*
93Ab688/B2601* 98Ab12399/Drummond*
98Ab12362/M107* 98Ab12362/6B95-2089*
27
A series of spring two-rowed and six-rowed barley parents are currently being grown for
crossing in the 2001-02 greenhouse. The greenhouse parents include several entries
with promising agronomic and malting quality characteristics as well as barley stripe rust
resistance.
BARLEY QUALITY
Quality evaluations in support of the barley breeding and genetics programs at Aberdeen
are generously conducted by the USDA-ARS Cereal Crops Research Unit - Madison,
Wisconsin, and reported in detail in their publications.
SPECIAL PROJECTS
NATIONAL SMALL GRAINS COLLECTION BARLEY GERMPLASM
EVALUATIONS
H.E. Bockelman, C.A. Erickson, and D.M. Wesenberg
Agricultural Research Service - USDA
National Small Grains Germplasm Research Facility, Aberdeen, Idaho
Cooperation University of Idaho
The systematic evaluation of barley accessions in the USDA-ARS National Small Grains
Collection (NSGC) and other elite germplasm is coordinated by National Small Grains
Germplasm Research Facility (NSGGRF) staff at Aberdeen. Cooperative barley
evaluations continued for reaction to barley stripe rust; spot and net blotch of barley;
Fusarium head blight; and barley stripe mosaic virus, as well as evaluations of betaglucan,
protein, and oil content of NSGC barley accessions. Specific Cooperative
Agreements or within ARS Fund Transfers involving cooperative evaluations and related

28
research for all small grains involve over 20 University and ARS projects in at least 15
states.
Data obtained from evaluations of NSGC germplasm are entered in the Germplasm
Resources Information Network (GRIN) system by the NSGGRF staff in cooperation
with the ARS National Germplasm Resources Laboratory, Beltsville, Maryland. Data
available on GRIN for barley are summarized below.
Descriptors with data on the Germplasm Resources Information Network.
Descriptor Testing Location(s) No. Evaluated
ALEURONE COLOR Aberdeen, ID; Mesa, Maricopa, AZ 7,922
AWN DECIDUOUSNESS Aberdeen, ID; Mesa, Maricopa, AZ 6,867
AWN ROUGHNESS Aberdeen, ID; Mesa, Maricopa, AZ 9,307
AWN TYPE Aberdeen, ID; Mesa, Maricopa, AZ 15,025
BETA GLUCAN Madison, WI 11,392
BSMV FREE Fargo, ND 17,709
BYDV Davis, CA; Urbana, IL 4,731
CEREAL LEAF BEETLE Michigan 8.968
DAY TO ANTHESIS Aberdeen, ID; Mesa, Maricopa, AZ 10,146
GROWTH HABIT Aberdeen, ID 24,663
HULL COVER Aberdeen, ID 18,737
KERNEL PLUMPNESS Aberdeen, ID 6,908
KERNELS PER SPIKE Aberdeen, ID 4,322
LEAF RUST Fargo, ND 2,696
LEMMA COLOR Aberdeen, ID 10,989
LIPID Madison, WI 3,885
LODGING Aberdeen, ID; Mesa, Maricopa, AZ 6.940
NECK BREAKAGE Aberdeen, ID 4,791
NET BLOTCH Fargo, Langdon, ND 14,101
PLANT HEIGHT Aberdeen, ID; Mesa, Maricopa, AZ 8,478
PLOIDY Aberdeen, ID 686
PROTEIN Madison, WI 10,390
RACHILLA HAIR LENGTH Aberdeen, ID 8.983
RUSSIAN WHEAT APHID Stillwater, OK 24,478
SCALD Langdon, ND 1,186
SHATTERING Aberdeen, ID; Mesa, Maricopa, AZ 4,797
SPIKE ANGLE Aberdeen, ID; Mesa, Maricopa, AZ 6,938
SPIKE ROW NUMBER Aberdeen, ID 20,157
SPOT BLOTCH Fargo, ND 20,212
STRAW BREAKAGE Aberdeen, ID; Mesa, Maricopa, AZ 4,791
STRIPE RUST Cochabamba, Bolivia 26,816
TEST WEIGHT Aberdeen, ID 6,145
YIELD Aberdeen, ID 6,932
The barley germplasm evaluation program concerned with barley stripe rust continued in
2001. Barley stripe rust evaluations were initiated in Cochabamba, Bolivia in 1990 under
the direction of Colorado State University plant pathologists W.M. Brown, Jr., Vidal
Velasco, and J.P. Hill. Nearly all cultivated barley accessions in the NSGC were
evaluated for reaction to barley stripe rust in Cochabamba, with resistant or moderately
resistant reactions recorded for nearly 500 accessions. Fifty percent of the barley stripe

29
rust resistant NSGC accessions originated in Ethiopia, with the balance coming from 45
other countries. Testing for reaction to barley stripe rust continued at Cochabamba
through 1996, with the focus on these barley stripe rust evaluation trials moving to
Toluca, Mexico; Mt. Vernon, Washington; and other locations in subsequent years. In
addition to the evaluation of NSGC germplasm, barley stripe rust evaluation trials at
Cochabamba, Bolivia and other locations have also included a number of barley varieties
and elite lines from several cooperators. Cooperators that have submitted entries for
evaluation in recent years include Busch Agricultural Resources, Inc.; Coors Brewing
Company; Montana State University; North Dakota State University; Oregon State
University; Plant Breeders 1; Texas A&M University; University of California - Davis;
University of Minnesota; USDA-ARS Aberdeen, Idaho; Utah State University;
Washington State University; and Western Plant Breeders.
*The authors wish to acknowledge the important contributions of the NSGGRF staff in
this germplasm evaluation effort, with special thanks to Glenda B. Rutger, Greg G. Laine,
Carol S. Truman, Judy Bradley, Kay B. Calzada, Karla Reynolds, and Dave E. Burrup.
BARLEY PUBLICATIONS
Wesenberg, D.M., Burrup, D.E., Whitmore, J.C. and Liu, C.T. 2000. Registration of
'Garnet' Barley. Crop Sci. 40(3):851.
PERSONNEL – 2001
USDA-ARS, Aberdeen
Charles A. Erickson Agronomist, Lead Scientist ARS-USDA
Darrell M. Wesenberg Research Agronomist ARS-USDA, ret.
Dave E. Burrup Biological Science Technician ARS-USDA
Kay B. Calzada UI Technical Aide UI Aberdeen R&E
Center
Karla Reynolds UI Technical Aide UI Aberdeen R&E
Center
James C. Whitmore Superintendent (Cooperating) UI Tetonia R&E
Center
Harold E. Bockelman Agronomist (Cooperating) ARS-USDA
Phil Bregitzer Research Geneticist ARS-USDA
An Hang Research Geneticist ARS-USDA
David L. Hoffman Research Geneticist ARS-USDA
Victor Raboy Research Geneticist ARS-USDA
Allen Cook Agricultural Research Science Technician ARS-USDA, ret.

Cooperating - University of Idaho
Idaho Barley Variety Enhancement and Selection Program
Larry D. Robertson Professor UI, Aberdeen
Karen Dempster Scientific Aide UI, Moscow
30
The Idaho Barley Variety Enhancement Program continued extensive testing of
numerous advanced selections in 2001 in important dryland environments in Idaho as
well as under irrigation at Kimberly and Parma. This extensive testing program was
initiated in northern Idaho in 1992. The program is concerned with the evaluation of a
diversity of barley germplasm throughout Idaho.

31
DEVELOPMENT OF SIX-ROWED MALTING BARLEY GERMPLASM FOR THE
WESTERN US USING CONVENTIONAL AND MARKER-ASSISTED SELECTION
TECHNIQUES
David Hoffman & An Hang
Agricultural Research Service - USDA
National Small Grains Germplasm Research Facility
Aberdeen, Idaho
INTRODUCTION AND OBJECTIVES
Malting barley is an economically important crop in Idaho and in other western states.
The construction of two new malting facilities in southeastern Idaho will significantly
increase the demand for malting barley in the West. The barley improvement program at
Aberdeen, Idaho has, as its primary objective, to develop improved two-rowed and sixrowed
spring and winter malting barley cultivars and germplasm lines adapted to
irrigated and dryland conditions. More emphasis has been placed on irrigated spring
barley. Much progress has been made in the development of two-rowed spring malting
barleys as evidenced by the release of ‘Klages’, ‘Crystal’, and ‘Garnet’ from Aberdeen,
and ‘Crest’, ‘Chinook’, ‘B1202’, and ‘Galena’ from other public and private institutions,
as examples.
The development of spring six-rowed malting barleys for the western U.S. has been more
challenging than that of two-rowed barley. It has been difficult to keep seed yields high
along with acceptable malting quality. Low gains in malting quality may be due to low to
moderate heritabilities of the traits (Foster et al., 1967), and the limitation on sample size
due to the difficulty and cost of malt tests. The problem may lie in the number of genes
controlling these quantitatively inherited traits and the number of segregants required to
combine all of the favorable alleles. Linkage drag and epistatic effects also may interfere
with the desired outcome of high yield and high malting quality. For instance, 'Colter' is a
spring six-rowed malting cultivar that performs very well agronomically in western
irrigated environments (Wesenberg and Burrup, 1998). As for malting quality, Colter is
consistently low in diastatic power (DP), yet has high malt extract levels in most
environments.
Modern molecular techniques have enabled the detailed genetic mapping of several
barley populations (GrainGenes - http://wheat.pw.usda.gov/ggpages/aps.html#barley).
The barley maps have been used for detailed QTL-association studies (Hayes et al.,
1993). Two important genome regions (one on chromosome 1 and the other on
chromosome 4) have been identified in the 'Steptoe'/'Morex' mapping population to have
overlapping alleles for several malting traits and these have been consistent over several
environments (Hayes et al., 1993). Surveys of marker polymorphisms among six-rowed
malting cultivars of the U. of Minnesota breeding program have identified additional
candidate markers/regions for malt quality QTL (Hayes, et al., 1997; Hoffman and
Dahleen, 1997). Alternatively, high grain yield QTL have been transferred from high
yielding two-rowed feed barley 'Baronesse' to two-rowed, malting barley 'Harrington

32
with markers, although marker-mediated-transfer of yield QTL has not been as successful
with six-rowed barley (A. Kleinhofs, personal communication).
The objectives of this study will be to 1) survey potential six-rowed malting barley
parents for variation of candidate marker alleles to key malting barley DP QTL, 2)
concurrently conduct conventional and marker-assisted selection for desirable malting
quality in a good agronomic background, and 3) compare marked donor regions as to
their contributions toward enhanced malting quality. It is anticipated that the information
and germplasm obtained from this work will lead to the development of new and
improved six-rowed malting barley cultivars for the Western intermountain region of the
U.S. The initial emphasis will be placed on DP QTL polymorphisms and polymorphisms
among Colter and the six-rowed malting standard 'Morex'.
METHODS
Genomic DNA was extracted from the above cultivars using a miniprep procedure that
was adapted from the DNA extraction protocol given in the 1994 ITMI Wheat Mapping
Workshop Laboratory Manual pp. 19-21. In addition to Colter and Morex, the mapping
parent 'Steptoe' will be added to the survey gels as a control to to validate mapped bands.
Techniques for PCRRAPD followed that of Hoffman and Bregitzer (1996); for AFLP,
that of Hoffman et al. (2000); and for PCR-STS, that of Tragoonrung et al. (1992).
PROGRESS TO DATE
Marker Analyses
In 2000, we reported on the polymorphism pattern of PCR-RAPD marker AB11M950 that
maps near RFLP locus VATP57A which is in between Brz and Amy2, a region where DP
QTL is found along with several of other malting and agronomic QTL. Last year, a
similar, but not identical polymorphism pattern to AB11, was found for the lower
molecular weight band generated by the PCR-STS primer pair KV1, KV9. This primer
pair was designed in Dr. Tom Blake's laboratory (Tragoonrung et al., 1992) from
sequences of B1-Hordein encoded by Hor2 (Shewry et al., 1988) which maps to the short
arm of barley chromosome 5(1H) where a DP QTL was identified by Hayes et al. (1993).
No other major malting or agronomic QTL have associated with this chromosomal
region. So far, none of the AFLP markers that mapped in DP QTL regions have yielded
useful polymorphism patterns (polymorphic between Morex and Colter and between
Morex and Steptoe, and band-positive for Morex) as described for AB11M950
(chromosome 1) and KV1, KV9 (chromosome 5).
Crosses Made and Segregants Tested
The first cross to be made for marker analysis was Colter/Morex. An F1 from this cross
was backcrossed several times to Colter. The idea is to extract malting quality QTLs
such as DP from Morex and place these in an adapted background such as Colter. The
resulting BC1F1s were tested with the KV1, KV9 primers which amplify sequences in
Hor2 on the short arm of chromosome 5. Both phenotypic and marker selection is to be
employed. The BC1F1s with the Morex band were backcrossed again to Colter and
screened for KV1, KV9 segregation. The resulting BC2F1s were again screened for
KV1, KV9 segregation and ones with Morex alleles were selected for selfing and further

33
backcrossing. The selfed lines will be selected for the Morex allele and advanced to the
BC2F2:3 generation for initial field selection and testing of DP. This first BC population
did not segregate for chromosome 1 marker AB11M950. It is thought that some intravariety
variability is present in Morex.
A new parent from the mapping stock was chosen to derive a new backcross series. The
new series is now in the BC1 generation and will allow selection with KV1, KV9 and
AB11M950. This study has been expanded to include additional 6-rowed varieties and
germplasms for tests of DP QTL in different backgrounds across several environments.
This expanded version will include the Bmy1 region of chromosome 4 and has received
one-year funding from the North American Barley Genome Project.
REFERENCES
Foster, A E., G.A.Peterson, and O.J. Banasik. 1967. Heritability of factors affecting
malting quality of barley. Crop Sci.7:611-613.
Hayes, P.M., B.H. Liu, S.J. Knapp, F. Chen, B. Jones, T. Blake, J. Franckowiak, D.
Rasmusson, M. Sorrells, S.E. Ullrich, D. Wesenberg, and A. Kleinhofs. 1993.
Quantitative trait locus effects and environmental interaction in a sample North
American barley germplasm. Theor. Appl. Genet. 87:392-401.
Hayes, P.M., J. Cerono, H. Witsenboer, M. Kuiper, M. Zabeau, K. Sato, A. Kleinhofs,
D.Kudrna, A. Kilian, M. Saghai-Maroof, D. Hoffman, and the North American Mapping
Project. 1997. Characterizing and exploiting genetic diversity and quantitative traits in
barley (Hordeum vulgare) using AFLP markers. J. Agric. Genomics V.3, Art.2.;
(online:http://www.ncgr.org/research/jag/).
Hoffman, D.L., and P. Bregitzer. 1996. Identification of reproducible PCR-RAPD
markers that allow the differentiation of closely-related six-rowed six-rowed malting
barley cultivars. J. Amer. Soc. Brew. Chem. 54:172-176.
Hoffman, D.L., and L.S. Dahleen. 1997. Markers polymorphic among closely-related
barley cultivars link to quantitative trait loci. Proc. 31 st Barley Imp. Conf. pp. 76-86.
Hoffman, D.L., A. Hang, and C. Burton. 2000. Interval mapping of AFLP markers in
barley with a subset of doubled haploid lines. J. Agric. Genom. v.5, Art.4; online:
(http://www.ncgr.org/research/jag/).
Shewry, P.R., S. Parmar, and J. Franklin. 1988. Recombination within the Hor2 locus of
barley. Barley Genetics Newsletter 18:14 (http://wheat.pw.usda.gov/ggpages/bgn/18/a18-
14.html)
Tragoonrung, S., V. Kanazin,, P.M. Hayes, and T. K. Blake. 1992. Sequence-taggedsite-facilitated
PCR for barley genome mapping. Theor. Appl. Genet. 84:1002-1008.
Wesenberg, D.M., and D.E. Burrup. 1998. Breeding, germplasm development, and
genetics of improved spring and winter malting barley. <strong>Annual</strong> <strong>Progress</strong> <strong>Report</strong> on
Malting Barley Research, American Malting Barley Association, Inc., Milwaukee,
Wisconsin, pp.11-37.

34
RECENT PUBLICATION
Hoffman, D.L. and L.S. Dahleen. 2002. Markers polymorphic among barley cultivars of
a narrow gene pool associate with key QTL. Theor. and Appl. Genet. In press.
PERSONNEL
David Hoffman, Research Geneticist
An Hang, Research Geneticist
Robert Campbell, Biological ScienceTechnician (Plants)
Charlotte Burton, Biological Science Technician (Plants)
Irene Shackelford, Biological Science Technician (Plants)
Kathy Satterfield, Biological Science Technician (Plants)

35
MINNESOTA BARLEY IMPROVEMENT PROJECT
Kevin P. Smith
Department of Agronomy and Plant Genetics
University of Minnesota
St. Paul, MN 55108, U.S.A.
Barley improvement at the University of Minnesota is a cooperative effort of the
Department of Agronomy and Plant Genetics, the Department of Plant Pathology, and the
Research and Outreach Centers of the University of Minnesota. The primary objective is
to develop high yielding disease resistant varieties that have favorable malting and
brewing characteristics. With the Fusarium head blight (FHB) epidemic of the last eight
years, developing varieties with resistance to this disease is the number one priority.
Secondary objectives are to enhance germplasm for future uses, and to generate breeding
and genetic information that will be helpful in achieving future progress.
Barley growers in the Midwest were again confronted with the continuing epidemic of
FHB. Disease was sporadic and influenced by weather patterns and planting dates. Early
planted fields had a greater tendency to escape disease since they avoided more
conducive weather occurring later in July. Grain harvested from yield trials in Crookston
had only trace amounts of deoxynivalenol (DON). Overall, yields in Minnesota were
down from last year with average yields reported to be 55 bushels per acre compared
with 64 bushels last year. This was likely due, in part, to exceptionally dry weather
during the first half of the growing season.
Varieties and breeding lines were evaluated in yield trials in five locations in Minnesota
(St. Paul, Morris, Crookston, Stephen, and Roseau). Advanced yield trials were
conducted at all five locations, intermediate trials at three locations (St. Paul, Morris, and
Crookston) and preliminary yield trials at two locations (St. Paul and Crookston). The
Mississippi Valley Nursery was grown at Morris and Crookston and both locations were
submitted for malting quality analysis at the USDA-ARS Cereal Crops Research Unit.
The Canadian Six-Row Coop Test was grown at Crookston and included the Minnesota
variety Lacey as an entry in 2001.
CURRENT VARIETIES
Robust continues to be the dominant barley variety in the Midwest occupying 78 % of the
acreage in 2000 (MN, ND, and SD). The acreage percentages for Minnesota in 2001
were: Robust 74%, Lacey 10%, Stander 7%, Morex 2%, Royal 2%, Conlon 1% and
others 4%. The latest release from the Minnesota Agricultural Experiment Station,
Lacey, was added to the AMBA list of approved malting varieties. Miller Brewing Co.
has rated it satisfactory in two years of testing while Anheuser-Busch is continuing
testing of Lacey with the 2001 crop.
Relative grain yield data for the current varieties is reported in Table 1 as the percent of
the mean of the varieties with the mean of the varieties reported in Bu/A at each location
as well as the state mean. Stander was the highest yielding variety in the state 3-year

36
average. The three new varieties Lacey, Legacy and Drummond all yielded above
Robust, with Lacey yielding about 11% better than Robust.
Other Agronomic characteristics for the varieties is reported in Table 2. All three of the
newer varieties appear to be about a half a percent lower in grain protein compared to
Robust. Most of the varieties have similar heading dates to Robust, while Stander and
Legacy appear to head a little later. Stander and Drummond have the best lodging
resistance.
Table 1. Grain yield (percent of the mean of the varieties) in Minnesota trials from
1999-2001.
Crookston Morris Stephen St. Paul Roseau Mean
3 1 3 2 3 3 14
Morex 92 87 87 87 88 89
Robust 97 95 96 97 88 96
Stander 108 101 114 104 109 110
Foster 98 100 109 105 106 101
MNBrite 100 105 92 93 97 95
Lacey 107 108 105 107 104 105
Drummond 97 96 82 107 101 98
Legacy 96 101 109 99 -- --
Mean (Bu/A) 101 91 67 81 90 87
1 number of trials.
Table 2. Description of barley varieties; 1999-2001.
Variety
Heading
(DAP)
Height
(inches)
Lodging
(%)
Plump
(%)
Protein
(%)
8 1 9 5 6 3
Morex 57 35 38 70 13.2
Robust 58 36 34 77 13.5
Stander 59 32 28 82 12.6
Foster 57 34 29 80 12.0
MNBrite 58 35 39 75 13.8
Lacey 58 33 29 79 12.9
Drummond 58 34 25 74 12.9
Legacy
1
number of trials
60 34 34 67 13.1

37
ADVANCED LINE EVALUATION
Varieties currently grown in Minnesota were evaluated with 17 new experimental lines in
advanced trials conducted at St.Paul, Morris, Crookston, Stephen and Rosseau. Two
lines were submitted for their first year of pilot scale testing M109 (M95/Lacey) and
M110 (M932-117/M95). These lines were also included in the Mississippi Valley
Nursery. Both lines M109 and M110 show a significant yield advantage over the
varieties Stander and Lacey based on two years of advanced yield trial data (Table 3).
Maturity is similar to Robust while height is between Lacey and Stander. In terms of
malting quality these lines are distinctly lower than Robust in grain protein, similar in
wort protein, and slightly higher in alpha amylase (Table 4).
Table 3. Agronomic comparisons of new AMBA pilot entries M109 and M110 to check
varieties 2000-2001.
Variety
Yield
Bu/A
8 1
Heading
(Days after 5/31)
Height
(cm)
Lodging
(0-9)
Maturity
(1-5)
4 6 2 3
M109 98.1 20.0 83.4 4.2 3.0
M110 99.4 20.5 81.7 3.5 3.0
Robust 82.6 20.6 93.8 4.4 3.0
Stander 93.6 20.5 82.7 3.2 2.7
Lacey
1
number of trials
92.4 20.3 85.4 3.2 2.8
Table 4. Malt Quality 1 of M109 and M110 compared to other varieties grown in
Minnesota based on 8 trials grown in 2000-2001.
Variety/Line Barley
Protein
(%)
Plump
Kernels
(%)
Malt
Extract
(%)
Wort
Protein
(%)
S/T
(%)
Dias.
Power
(ºL)
Alpha
amylase
(20º DU)
M109 12.8 81.7 79.3 5.5 45.0 147 58.0
M110 12.6 85.7 78.9 5.3 45.0 141 55.0
Robust 14.0 81.9 77.6 5.6 40.9 150 48.4
Stander 13.1 87.3 79.3 6.5 51.8 133 77.4
Lacey 13.2 86.4 78.6 5.4 42.6 145 54.5
1 Data courtesy of the USDA-ARS Cereal Crops Research Unit, Madison, WI.

38
INTERMEDIATE AND PRELIMINARY YIELD TRIALS
Thirty-one lines were grown and evaluated in our intermediate yield trials at 3 locations
(St. Paul, Morris, and Crookston). 4 of these lines have partial resistance to FHB. Our
preliminary yield trials grown at St. Paul and Crookston included 190 lines. Of these
lines 60 came from our FHB screening program and have partial resistance to FHB.
EARLY GENERATION LINES
34 F5 populations were grown at Crookston and St. Paul. The crosses were largely of two
types, i.e., traditional elite by elite and elite by FHB resistant. Fourteen of the F5
populations (elite by elite) were advanced through the F3 and F4 generations via single
seed descent (SSD) in greenhouses in fall and winter 1999-00. These populations were
planted as head rows in Crookston. Selection was based on heading date, height, lodging,
kernel plumpness, and spike characteristics. Heads were harvested from selected rows
and planted in a winter nursery in Arizona. Rows of selected lines were harvested for
malting quality evaluation in Madison,WI. Sixteen of the FHB populations were
advanced by SSD to the F3 generation in the greenhouse in St. Paul and sent to an
Arizona winter nursery to be advanced to the F4 generation. Individual F4 plants were
harvested in Arizona to permit us to evaluate F4:5 lines in FHB nurseries (2 locations and
2 reps). Selection was based on FHB resistance, heading date, and plant phenotype.
Rows were harvested from selected lines for DON analysis. Based on these data, 70 lines
were selected for entry to preliminary yield trials in 2002. A total of 55 F2 and F3
populations were grown at St. Paul and Crookston. Twenty-eight of these populations
were from crosses that included a FHB resistant parent.
DISEASES
FUSARIUM RESISTANCE BREEDING
Significant progress has been made in breeding for resistance to FHB. Expanded funding
through the US Wheat and Barley Scab Initiative and the Minnesota State Legislature
have allowed us to increase our efforts in disease resistance breeding and studies aimed to
understand and exploit the genetics of disease resistance. Our expanded FHB screening
effort is done in collaboration with Dr. Ruth Dill-Macky. Our field scab nurseries have
increased in size from 3900 rows in 1997 to over 8000 rows in 2001. We conducted 3
greenhouse studies in fall and winter to screen for resistance in elite material and to
evaluate genetic studies. Over the past two years the breeding program has evaluated and
made crosses to over 30 sources of resistance; mostly two-rowed barley that are poorly
adapted to Minnesota. We are continuing to make crosses with new sources of resistance
as they are identified. Recently, these have included crosses with lines identified by
Brian Steffenson as part of a project to screen the world collection of six-rowed spring
barleys for FHB resistance. In a parallel effort, we are assessing the genetic diversity of
these new sources using SSR markers (see Other Studies) to identify parents that will be
more likely to carry FHB resistance genes different from those we have already used. All
the germplasm currently in the FHB resistance program trace back to nine different
resistant parents. Some of the material in our program represents 3 rd or 4 th cycle breeding
material and is making its way into advanced yield testing.

39
OTHER BARLEY DISEASES
In 2001, resistance screening was conducted for six diseases in addition to FHB (kernel
discoloration, spot blotch, net blotch, septoria speckled leaf blotch, powdery mildew, and
loose smut). The number of populations and size of population varied depending on the
disease. Screening was done in the field or in glasshouse for the six diseases. Screening
of kernel discoloration, net blotch, powdery mildew, and loose smut was done in
collaboration with Dr. Ruth Dill-Macky. Screening for resistance to spot blotch and
septoria speckled leaf blotch was done in collaboration with Dr. Brian Steffenson and Dr.
Toubia-Rahme, respectively.
QUALITY EVALUATIONS
Approximately several hundred samples from lines in advanced stages of testing have
been submitted to the Cereal Crops Research Unit for quality evaluation. Data received
in December was of good quality and was used to select among crosses made in our fall
greenhouse. In addition, this data allowed us to identify entries for 2002 advanced yield
trials. Quality data from F5 head rows is expected soon and will be used to select lines
for this years preliminary yield trial entries.
OTHER STUDIES
1. Assessing Genetic Diversity of FHB resistant sources Graduate student Bill
Wingbermuehle is characterizing seven sources of resistance from unadapted two-rowed
barley with a set of 60 SSR markers and conducting cluster analyses. Three of the seven
sources of FHB resistance (CIho4196, PFC88209, and HOR211) were different from
previously characterized sources based on genetic distance estimates. Technician Kelley
Belina is conducting a similar analysis on six-rowed spring lines from the world
collection that posses some resistance to FHB. These data sets, as well as the set of data
characterizing the Minnesota elite breeding germplasm (described below), are being
combined to help develop strategies for retaining desirable genes assembled over 50
years of plant breeding and introgressing new genes for disease resistance.
2. Evolution of SSR Allelic Diversity in the Development of Elite Minnesota Barley
Germplasm. Graduate student Federico Condon is testing the effect of breeding within a
narrow germplasm on genetic diversity. He has examined coancestry coefficients and
SSR allelic diversity in a set of 58 variety candidates developed over a 40+ year period in
the University of Minnesota breeding program, 26 of the 31 direct ancestors to the
breeding program, and 9 of the founders of the Midwestern six row barley germplasm.
Using 30 SSR's distributed across seven chromosomes, the average number of alleles per
locus was 6.8 in the direct ancestors. The average number of alleles in the varieties
candidates developed in four consecutive decades from 1958 to the present was 3.7, 2.6,
2.0 and 2.6 respectively. The rate of change in the number of alleles was not constant
across the genome. For example, Bmag613, in chromosome 7 changed from 6 alleles to 1
and Bmag872 on chromosome 5 changed from 5 to 4. These changes in allelic diversity
will be examined with respects to traits under selection in the breeding program and map
positions of known genes/QTL's conditioning those traits.

40
3. Mapping New Sources of Resistance to Fusarium Head Blight. The mapping
populations Atahualpa x M81 and Hor211 x Lacey have been set up to identify new QTL
for FHB resistance. In breeding populations, we have identified resistant progeny from
crosses to both of these sources giving us confidence that genetic investigation will reveal
novel genes for disease resistance. Research sponsored through the U.S.Wheat and
Barley Scab Initiative has shown that Atahualpa and Hor211 are genetically different
from other sources of FHB resistance. This research has also demonstrated that
previously identified genetic markers, linked to established FHB resistance QTL, do not
account for much or any of the variation for resistance in these sources of resistance.
Therefore further research with these new sources of resistance will likely lead to novel
genes and DNA markers that should be useful marker assisted breeding efforts to develop
new disease resistant barley varieties.
4. Near Isogenic Lines and for FHB QTL. Post Doc Lex Nduulu continues to develop
near isogenic lines (NIL) for FHB QTL regions identified in the Chevron x M69
population (de le Pena et al, 1999) on chromosome 2 and 6 and the Frederickson x
Stander population (Mesfin et al, in preperation) for chromosome 2. We have completed
4 and 2 backcrosses utilizing marker assisted selection (MAS) for the Chevron and
Frederickson derived lines, respectively. We will also be using these NIL to generate
fine maps in these regions to more precisely define the location of the QTLs.
5. Marker Assisted Selection (MAS) in Lacey background. To better utilize Chevron
FHB resistance alleles at FHB QTL on chromosomes 2 and 6 we have evaluated MAS
for these regions in crosses with our newest variety Lacey. Simple sequence repeat
(SSR) markers were used to conduct selection on individual F2 plants in the summer of
2000. These lines have been advanced by single seed decent and F4:5 lines were
evaluated in 2001. MAS for the Chevron allele on chromosome two resulted in a 43%
reduction in FHB severity compared to the random control. MAS for the Chevron allele
on chromosome six resulted in a 11% reduction in FHB severity.
6. Mapping Disease Resistance in Hordeum vulgares subsp. Spontaneum. Post Doc
Laszlo Gyenis continues to develop a map and collect disease data in a cross between a
spontaneum accession (OUH602) and Harrington. The wild parent has disease resistance
to four diseases; spot blotch, net blotch, scald, and septoria. This work is being done in
collaboration with Gary Muehlbauer and Brian Steffenson.
Publications
Rasmusson, D. C., K. P. Smith, R. Dill-Macky, E. L. Schiefelbein, and J. V. Wiersma .
2001. Registration of 'Lacey Barley'. Crop Sci 41: 1991.
Kolb, F.L., G-H. Bai, G.J. Muehlbauer, J.A. Anderson, K.P. Smith, and G. Fedak. 2001.
Host Plant Resistance Genes for Fusarium Head Blight: Mapping and Manipulation with
Molecular Markers. Crop Sci 41: 611-619.
Simon, H. M., Smith, K. P., Dodsworth, J. A., Guenthner, B., Handelsman, J. and
Goodman, R. M. 2001. Influence of Tomato Genotype on Growth of Inoculated and
Indigenous Bacteria in the Spermosphere. Appl. Environ. Microbiol. 67:514-52

41
Wingbermuehle, W. J., K.M. Belina, and K.P. Smith. 2001. Assessing the Genetic
Diversity of Fusarium Head Blight Resistant Sources in Barley. Proceedings of the 2001
National Fusarium Head Blight Forum. Erlanger, KY 12/8/01 - 12/10/01.
Gustus, C. and K. P. Smith. 2001. Evaluating Phenotypic and Marker Assisted
Selection in the F2 Generation for Chevron-derived FHB Resistance in Barley.
Proceedings of the 2001 National Fusarium Head Blight Forum. Erlanger, KY 12/8/01 -
12/10/01.
Smith, K. P. 2001. Variety Development and Uniform Nurseries: FHB Resistance in
Barley. Proceedings of the 2001 National Fusarium Head Blight Forum. Erlanger, KY
12/8/01 - 12/10/01.
A. Mesfin, P. Canci, R. de la Pena, R. Dill-Macky, K. Smith and G.J. Muehlbauer. 2001.
Barley chromosome 2: does it carry FHB resistance? 2001 Plant and Animal Genome
Meeting, San Diego, CA. 1/13/01 - 1/17/01.
Smith, K., Schwarz, P., and Barr, J. 2001. Impact of Fusarium Head Blight disease
management on malting barley. Brewers Digest 76(3):41.
Smith, K. P. 2001. Breeding FHB Resistant Malting Barley in Minnesota. Barley
Improvement Conference. San Antonio, TX.
PERSONNEL ENGAGED IN BARLEY RESEARCH
Barley Project
Kevin P Smith, Assistant Professor
Edward Schiefelbein, Research Scientist
Guillermo Velasquez, Plot Technician
Charles Gustus, Research Scientist
Asfaw Mesfin, Post Doc
Laszlo Gyenis Post Doc (Muehlbauer, Steffenson)
Lex Nduulu, Post Doc
Bill Wingbermuehle, Research Assistant
Federico Condon, Research Assistant
Kelley Belina, Junior Research Scientist
Collaborators
Ruth Dill-Macky, Associate Professor, Department of Plant Pathology, University of
Minnesota
Gary J. Muehlbauer, Department of Agronomy and Plant Genetics, University of
Minnesota
John V. Wiersma, Northwest Research and Outreach Center, University of Minnesota
George A. Nelson, West Central Research and Outreach Center, University of Minnesota
Brian J. Steffenson, Department of Plant Pathology, University of Minnesota
Halla Toubia-Rahme, Department of Plant Pathology, University of Minnesota

42
MANAGEMENT AND EPIDEMIOLOGY OF DISEASES IN BARLEY
Ruth Dill-Macky, Kent Evans, and Bacilio Salas
Department of Plant Pathology
University of Minnesota
St. Paul, MN 55108, U.S.A.
Introduction
This is an applied research program directed to the control of the plant diseases of
greatest importance to the barley industry in Minnesota. Over the last seven years
Fusarium head blight has presented the greatest threat to barley production in Minnesota
and research on Fusarium head blight has been the focus of the program.
Barley Pathology Research at the University of Minnesota is a cooperative effort of the
Department of Plant Pathology, the Department of Agronomy and Plant Genetics and the
branch experiment stations of the University of Minnesota
Objectives
The project incorporates three interrelated objectives:
1. To improve the management and control of diseases in barley. Emphasis is placed on
the control of the foliar diseases of greatest impact on commercial barley production,
through the use of host resistance and cultural control practices.
2. Develop effective screening methodologies in the greenhouse and field, identify
sources of resistance and determine the character and inheritance of host resistance
with the ultimate goal of improving genetic resistance. Examine the epidemiology of
disease development and determine effective cultural control practices to manage
plant diseases.
3. The program also aims to provide support to the barley breeding program as part of
ongoing collaborative efforts to develop and maintain germplasm with improved
resistance to Fusarium head blight, net blotch, spot blotch, stem rust, leaf rust,
powdery mildew, kernel discoloration, loose smut and Septoria diseases.
Fusarium Head Blight Research
Field Screening Program: A large effort is maintained each field season assessing
Fusarium head blight reactions within plots at the three screening locations of St. Paul,
Morris, and Crookston. The pathology effort supported by this project involves the
oversight of inoculum preparation during the winter and spring, summer plot
maintenance including the operation of the mist-irrigation system, and spray applications
of inoculum within screening nurseries of the respective breeding programs during the
summer field season.
The number of lines (or field rows) evaluated has increased 5-10% each year for the past
three years. In the 2001 field season a total of 10,514 rows were screened in breeding
nurseries at the St Paul (4,751 rows), Morris (782 rows) and Crookston (4,981 rows) field
sites.

43
In 2001 winter/spring, over 300 liters of macroconidial inoculum (800,000 macroconidia
ml -1 ) were produced in our lab for use by the barley and wheat improvement programs.
This is enough inoculum to inoculate about 60 miles of row… and we used it all! Much
of the material that was screened at St. Paul was also included in the Morris and
Crookston nurseries where either macroconidial- or colonized-corn-seed-inoculum was
used.
Greenhouse Screening Program: During the fall, winter, and spring seasons greenhouse
tests for resistance to Fusarium head blight are conducted in the greenhouse. Testing of
200 barley lines was completed in 2001. The inoculation of barley plants in the
greenhouse is conducted using an airbrush sprayer to deliver macroconidial inoculum to
one face of the barley spike. Point inoculations have not differentiated between the
currently available sources of resistance and susceptible varieties.
Effect of burning wheat and barley residues on the survival of Fusarium
graminearum and Cochliobolus sativus: The amount of cereal residues left in fields
after harvest has increased in recent years largely because of the widespread adoption of
minimum tillage practices. Since residues decompose slowly in the Upper Midwest crop
residues are suspected to be the principal reservoir of the primary inoculum of F.
graminearum in the recent severe epidemics of Fusarium head blight. Fungi such as F.
graminearum, are able to survive in wheat residues for at least two years therefore, any
practice that enhances residue decomposition or eliminates these residues may aid in the
management of this destructive disease. This study examined the effect of residue
burning on the survival of F. graminearum and C. sativus the causal agents of two
important diseases of barley (Fusarium head blight and spot blotch) in the Midwest cereal
producing areas.
The field study was established at the University of Minnesota’s Research and Outreach
Center, Crookston, MN. On September 15-16, 2000, two quadrats (0.91 m 2 ) were burned
in a field of barley residue using a flame thrower. Two additional non-burned quadrats
were used as controls. Residues left after burning, and all residues in control plots were
collected and stored at -10°C until isolations were made. To quantify the effect burning
on the amount of residue, the total number of nodes recovered was determined for each
experimental plot. To examine the mycoflora present in node tissues, nodes were excised
from the straw, and split in two, surface sterilized and each half node was plated
separately on one of two media. A total of 48 node-segments from each control quadrat,
and all available node segments from burned-quadrats were plated. Node segments were
plated (eight/petri dish) on petri plates containing acidified half strength PDA or a
Fusaria selective medium (Komada). Culture plates were incubated with 12 hr
photoperiod using a combination (3:1) of cool white and UVA light. Fusarium
graminearum was identified based on perithecia formed on Carnation Leaf Agar.
Cochliobolus sativus and other fungi were identified based on morphological
characteristics. Data obtained in this study were analyzed by ANOVA (SAS, GLM
Procedure). When needed, as appropriate, treatment means were separated using Fisher’s
protected least significant differences (P = 0.05).
There was a significant reduction in the number of nodes recovered from burned
treatments (339 nodes) as compared to the unburned controls (515 nodes). Isolations

44
made on culture media showed that barley nodes recovered, irrespective of treatment,
were readily colonized by F. graminearum and C. sativum. Other pathogenic and nonpathogenic
Fusarium species were also recovered from nodes, but less frequently.
Recovery of F. graminearum (FG) and C. sativus (CS) were significantly (P>0.01)
reduced in burned residues (FG, 6%; CS, 4%) in comparison with the non-burned
residues (FG, 42.7%; CS, 13%). Recovery of both pathogens was almost nil from
visually charred residues. Recovery of F. culmorum, F. avenaceum, and F.
sporotrichioides, and other fungi followed a similar pattern. Our data show that residue
burning can reduce the inoculum potential of pathogens present in residues, and may
assist in the management of destructive diseases such as Fusarium head blight.
Our data shows that wheat and barley residues left on the field after harvest were
colonized by F. graminearum, and C. sativus, which indicates that crop residues provide
a suitable reservoir for the survival of these pathogens between growing seasons. The
practice of burning residue reduced the amount of residues left on the soil after harvest by
two thirds, and substantially reduced the population of F. graminearum, and C. sativus
present in straw. These results agree with those found by Reis and Abrao (1983; Plant
Disease, 76:1088-1089) and Bateman et al. (1988; Ann Appl. Biol., 132:35-47). These
findings confirm the role that residues play in the epidemiology of foliar diseases of
barley and suggest that residue burning, even where residues are not totally destroyed,
may be useful in the management of destructive diseases such as Fusarium head blight.
Influence of Mist-Irrigation on Fusarium Head Blight and Seed Characteristics of
Barley:
Fusarium head blight levels in field nurseries are often variable and this variation
complicates our ability to obtain consistent results when screening parental sources of
barley and segregating populations in search of resistant germplasm. Field experiments
were conducted in 2000 and 2001 to evaluate examining the management of
supplemental moisture volume in FHB screening nurseries. The main objective of this
study was to determine the effect of different daily mist-irrigation volume treatments on
several variables that are used to estimate FHB-severity. These data were compared to
inoculated barley plots with no mist irrigation, relying only on seasonal dew and rainfall
to promote FHB infection.
The experimental design was a randomized complete split-block for each mist-irrigation
(MI) volume treatment. Split-block treatments were inoculated vs. non-inoculated. The
barleys examined were MNS 93 (resistant, R), MNBrite (moderately resistant, MR),
Robust (susceptible, S), and Stander (S). Row spacing was 0.3 m and length of a plot-row
was 2.4 m with two rows per plot. At heading, a suspension, containing 100,000
macroconidia ml-1 was applied using a CO2 powered backpack sprayer with TeeJet
SS80015 flat-fan nozzles operating at a pressure of 2.8 kg cm -2 . Inoculum suspensions
were mixed with 2 ml Tween-20 l -1 . Following the initial inoculation, mist irrigation was
applied each day beginning at 5 PM and concluding the following morning at 8 AM with
a total of 8 mist irrigation intervals per night. Water volume dispensed in each of the
blocks was 2, 4, and 8 mm of water, respectively. Mist irrigation was terminated the day
FHB assessments were conducted. Disease assessments were made in the field 14-18
days post-inoculation (PI) and plot seed was harvested at crop maturity. FHB disease

45
assessments were made by counting the percentage of FHB infected spikelets on 20
heads per plot. FHB incidence (FHBI) was measured as the percentage of infected spikes
of the twenty that were assessed. FHB severity (FHBS) per plot was then derived as the
product of FHBI and FHB severity of infected spikes. Discolored kernels were measured
as the percentage of seed that were obviously discolored compared to normal bright
colored barley from a 100-seed sample from each plot. Deoxynivalenol concentration
(parts-per-million, ppm) in grain was determined following procedures established by
Mirocha et al. (1998; J. Agric. and Food Chem., 46:1414-1418).
In 2000 mean FHBI was 25, 36, 50, and 75 (l.s.d.0.05=18) without MI for the barleys
MNS 93, MNBrite, Robust and Stander, respectively. The high FHBI for MI treatments
of 2-8 mm per day for barley readily confirmed MNS 93 to be more resistant than the
other barleys. Differences of FHBS were significant for barley but differences among
entries were less variable at the no-mist and 2 mm MI treatments. Differences of FHBS
were pronounced between MNS 93 and Stander barley over all MI volume treatments
and no different between Robust and MNBrite. Differences among the barley entries for
the percentage of discolored kernels were most apparent at 2 and 8 mm volume
treatments. Differences of deoxynivalenol (DON) concentration in grain among the four
barley cultivars were consistent over each of the mist-irrigation treatments.
Differentiation among susceptible and resistant barley cultivars was possible under the
no-mist treatment for FHBI. Drier weather in 2001 diminished our ability to differentiate
barley entries. Differences among barley cultivars for FHBS were apparent over the four
MI treatments tested and was clearest for discolored kernels at the highest MI treatment.
These preliminary data suggest that reducing mist-irrigation volume does not inhibit
investigators from differentiating resistant and susceptible germplasm. Reducing MI
volume could also minimize the likelihood of inducing too severe FHB levels. This
would then facilitate the production of higher quality seed within breeding lines that are
less severely infected with FHB and provide higher quality seed for later experiments.
These data also demonstrate that investigators are not necessarily bound to locations with
irrigation facilities. Useful information could be obtained by inoculating plots in
locations without water thus increasing the number of locations that could be utilized to
screen promising germplasm. Lower disease severity conditions may more realistically
reflect conditions observed in farmers’ fields as well. Additional studies are underway to
further examine the effects of reducing mist-irrigation in FHB screening nurseries.
Plant Pathology Research on other Barley Diseases
This program also provides support to the barley breeding program as part of ongoing
breeding efforts to develop improved germplasm with improved disease resistance.
Screening breeding germplasm for resistance to kernel discoloration, leaf rust, loose
smut, net blotch, powdery mildew, and spot blotch are conducted on an ongoing basis
with the barley breeding program.
In 2001 evaluations were conducted to identify resistant germplasm in breeding
populations following natural infections of leaf spot diseases at the Northwest Research
and Outreach Center, Crookston and the West Central Research and Outreach Center,

46
Morris. Greenhouse tests for resistance to net blotch, powdery mildew, and loose smut
were also conducted.
Publications
Dill-Macky, R., and Salas, B. (2001). Effect of burning wheat and barley residues on the
survival of Fusarium graminearum and Cochliobolus sativus. In: Proceedings of the
2001 National Fusarium Head Blight Forum, Erlanger, Kentucky, USA, December 8-10,
2001, p. 112.
Dill-Macky, R., Evans, C.K., and Culler, M.D. (2001). Manipulating artificial epidemics
of Fusarium head blight in wheat with inoculum concentration. In: Proceedings of the
2001 National Fusarium Head Blight Forum, Erlanger, Kentucky, USA, December 8-10,
2001, p. 113.
Evans, C.K., and Dill-Macky, R. (2001). Influence of mist-irrigation on Fusarium head
blight and seed characteristics of wheat and barley. In: Proceedings of the 2001 National
Fusarium Head Blight Forum, Erlanger, Kentucky, USA, December 8-10, 2001, p. 234.
Dill-Macky, R., and Salas, B. (2001). Effect of burning wheat and barley residues on the
survival of Fusarium graminearum and Cochliobolus sativus. Phytopathology, 91:S23.
Evans, C.K., and Dill-Macky, R. (2001). Influence of mist-irrigation on Fusarium head
blight and seed characteristics of wheat and barley. Phytopathology, 91:S27.
Personnel
Ruth Dill-Macky, Associate Professor, Project Leader
C. Kent Evans, Research Associate
Bacilio Salas, Research Associate
Amar M. Elakkad, Research Plot Technician
Christine C. Newby, Junior Laboratory Technician
Karen J. Wennberg, Assistant Scientist
Cooperators
Kevin P. Smith and Gary J. Muehlbauer - Department of Agronomy and Plant Genetics,
University of Minnesota, St Paul, MN 55108
Roger K. Jones and Brian J. Steffenson - Department of Plant Pathology, University of
Minnesota, St Paul MN 55108
John V. Wiersma - Northwest Research and Outreach Center, University of Minnesota,
Crookston, MN 56716
George A. Nelson - West Central Research and Outreach Center, University of
Minnesota, Morris, MN 56267

47
IDENTIFICATION OF NOVEL DISEASE RESISTANCE GENES FOR BARLEY
Brian J. Steffenson
Department of Plant Pathology
University of Minnesota
St. Paul, MN 55108
OBJECTIVES
The primary mission of the Cereal Disease Resistance Project at the University of
Minnesota is the control of economically important barley (Hordeum vulgare) diseases.
For many diseases, this goal is best achieved through the development of cultivars with
genetic resistance. Wild barley species are rich sources of disease resistance genes for
cultivated barley. Thus, one of the main objectives of this research is to evaluate selected
accessions of H. vulgare subsp. spontaneum (wild barley) and H. bulbosum (bulbous
barley grass) for resistance to the major diseases in the Upper Midwest. Ancillary
objectives related to this project and Upper Midwest barley breeding programs include,
surveying commercial barley fields for diseases in Minnesota; collecting pathogen
isolates from infected barley cultivars and assessing their virulence; and increasing and
maintaining pathogen stocks for testing breeding lines and wild species for resistance.
Evaluation of Hordeum vulgare subsp. spontaneum accessions for disease resistance.
A total of 391 H. v. subsp. spontaneum accessions collected across the Fertile Crescent
were evaluated to Fusarium head blight (FHB) (caused by Fusarium graminearum) in
Hangzhou, China. A high degree of genetic diversity was observed for FHB reaction as
disease severities ranged from

48
were resistant to all six diseases. These accessions have been crossed to a susceptible
malting barley cultivar to study the genetics of multiple disease resistance.
Evaluation of H. vulgare × H. bulbosum recombinants. Dr. Richard Pickering of Crop
and Food Research in New Zealand has established a world-renown gene introgression
program to develop H. vulgare × H. bulbosum recombinants. We routinely screen
recombinants developed by Dr. Pickering for disease resistance. FHB data were obtained
on 26 introgression lines planted in China. Only two lines (38P18/22/3 and 219W3)
exhibited less than 10% FHB incidence and severity under light disease pressure. It is
not known whether the lower disease severity in these lines was due to the H. bulbosum
introgression because the H. vulgare parent also exhibited relatively low disease levels.
Further evaluation tests are planned to assess the FHB resistance and DON accumulation
of these recombinants.
Barley disease survey. A survey was made in the northwestern part of Minnesota (the
primary barley production area in the state) to ascertain the prevalence and importance of
barley diseases in 2001. Eleven fields were visited during the survey conducted on July
24-26. FHB and net blotch were detected in every field; the observed severities ranged
from trace to 4% and trace to 80%, respectively (Table 1). Stem rust, SSLB, and loose
smut were the next most common diseases observed in commercial fields. Other diseases
identified in the survey (listed in decreasing order of prevalence) were crown rust, barley
yellow dwarf, and bacterial blight. Net blotch has been and continues to be a major
disease problem on six-rowed barley in the region. FHB and SSLB have historically
been minor diseases of barley in the Upper Midwest. However, since 1993, these
diseases have increased significantly in importance, and epidemics have occurred in some
regions of the state. It is not uncommon to find loose smut in Minnesota barley fields,
but the incidence is usually very low. Producers who save seed from the previous year’s
crop often have higher levels of loose smut in their fields. Crown rust, barley yellow
dwarf, and bacterial blight are common diseases in Upper Midwest barley fields—the
severity of each disease in any one year can vary dramatically. For example, barley
yellow dwarf is usually found in trace amounts in just a few fields each year. However,
in 1999, a severe epidemic occurred, and the disease was found in over 80% of fields and
in severities of up to 50%.
Collecting and maintaining a library of pathogen isolates. It is essential to keep a
library of characterized pathogen isolates to conduct disease evaluation tests of breeding
material and other germplasm. Additional isolates of F. graminearum, P. t. f. teres, C.
sativus, and P. g. f. sp. tritici were collected during the 2001 barley disease survey.
These isolates were purified and then stored for future virulence evaluations.
Additionally, eleven isolates of P. hordei were received from cooperators around the
United States in 2001. Due to severe insect infestations in the greenhouse, none of the
isolates was successfully increased.
Evaluation of barley breeding germplasm for resistance to foliar disease. One
hundred and forty-four barley lines from the University of Minnesota Barley
Improvement Program were inoculated with the spot blotch pathogen in the field at St.
Paul. This evaluation was done to ensure that the durable spot blotch resistance in sixrowed
germplasm was maintained in populations derived from non-elite parents.

49
Unfortunately, disease levels in the nursery were too low to assess due to the extremely
dry weather after inoculation. Instead, seedling plants will be inoculated and evaluated
for spot blotch resistance in the greenhouse.
Personnel:
Brian J. Steffenson, Associate Professor/Project Leader
Chris Kavanaugh, Junior Scientist
Hilda Salas, Junior Scientist
Gacheri Kimanthi, Postdoctoral Research Associate
Shaobin Zhong, Postdoctoral Research Associate
Cooperators:
Michael C. Edwards, USDA-ARS, Fargo, ND
Paul B. Schwarz, NDSU, Fargo, ND
Jerome D. Franckowiak, NDSU, Fargo, ND
Richard D. Horsley, NDSU, Fargo, ND
Kevin Smith, UM, St. Paul, MN
Gary Muehlbauer, UM, St. Paul, MN
Ruth Dill-Macky, UM, St. Paul, MN
Darrell Wesenberg, USDA-ARS, Aberdeen, ID
Harold Bockelman, USDA-ARS, Aberdeen, ID
Andy Kleinhofs, WSU, Pullman, WA
Richard Pickering, Crop & Food Research, Christchurch, New Zealand
Patrick M. Hayes, OSU, Corvallis, OR
Recent Publications:
Zhong, S., and Steffenson, B. J. 2001. Genetic and molecular characterization of mating
type genes in Cochliobolus sativus. Mycologia 93:852-863.
Zhong, S., and Steffenson, B. J. 2001. Virulence and molecular diversity in Cochliobolus
sativus. Phytopathology 91:469-476.
Schwarz, P. B., Schwarz, J. G., Zhou, A., Prom, L. K., and Steffenson, B. J. 2001. Effect
of Fusarium graminearum and F. poae infection on barley and malt quality. Monatsschr.
Brauwissenschaft 54:55-63.
Edwards, M. C., Fetch, T. G. Jr., Schwarz, P. B., and Steffenson, B. J. 2001. Effect of
Barley yellow dwarf virus infection on yield and malting quality in barley. Plant Dis. 85:
202-207.
Steffenson, B. J. 2001. Exploiting wild Hordeum species for barley improvement. Pages
28-33 in: Proc. 33 rd Barley Improvement Conference. San Antonio, TX.

Table 1. Results of the 2001 barley disease survey (11 fields visited) in Minnesota.
Disease/Pathogen
Fusarium head blight
(Fusarium spp.)
Net blotch
(Pyrenophora teres f. teres)
Wheat stem rust
(Puccinia graminis f. sp. tritici)
Septoria/Stagonospora speckled leaf blotch
(Septoria and Stagonospora spp.)
Loose smut (incidence)
(Ustilago nuda)
Crown rust
(Puccinia coronata var. hordei)
Barley yellow dwarf
(Barley yellow dwarf virus BYDV)
Bacterial blight
(Xanthomonas translucens pv. Translucens)
50
Number of fields
with disease
Disease severity or
incidence
11 Trace to 4%
11 Trace to 80%
8 Trace to 3%
7 Trace to 5%
7 Trace
8 Trace to 15%
4 100%
1 20%

51
BARLEY TRANSFORMATION
G.J. Muehlbauer, C.A. Mackintosh, D.T. Tuong and N. Al-Saady
Department of Agronomy and Plant Genetics, University of Minnesota
St. Paul, MN 55108
Summary
1. We have modified a protocol for transforming the elite barley variety Conlon in our
laboratory. 1500, 1105 and 40 callus pieces have been bombarded with gold particles
coated with DNA encoding the following antifungal proteins (AFP): a wheat thaumatinlike
protein 1 (tlp-1), a barley chitinase and a barley glucanase. Many regenerating
plantlets are in the final stages of tissue culture. Further bombardments using the barley
glucanase and other AFP genes are on-going.
2. We have obtained affinity purified polyclonal antibodies to the following proteins; a
barley ribosome inactivating protein (RIP), a wheat tlp-1, a barley chitinase and a
barley glucanase. Protocols for using these antibodies in Western blotting to determine
AFP expression are being developed using transgenic wheat. Thus, we will be able to
determine the presence of these proteins in our transgenic barley as soon as the plants are
growing in soil.
3. We are developing in vitro fungal growth assays in order to identify antifungal
properties of transgenic barley seedlings. This will allow us to determine potential
antifungal effects before the plants reach flowering, the growth stage at which Fusarium
graminearum-glasshouse disease screening occurs.
4. We have continued to characterize barley plants transformed with the GUS gene
driven by the sugarcane badnavirus (ScBV) promoter. As described in the AMBA 2000-
2001 progress report, we developed twelve transgenic lines of barley expressing GUS
driven by the ScBV promoter. In the T1 generation we showed that the ScBV promoter
drives expression in all barley tissues in all twelve transgenic lines. Our work within the
last year was directed at determining the stability of GUS expression over generations.
Therefore, we have evaluated T2 generation lines. In the T2 generation, GUS was
expressed in eight of the 12 transgenic lines and appeared to be silenced in the other four
lines.
Introduction
Genetic transformation promises to be a powerful tool for improvement of crop plants.
Genetic manipulations that are not possible using conventional breeding methods are
attainable with plant transformation. Barley transformation has been accomplished via
particle bombardment into immature zygotic embryos, callus-derived immature embryos
and microspore-derived embryos (Wan and Lemaux, 1994).
Fusarium head blight (FHB), a fungal disease of small grain crops caused by Fusarium
graminearum, threatens to reduce barley to an economically unviable crop in Minnesota
and many other states. Currently, large breeding efforts at the University of Minnesota
and around the country are focused on developing FHB resistant cultivars. However,

52
there is a question about whether there is sufficient genetic variation for FHB resistance
in these programs.
To increase FHB resistance in barley, my laboratory is developing transgenic barley
carrying antifungal protein (AFP) genes. Our long-term goal is to develop a large set of
transgenic barley lines carrying a diverse array of AFP genes.
This novel germplasm will be screened for resistance to scab as well as against other
fungal pathogens in the glasshouse. However, glasshouse screening of transgenic barley
lines takes some time, with screening typically not occurring before T2 generation plants.
Therefore, we aim to develop in vitro assays which will allow us to determine whether or
not the desired antifungal effect is likely to be produced by our transgenic barley lines in
the glasshouse and field.
There are a limited number of useful promoter sequences for barley transformation.
Therefore, there is a need to develop and test new promoter sequences for use in barley
transformation. The sugarcane badnavirus (ScBV) promoter exhibits a high level of
constitutive expression in oat (Tzafrir et al., 1998). We have continued to investigate the
usefulness of the sugarcane badnavirus promoter in barley.
1. PLANT TRANSFORMATION AND CHARACTERIZATION
1.1 Materials and Methods
1.1.1 Plant material
Previously, we were using the cultivar Golden Promise in our transformation system and
were able to produce several lines of barley over-expressing a barley RIP gene.
However, at the beginning of this funding period, we decided to transform the cultivar,
Conlon. Conlon exhibits good agronomic and malting quality characteristics. Also,
screening of Golden Promise against FHB was known to be notoriously difficult. Thus,
we felt our efforts were better concentrated on developing a transformation system in our
laboratory for the variety Conlon. Consequently, our efforts have largely invested in this
area and our previously produced lines of Golden Promise carrying RIP have received
little attention.
Conlon stock plants are grown in growth chambers at 20°C under a 16-h light period and
at 18°C for an 8-h dark period.
1.1.2. Available antifungal protein genes and promoters
Currently, we have the following antifungal protein genes available: alfalfa acidic ß-1,3glucanase,
barley chitinase, Arabidopsis thaliana PR5, rice chitinase, oat thaumatin-like
protein 1, oat thaumatin-like protein 4, wheat α-thionin, wheat thaumatin-like protein 1,
barley Type I ribosome inactivating protein (RIP) gene, and the barley class-II ß-1,3glucanase
gene. In general, the mode of action for all of these AFPs is to degrade or
disrupt either the cell wall or membrane of F. graminearum. The exception is the RIP
gene, which encodes a protein that inhibits fungal protein synthesis. We are using the
constitutive promoter from the maize ubiquitin gene to drive expression of the AFP
genes.

53
We have available the sugarcane badnavirus (ScBV) promoter which exhibits constitutive
expression in oat (Tzafrir et al., 1998). We obtained a plant transformation plasmid with
the ScBV promoter fused to the ß-glucuronidase (GUS) gene.
All of the plant transformation plasmids described above were obtained from either Drs.
Ann Blechl (USDA-ARS) or Richard Zeyen (University of Minnesota).
1.1.3 Selectable markers and reporter gene plasmids
pAHC25 contains the maize ubiquitin promoter driving the expression of the bar and
GUS genes. The pAHC20 plasmid contains the maize ubiquitin promoter driving
expression of the bar gene. Expression of bar in plant cells confers resistance to the
herbicide bialaphos. Expression of GUS confers a blue precipitate upon incubating the
tissue in X-Gluc (Jefferson et. al., 1987).
1.1.4 Explant sources
Barley spikes are harvested approximately 12-14 days after anthesis. Developing kernels
are sterilized according to Tingay et al., 1997. Immature embryos are excised from the
kernels and placed scutellum-side up on callus induction media. A few days later, the
root and shoot axis is removed from the embryo and the embryo halved, the two halves
being placed on fresh callus induction media. The embryo pieces are then left in the dark
at 24°C for approximately two weeks.
1.1.5 Biolistic transformation
Small callus pieces are placed on osmoticum media overnight in the dark at 24°C before
being bombarded with gold-coated DNA according to Wan and Lemaux (1994). All
bombardments contain a plasmid carrying the maize ubiquitin promoter driving the
expression of an AFP gene as well as the pAHC25 plasmid.
1.1.6 Selection and regeneration of transgenic plants
We have modified a method for selection and regeneration of transgenic barley
developed by L. Dahleen, USDA-ARS. After particle bombardment, the callus pieces are
transferred to callus induction media supplemented with 5 mg/l bialaphos. Selection of
embryogenic callus takes six weeks and takes place in the dark at 24°C. Callus pieces are
then placed on maintenance media for 4 weeks, supplemented with 5 mg/l bialaphos, and
placed at 24°C under dim light for 16 h/day. To regenerate plantlets, resistant
embryogenic callus are transferred to regeneration media supplemented with 5 mg/l
bialaphos, and incubated at 24°C under bright lights (16 h/day). This process takes 4
weeks. To induce rooting, plantlets will be placed on rooting media with 3 mg/l
bialaphos. After roots develop, plantlets will be transferred to soil and grown to maturity.
1.1.7 Characterization of transgenic plants
GUS activity will be monitored histochemically with X-Gluc substrate as described in
Jefferson et al., 1987. Antifungal gene expression will then be determined by RT-PCR.
Antifungal protein expression will be determined by Western blotting using our affinity
purified polyclonal antibodies. Further molecular characterization of the transgenic
plants will be conducted by RNA and DNA gel blot analysis. Expression analysis of
AFP genes will be conducted in the T0, T1 and T2 generations.

54
1.2 Results
Most of this year was spent establishing a transformation protocol for Conlon in our
laboratory. So far, 1500, 1105 and 40 callus pieces have been bombarded with DNA
encoding tlp-1, chitinase and glucanase. Numbers of regenerating callus pieces showing
the presence of green shoots are shown in Table 1. Many of these plantlets are due to be
moved onto rooting media on March 7 th 2003. Thus, we are very close to having plants
in soil and beginning the characterization work. Particle bombardments are continually
on-going in our laboratory.
Table 1: Conlon transformation tissue culture
Gene Total number of
regenerating plantlets
Number of unique
regenerating events
Bombarded callus pieces
not yet at regeneration
stage
Tlp-1 164 101 n/a
Chitinase 131 123 675
Glucanase n/a n/a 40
2. ANTIBODY PRODUCTION
Polyclonal antibodies were produced by Quality Controlled Biochemicals, Hopkinton,
MA. From the translated sequences of our barley glucanase and barley RIP genes, we
were able to choose one peptide sequence which was then synthesized, purified by HPLC
and its sequence verified by mass spectrometry. Each peptide was then conjugated to a
carrier protein and used to immunize two rabbits. For each animal, a pre-immune bleed
was taken, followed by four production bleeds at various intervals after a total of five
immunizations.
For our barley chitinase and our wheat tlp-1 proteins, two peptides were synthesized for
each antibody produced and used together for the immunization of each of the two
animals. The proteins were less immunogenic and thus, two peptides were chosen to
increase the likelihood that the animals would produce antibodies.
Each bleed was tested in our laboratory against the purified peptide and detection of the
protein by the antibodies was successful. The bleeds were then affinity purified to
produce purified polyclonal antibodies.
These antibodies will be used to detect the corresponding proteins in the transgenic
barley plants.
3. IN VITRO FUNGAL GROWTH ASSAYS
3.1 Materials and Methods
We have tried several different methods in an attempt to develop an in vitro assay which
would allow us to test whether or not a transgenic plant was likely to produce an
antifungal effect in glasshouse screening of F. graminearum.
Firstly, we tried to infect plant leaves floating on water in a Petri dish, by placing drops
of F. graminearum spore suspensions on the leaf surfaces. However, we could not get
reproducible fungal hyphae growth on plant leaves.

55
Secondly, we tried to grow F. graminearum on liquid carrot juice with or without plant
tissue extracts. However, we were not able to quantify the fungal growth since the
hyphal spread over the surface of the carrot juice occurred over the entire surface of the
well. It did not occur from the center outwards or from the circumference inwards.
Thirdly, we tried to grow F. graminearum on solid carrot juice with or without plant
tissue extracts. This method may be useful, but there are problems associated with in
vitro fungal growth on solid media. Often, the mycelium can grow, avoiding the
antifungal substances incorporated into the media (Issac and Jennings, 1995). Similarly,
we tried allowing F. graminearum to grow on solid carrot juice from the center outwards.
Around the circumference of the dishes were filter paper pieces with extracts from plant
tissues. We had hoped that the fungal growth would be halted upon reaching a filter
paper with extract from a transgenic plant. However, we feel that the fungal growth was
too vigorous by the time it reached the filter paper pieces to have been affected by the
potential antifungal plant extracts. However, this method may be worthy of further
investigation.
We have developed a system for growing F. graminearum in liquid culture. Inhibiting
fungal growth in liquid culture is known to be more representative of the effect of the
plant extract on the fungus rather than growing the fungus on solid media with the plant
extract incorporated into it (Issac and Jennings, 1995). F. graminearum was maintained
on Potato Dextrose Agar (PDA) in the dark at 24°C. 250ml-flasks were sterilized and 70
mls of Yeast Peptone Glucose (YPG) media added to each flask. Varying concentrations
of EDTA were filter-sterilized and added to the flasks in duplicate to obtain final
concentrations of 10 mM, 1 mM, 100 µM, 10 µM, 1 µM and 100 nM EDTA. Control
flasks had water added only. Two 5-mm discs of fungal mycelium, each divided in half,
were added aseptically to the flasks and the fungus grown, shaking, at 24°C under a 16 h
photoperiod. After two days, the fungus was strained, and centrifuged at 3000 rpm for 15
minutes to remove excess liquid. The wet weights were recorded.
3.2 Results
Growth of F. graminearum was totally inhibited by 1 mM EDTA (Figure 1). EDTA was
chosen to develop the assay since it is known to inhibit the growth of F. graminearum
(R. Skadsen, personal communication). We feel that the assay will be suitable to test
plant extracts in vitro against the growth of F. graminearum.

7
6
5
4
3
2
1
0
56
Figure 1: Weight of Fusarium graminearum mycelium grown in liquid culture. Results are the
means ±SEM of duplicate measurements.
* indicates only one measurement made
Weight of Fusarium graminearum mycelium (g)
Control * 10 mM EDTA 1 mM EDTA 100 µM EDTA
Treatment
10 µM EDTA * 1 µM EDTA 100 nM EDTA
4. CHARACTERIZATION OF THE SCBV PROMOTER
From twelve T0 events, 5-10 T1 seeds were planted to produce T1 plants. These T1 plants were
analyzed for GUS expression. GUS was expressed highly in the roots, stems, leaves and all
reproductive tissues of the barley plants, the ovaries, anthers, bracts and rachis.
T2 seed from each of those twelve lines was then planted out and the T2 plants analyzed for GUS
expression. In eight of the lines, the expression remained constant, as it had in the previous
generation. However, in four of the lines, GUS expression was silenced. Nevertheless, these
data indicate that the ScBV promoter will be a useful promoter for transforming barley.
Future work - 1 st March 2002 - 30 th June 2002
Conlon stock plants are planted every two weeks. Therefore, an almost continuous supply of
explant material is available. In the remaining four months of this project, we will increase the
number of callus pieces bombarded with our barley glucanase gene from 40 to approximately
1000. We will also bombard approximately 1000 callus pieces with our barley RIP gene. The
success rate of barley transformation is approximately 1%, thus, 1000 callus pieces are necessary
to produce a significant number of transgenic events.
We will begin to characterize transgenic plants that are presently in the late stages of tissue
culture, utilizing our antibodies. We will begin to advance them to the next generation. We will
also test transgenics that express AFPs for in vitro antifungal effects.

57
References:
Issac, S. and Jennings, D.H. 1995. Microbial culture. Bios Scientific Publishing,
Oxford.
Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusion: ß-glucuronidase
as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6:3901-
3907.
Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang, M., Thornton, S. and Brettell, R.
1997. Agrobacterium tumefaciens-mediated barley transformation. Plant J. 11:1369-
1376.
Tzafrir, I., Torbert, K.A., Lockhart, B.E.L., Somers, D.A. and Olszewski N.E. 1998. The
sugarcane bacilliform badnavirus promoter is active in both monocots and dicots. Plant
Mol. Biol. 38:347-356.
Wan, Y. and Lemaux, P.G. 1994. Generation of large numbers of independently
transformed fertile barley plants. Plant Physiol. 104:37-48.
Personnel:
Caroline A. Mackintosh, Postdoctoral Research Associate
Dong T. Tuong, Technician
Nadiya Al-Saady, Ph.D. student
Collaborators:
Dr. Ann Blechl, USDA-ARS, Albany, CA
Dr. David Somers, University of Minnesota
Dr. Richard Zeyen, University of Minnesota

58
DEVELOPING IMPROVED MALTING BARLEY VARIETIES FOR MONTANA
Tom Blake
Department of Plant Science and Plant Pathology
Montana State University
Objectives
Barley is an important component of the Montana economy with barley sales accounting
for almost $80 million annually. With 1.1 million acres of barley seeded in 2001,
Montana ranked second in US barley production. Over the past three years, malting
varieties have accounted for 58% of the barley seeded in the state with Harrington as the
predominant malt variety. While Harrington has the market edge in Montana, we are
attempting to produce new lines which have a more stable agronomic performance yet
maintain and potentially improve malting quality. By utilizing high malting quality and
strong agronomically performing lines in our breeding program, we now have
experimental lines which can surpass Harrington for agronomic performance and may
challenge Harrington for malting quality. The major objective of our project was to
improve the agronomic performance and stability of our 2-rowed malting barley
germplasm base in Montana. We feel great advancements have been made toward this
objective through the use of improved parental materials and a strong breeding and
selection program as evidenced in this report.
Methodology
The Montana State University barley improvement program utilizes a rapid advancement
(3 generations/year) single seed descent approach to inbred line derivation. In addition to
our traditional breeding program, we have incorporated molecular genetic approaches
into our germplasm selection and improvement. Molecular mapping of Karl x Lewis
recombinant inbred lines has revealed QTL which are currenlty being used in the
development of stable, low grain protein lines.
The barley breeding program at Montana State is a collaborative effort with the Montana
Agricultural Research Centers. We have seven major research locations distributed
across Montana which are a major assett to our breeding program. From Huntley and
Sidney stations in the east to Moccasin and Havre in central Montana, Conrad and
Kalispell in the west, and Bozeman in south central, we can evaluate our lines under a
diverse range of environmental conditions in a given year. These seven research stations
allow observation under six dryland conditions and five irrigated conditions.
We make approximately 100 new crosses each year. During a 2 year process we advance
between 100 and 200 recombinant inbred lines per initial cross to the F5 generation, and
evaluate about 15,000 F5 headrows each spring. From these F5 headrows approximately
3000 plants are selected for seeding the following year in normal seeding density 2-row
plots. These 3000 plots are evaluated for agronomic desirability, and approximately 600
are hand-harvested. Grain from these F6 lines is evaluated for yield, protein content, and
test weight. Based on these paramters, the most promising 250 are advanced to our
Preliminary Yield Trial (PYT) conducted at two locations (Bozeman-irrigated and

59
Huntley-dryland) with two replications per location. The top 75 percent of these lines
ware sent to the CCRU Malt Quality Lab in Madison, WI. From the PYT results, 60
lines are selected and advanced to an Early Yield Trial (EYT). The EYT is conducted at
four locations (Moccasin, Bozeman, Havre, and Sidney) with three replications per
location. Based on agronomic performance and malting quality, around 25 lines from
the EYT are advanced to our intrastate trials. The intrastate trials are conducted at 11
locations (six dryland and five irrigated) with three replications per location. Grain from
the Bozeman location of the intrastate yield trial is also analyzed by the CCRU Malt
Quality Lab. Agronomic performance is combined with this information to determine if
a line merits further evaluation for potential release.
Results
Malting Quality: The use of Baronesse in our breeding program since the early 1990’s
has greatly improved the agronomic performance of our lines. Two Klages x Baronesse
derivatives, MT970110 and MT970116, and a Manley x Baronesse derivative,
MT960101, were entered into the 2001 crop AMBA Pilot Scale Malting Evaluation
Program. MT960101 passed its first year of pilot scale tests in 2000. MT960101 and
MT970116 have exhibited higher malting quality than Harrington produced under
irrigation in the 2001 growing season (Table 1).
While only MT970116 surpassed the malt quality score of the Harrington Malt Standard,
we have several experimental lines in development with comparable quality to the
recommended malting varieties when grown in the same environment (Table 1). We are
now employing recurrent selection practices for further improvement of these lines. This
approach will allow us to accumulate the favorable traits and continually enhance our
germplasm base through recombination and selection.
Agronomic Performance: Based on multiyear averages, Baronesse has greater yield than
any of the experimental lines with high malting quality scores in the 2001 trial (Table 2).
MT960099 had the greatest yield, averaging 0.6 bu/A less than Baronesse, but had a
malting quality score of 31. Other lines of great interest include MT981091 yielding 2.8
bu/A less than Baronesse averaged over all locations and 2.5 bu/A less on irrigated plots
(Tables 2 and 3). This line had a quality score of 38 and appears to have a good
complement of agronomic and malting qualities. Based on the multiyear data,
MT970116 yields 5.8 bu/A less than Baronesse averaged over all locations and 6.2 bu/A
less for irrigated locations. MT970116 had the highest quality score of our experimental
lines.
In addition to our intrastate evaluation, three experimental lines were entered in the 2001
Western Regional Spring Barley Trials. Averaged over nine high moisture or irrigated
locations in 2001, Baronesse yielded 108.5 bu/A while MT960099, MT960228, and
MT970116 yielded 114.5, 110.0, and 110.6 bu/A, respectively. Under four dryland
locations, MT960099, MT960228, and MT970116 yielded 102.5, 108.9, and 102.6 bu/A,
respectively, relative to Steptoe at 94.9 bu/A and Hector at 96.7 bu/A.
Based our intrastate yield trials, Western Regional trials, and the malting quality
information, we anticipate MT970116 to be an outstanding line for both malting quality
and agronomic performance in our region. Crosses between our most promising lines

60
have already been made in efforts to surpass the yield of Baronesse and the malting
quality of Harrington while maintaining environmental stability.
Table 1. Malting quality performance of high yielding experimental lines and
recommended malting varieties for Montana. Data from 2001 Irrigated Intrastate Trials
in Bozeman, MT.
Kernel on Barley Malt Barley Wort Alpha- Beta-
Variety or Weight 6/64" Color Extract Wort Wort Protein Protein S/T DP amylase glucan Quality
Selection (mg) (%) (Agtron) (%) Color Clarity (%) (%) (%) (°ASBC) (20°DU) (ppm) Score
Harrington 37.4 77.1 84 81.0 1.4 1 11.3 4.25 37.8 97 58.6 423 26
Morex 33.4 72.2 89 78.5 1.7 2 13.2 4.40 34.3 137 47.1 369 29
Legacy 33.3 75.7 84 78.3 1.5 2 13.0 4.88 37.9 145 62.3 318 32
Merit 41.8 89.3 76 79.8 1.4 1 12.5 4.46 35.6 105 61.4 212 35
Conlon 36.2 79.7 73 79.0 1.5 1 12.2 3.16 27.5 46 35.1 439 20
Garnet 39.9 84.6 82 81.9 1.6 1 12.6 4.77 38.0 135 70.4 279 33
MT970116 42.3 87.5 78 80.2 1.2 1 12.1 3.75 32.7 98 45.3 208 41
MT981039 38.1 85.5 88 81.8 1.5 1 11.4 4.25 39.7 99 57.6 109 38
MT981091 42.3 88.8 79 79.5 1.1 1 11.9 3.65 30.9 100 40.5 422 38
MT981238 46.5 93.5 83 79.7 1.5 1 13.3 4.74 35.7 117 67.4 428 38
MT981210 39.5 85.3 87 81.1 1.4 1 12.6 4.67 39.8 133 58.1 226 36
MT970155 41.4 88.0 78 79.7 1.4 1 13.2 4.47 36.4 101 63.6 286 35
MT960101 36.2 62.2 84 80.7 1.5 2 13.2 4.67 36.1 117 68.9 97 34
MT990023 36.7 69.6 84 79.3 1.3 1 12.7 3.95 31.3 116 48.5 131 32
MT990041 36.1 74.2 81 81.7 1.5 1 12.6 5.13 40.9 151 73.3 106 32
MT960099 37.7 60.7 75 80.0 1.6 1 12.5 4.52 38.0 116 59.7 179 31
MT970110 38.9 77.8 83 78.9 2.1 2 12.4 3.67 31.3 97 34.5 527 21
Harrington
(Check)
Harrington
(Check)
39.3 93.9 81 82.2 1.9 1 11.7 5.49 49.8 111 78.6 44 42
38.8 94.0 81 81.9 2.0 1 11.4 5.54 51.0 109 80.0 44 39

61
Table 2. 1992 thru 2001 Overall Summary for Montana experimental lines with high
malting quality scores, recommended malting varieties for Montana, and feed barley
checks. Data is averaged over dryland and irrigated locations.
ID
Pedigree
#
Yrs
Yield
(bu/ac)
#
Yrs
Test Wt
(lb/bu)
#
Yrs
Plump
(%)
#
Yrs
Heading
Date
#
Yrs
Plant Ht.
(in)
PI568246 Baronesse 101 103.0 100 51.1 97 78.4 96 178.0 99 28.6 81 12.1
MT960099 Manley/Baronesse 41 102.4 40 50.5 41 65.4 39 179.2 41 27.7 41 12.1
MT981091 MT851195/MT140523 21 100.2 20 51.7 21 83.2 19 175.9 21 28.6 21 12.0
MT960101 Manley/Baronesse 41 97.5 40 50.5 41 69.2 39 179.8 41 29.1 41 12.1
MT970116 Klages/Baronesse 32 97.2 31 52.4 32 84.3 30 175.7 32 33.5 32 12.2
MT970110 Klages/Baronesse 32 97.1 31 51.4 32 80.7 30 178.9 32 30.8 32 12.4
PI610264 Valier 51 96.9 50 51.5 51 76.1 47 178.5 50 30.9 51 12.7
MT981210 MT910150/Stark 21 96.9 20 51.7 21 88.8 19 177.1 21 30.8 21 12.4
MT970155 MT886610/MT140523 26 96.3 25 51.4 26 81.4 24 179.1 26 29.3 26 12.4
MT981238 ND112311/Lewis 21 96.0 20 52.6 21 87.0 19 174.2 21 32.2 21 12.6
MT910189 ND 7293/Bearpaw 83 93.6 82 51.5 81 83.4 78 174.3 81 29.7 76 11.9
SiteMean 101 93.5 100 50.8 97 77.5 96 176.0 99 30.2 81 12.5
PI491534 Gallatin (Check) 101 93.2 100 51.7 97 74.9 96 175.4 99 31.5 81 12.3
CI 15856 Lewis 101 92.8 100 51.8 97 77.2 96 176.5 99 31.4 81 12.9
2B914947 Merit 60 91.7 59 48.6 60 75.7 55 178.9 58 30.4 58 12.4
BA 1202 Busch Agr 1202 72 91.4 72 49.0 69 75.1 69 177.8 70 30.4 60 12.6
SK 76333 Harrington 101 90.8 100 49.6 97 75.2 96 177.7 99 30.6 81 12.3
MT981039 H5860219/Baronesse 21 90.3 20 51.4 21 81.2 19 179.3 21 29.2 21 12.5
6B932978 Legacy 21 87.8 20 48.0 21 71.9 19 175.2 21 32.7 21 12.7
CI 15773 Morex 65 80.0 64 48.9 65 72.5 62 172.7 64 35.4 59 12.7
#
Yrs
Protein
(%)

62
Table 3. 1992 thru 2001 Overall Summary in Irrigated Environments for Montana
experimental lines with high malting quality scores, recommended malting varieties for
Montana, and feed barley checks.
ID
Pedigree
#
Yrs
Yield
(bu/ac)
#
Yrs
Test Wt
(lb/bu)
#
Yrs
Plump
(%)
#
Yrs
Heading
Date
#
Yrs
Plant Ht.
(in)
PI568246 Baronesse 45 124.2 45 51.8 44 86.8 43 176.9 45 30.8 35 11.4
MT960099 Manley/Baronesse 19 123.8 19 51.2 19 76.5 18 178.1 19 29.9 19 11.3
MT981091 MT851195/MT140523 10 121.7 10 51.9 10 87.9 9 174.8 10 30.4 10 11.8
MT970110 Klages/Baronesse 15 119.7 15 52.0 15 90.5 14 178.0 15 33.3 15 11.5
MT970116 Klages/Baronesse 15 118.0 15 52.6 15 89.0 14 175.1 15 35.5 15 11.6
MT981238 ND112311/Lewis 10 117.3 10 52.6 10 90.2 9 173.7 10 34.7 10 12.4
PI610264 Valier 23 117.3 23 51.9 23 85.0 21 177.3 23 33.4 23 12.0
MT960101 Manley/Baronesse 19 117.1 19 51.0 19 77.6 18 178.7 19 31.5 19 11.4
MT981210 MT910150/Stark 10 116.3 10 51.7 10 92.8 9 176.3 10 32.9 10 11.9
MT970155 MT886610/MT140523 15 115.0 15 51.7 15 88.3 14 178.1 15 31.5 15 11.6
2B914947 Merit 26 113.8 26 49.2 26 86.8 24 177.5 26 32.8 25 11.6
SiteMean 45 112.8 45 51.1 44 84.0 43 174.8 45 32.3 35 11.8
PI491534 Gallatin (Check) 45 112.3 45 52.1 44 81.8 43 174.7 45 33.9 35 11.6
BA 1202 Busch Agr 1202 31 112.1 31 49.9 30 85.3 30 176.7 31 32.9 25 11.8
6B932978 Legacy 10 111.5 10 49.0 10 80.4 9 174.6 10 35.4 10 12.1
MT910189 ND 7293/Bearpaw 31 111.5 31 51.3 31 87.0 29 173.3 31 31.7 30 11.4
CI 15856 Lewis 45 110.2 45 52.0 44 83.1 43 175.2 45 33.9 35 12.3
SK 76333 Harrington 45 108.7 45 50.1 44 81.9 43 176.8 45 33.1 35 11.7
MT981039 H5860219/Baronesse 10 106.4 10 51.5 10 86.7 9 178.5 10 32.1 10 11.2
CI 15773 Morex 31 97.4 31 49.2 31 81.4 30 172.2 31 37.9 26 12.2
Other Research Projects and Future Research Direction
In addition to the research funded by the AMBA, we are working on a number of projects
focusing on barley improvement. We currently have a graduate student developing near
isogenic lines for a low grain protein QTL identified in a Lewis x Karl recombinant
inbred population. Fine mapping of this chromosome region will be an important
contribution to the barley community as we focus on identifing the genes conferring low
grain protein. A phenotypic and marker assisted selection program is also being
innitiated to more fully utilize the grain protein QTL identified in the Lewis x Karl
recombinant inbred population for our breeding program. Research on the use of barley
as a feed source for rangeland cattle is ongoing in our program. We anticipate this study
will lead to a prediction model which can be used for accurately predicting animal
performance based on grain quality parameters. <strong>Progress</strong> in SNP marker development
has also been made over the last year in our lab. We anticipate the focus of our future
research to remain on breeding improved malting varieties with increased agronomic
#
Yrs
Protein
(%)

63
performance for Montana farmers. This approach will allow farmers to grow barley
which can be sold for malting while not sacrificing yield if malt quality standards are not
reached in a given year.
Project Personnel
Tom Blake, Professor and Project Leader (Academic Leave 12/31/01 to 1/1/04)
Suzanne Mickelson, Adjunct Assistant Professor and Project Leader (1/1/02 to present)
Pat Hensleigh, Research Associate
Hope Talbert, Research Associate
Recent Publications
Benham J and Blake TK. 1999. Genographer: A graphical tool for automated AFLP
analysis. Journal of Agricultural Genomics vol 4. http://www.ncgr.org/ag/jag
Habernicht, D.K. and Blake T.K. 1999. Application of PCR to detect varietal purity in
barley malt. Journal of the American Society of Brewing Chemists 57(2):64-71.
Shan X. TK Blake and LE Talbert. 1999 Conversion of AFLP markers to sequencespecific
PCR markers in barley and wheat. Theor and Appl. Genet.98:1072-1078.
Regli DC, Bowman JGP, Blake TK, Borkowski JJ, Surber LMM, Rolando SJ, Robinson
BL, Roth NJ, Bockleman H. 1999. Digestibility characteristics of barley lines from the
world collection in rats. Proc. West Sect. Amer. Soc. An Sci. 50:79-82
Blackhurst TC, Bowman JGP, Surber LMM, Milner TJ, Daniels TK, Blake TK. 1999.
Feeding value of Lewis x Baronesse Recombinant Inbred Barley Lines for Finishing
Steers. Proc. West Sect. Amer. Soc. An Sci. 50:104-108
Boss DL, Bowman JGP, Surber LMM, Anderson DC, Blake TK. 1999. Feeding value of
two Lewis x Baronesse Recombinant Inbred Lines, LB13 and LB30, for Finishing Steers.
Proc. West Sect. Amer. Soc. An Sci. 50:119-126
Surber LMM, Bowman JGP, Blake TK, Blackhurst TC, Rolando SJ. 1999. Variation in
feed quality chatacteristics in the Lewis x Baronesse Recombinant Inbred Barley Lines.
Proc. West Sect. Amer. Soc. An Sci. 50: 241-246
Sayed-Tabatabaei B-E, Mano Y., Takaiwa F., Oka S., Blake TK, Komatsuda T. 1999
Sequence-Tagged-Sites for Barley Genome Mapping. Bull. Of the Nat. Ins. Of
Agrobiological Resources (Japan) 13:11-22.
Mano,Y. Sayed-Tabatabaei BE, Graner A, Blake T, Takaiwa F, Oka S, Komatsuda T.
2000. Map construction of sequence-tagged sites in barley. Theor. Appl. Genet. 99:937-
946.
Roy JK, Prasad M, Varshney RK, Balyan HS, Blake TK, Dhaliwal HS, Singh H,
Edwards KJ, Gupta PK.2000. Identification of a Microsatellite on Chromosome 6B and a
STS on 7D of Bread Wheat showing association with preharvest sprouting tolerance.
Theor. Appl. Genet. 99:336-340.

See, D., Kanazin V., Talbert H., Blake T. 2000. An Electrophoretic Single Nucleotide
Polymorphism Detection System. Biotechniques 28:710-716.
64
Blackhurst, T. C., J.G.P. Bowman, L.M.M. Surber, T. K. Daniels, and T. K. Blake. 2000.
Improving the feed value of barley for finishing steers. Proc. West. Sec. Am. Soc. Anim.
Sci. 51:30-33.
Surber, L.M.M., J.G.P. Bowman, T. K. Blake, D. D. Hinman, D. L. Boss, and T. C.
Blackhurst. 2000. Prediction of barley feed quality for beef cattle from laboratory
analyses. Proc. West. Sec. Am. Soc. Anim. Sci. 51:295-298.
Surber, L.M.M., J.G.P. Bowman, T. K. Blake, V. E. Nettles, A. L. Grindeland, M. T.
Stowe, R. L. Endecott, K. N. Robinson, B. L. Robinson, and D. R. See. 2000.
Determination of genetic markers associated with forage quality of barley for beef cattle.
Proc. West. Sec. Am. Soc. Anim. Sci. 51:454-457.
Bowman JGP, Blake TK, Surber LMM, Habernicht DK, and Bockleman H. 2001. Feed-
Quality Variation in the Barley Corel Collection of the USDA National Small Grains
Collection. Crop Sci. 41:863-870.
Surber LMM, Stowe MT, Bowman JGP, Cash SD, Hensleigh PF, and Blake TK. 2001.
Variation in forage quality characteristics of barley. Proc. West. Sec. Am. Soc. Anim.
Sci. 52: 353-356.
Surber LMM, Bowman JGP, Blake TK, Robison DN, Endecott RL, and Robison BL.
2001. Identification of genetic markers associated with forage quality characteristics in
Lewis x Karl barley lines. Proc. West. Sec. Am. Soc. Anim. Sci. 52: 292-295.
Endecott, R. L., J.G.P. Bowman, L.M.M. Surber, D. L. Boss, K. N. Robison, and T. K.
Blake. 2001. Feeding value of Lewis and Baronesse barley lines for finishing steers.
West. Sec. Am. Soc. Anim. Sci. 52:551-554.
Kincheloe, J. J., J.G.P. Bowman, L.M.M. Surber, K. A. Anderson, K. N. Robison, R. L.
Endecott, B. L. Robinson, and T. K. Blake. 2001. Feeding value of Morex, Steptoe, and
two experimental backcross barley lines for finishing steers. West. Sec. Am. Soc. Anim.
Sci. 52:555-558.
Sherman JD, Smith LY, Blake TK, Talbert LE. 2001. Identification of barley genome
segments introgressed into wheat using PCR markers. Genome 44: 1-7.
Beecher B, Smidansky ED, See D, Blake TK Giroux MJ. 2001. Mapping and sequence
analysis of barley hordoindolines. Theor. Appl. Genet. 102:833-840.
Kanazin, V. Talbert H., See, D., Blake T. in press. Discovery and Manipulation of Single
Nucleotide Polymorphisms in Barley. Plant Mol. Biol. March 2002.
See D. Kanazin V. Kephart K., Blake T. in press. Mapping genes controlling variation in
barley grain protein content. Crop Science. May 2002.
Blake, T. Bowman, J. Hensleigh P., Boss D., Carlson G., Kushnak G., Eckhoff J.
submitted. Release of ‘Valier’ Barley. Crop Science. Accepted for publication 11/01.

65
Blake, T,. Hensleigh P., Boss D., Carlson G., Kushnak G., Eckhoff J. submitted. Release
of ‘H3860224’ Barley. Crop Science. Accepted for publication 11/01.
Beecher B, Bowman J, Martin J, Bettge A, Morris CF, Blake TK and Giroux MJ.
submitted. Hordoindolines are associated with a major endosperm texture QTL in Barley
(Hordeum vulgare L.). Genome. Accepted for publication 1/02.
Fischer, AM, Meyer, FD, Garmenr JP, and Blake TK. submitted. Mapping of QTLs
associated with nitrogen storage and remobilization in barley (Hordeum vulgare L.)
leaves. J. Expt. Bot. In review.

66
EPIDEMIOLOGY AND CONTROL OF BARLEY LEAF DISEASES CAUSED BY
FUNGAL PATHOGENS
M. R. Johnston
Department of Plant Sciences and Plant Pathology
Montana State University
Bozeman, MT 59717
OBJECTIVES
Continue to develop a catalog of mapped stripe rust resistance loci with special emphasis
on temperature sensitive and growth stage dependant disease resistant reactions in order
to implement a rational and durable stripe rust management strategy for the western U.S.
In connection with objective we will also investigate resistance to several other fungal
leaf diseases in segregating populations. The final goal would be the release of barley
cultivars with multiple resistances.
The popular demand for pesticide free food will make chemical control of diseases more
undesirable in future. Environmental aspects are expected to further restrict the
application of chemicals in agriculture. The use of resistant cultivars as opposed to
susceptible ones will give barley growers more competitive in such a marketplace.
Moreover resistant cultivars are considered an economical means of disease control. In
the past single genes for disease resistance have been rendered ineffective by the everchanging
pathogens. Strategies such as the employment of numerous genes in one
cultivar or the use of nontraditional resistance genes (temperature sensitive genes, adult
plant resistance) are advisable.
METHODS
Selected lines from the Oregon State breeding program (as well as other interested
programs) will be inoculated with pure cultures of barley stripe rust and subjected to
different temperature profiles. The normal evaluation environment will be 65°/70°F
(dark/light). 54°/60°F (dark light) will represent the cool environment. Plants are grown
under these conditions prior to inoculation. A measured amount of urediniospores will be
shot up into a settling tower with a CO2 gun and allowed to settle for 4 min. Resulting in
an even distribution of inoculum. Inoculated plants are then placed in a dew chamber at
10°C for a minimum of 24 hrs. and subsequently returned to their respective
environments. Disease reactions will be evaluated 10 or 18 days after inoculation
depending on the temperature regime used. The reaction type will be determined on the
first and second leaves.
We will also subject the materials to the disease at different developmental stages. Adult
plant resistance in wheat to stripe rust is well documented. Plants with already
determined seedling reaction will be grown to maturity under evaluation environment
which resembles natural conditions during the summer months (15°/24°C). Plants will be
inoculated when the stems elongate and a second time when the flag leaf is fully
developed. Plants will be dusted with a mixture of talcum and urediniospores since the

67
settling tower cannot accommodate larger plants. Inoculated plants will be treated as
described above.
RESULTS
Qualitative and quantitative resistance genes to barley stripe rust have been pyramided in
numerous two-row spring barley lines in the Oregon program. Sixty-three of these with
documented field resistance to barley stripe rust were evaluated for seedling resistance
under two different temperature profiles (Tab. 1 and 2). The majority of the lines also
exhibited a highly resistant or intermediate reaction as seedlings. Few lines were scored
highly susceptible. Differences between the two temperature profiles used were small.
Some plants were allowed to develop to the boot stage. These were reinoculated and
evaluated for resistance. A number of lines showed considerable differences between
seedling and adult stage reaction to barley stripe rust.
Forty-four spring and winter barley breeding lines from the Oregon program with field
resistance to barley stripe rust were evaluated in the described fashion (Tab. 3). As
described above, most of these lines were also resistant as seedlings under both
temperature profiles. This set, however, seems to contain a few entries with a highly
temperature dependent reaction to the pathogen (Stab 7, Icaro/Acuario34 and
Ajo/He6890 18-3). One entry (Stab 113) was segregating (3:1) for resistance to stripe
rust.
Table. 1: Seedling reaction to stripe rust of 18 two-row spring barley lines with adult
plant resistance.
Line Pedigree
Stripe Rust Reaction
Night/day temp.
65/70F 55/60F
OPS 35 BCD12xD3-6/B61(Ops) 5 8
OPS 36 BCD12xD3-6/B61(Ops) 2n 2n
OPS 37 BCD12xD3-6/B61(Ops) 2n 1
OPS 38 BCD12xD3-6/B61(Ops) 5 7
OPS 41 BCD12xD3-6/B61(Ops) 2n 1n
OPS 42 BCD12xD3-6/B61(Ops) 6 7
OPS 43 BCD12xD3-6/B61(Ops) 7 7
OPS 44 BCD12xD3-6/B61(Ops) 7 8
OPS 46 BCD12xD3-6/B61(Ops) 2n 1
OPS 48 BCD12xD3-6/B61(Ops) 1 1
OPS 49 BCD12xD3-6/B61(Ops) 2n 1
OPS 54 BCD12xD3-6/B61(Ops) 8 8
OPS 68 BCD12xD3-6/B61(Ops) 1n 1n
OPS 73 BCD12xD3-6/B61(Ops) 1n 1n
OPS 79 BCD12xD3-6/B61(Ops) 2n 1n
OPS 80 BCD12xD3-6/B61(Ops) 1 1
OPS 83 BCD12xD3-6/B61(Ops) 2n 2n
OPS 85 BCD12xD3-6/B61(Ops) 1 1

68
Table 2. Seedling reaction to stripe rust of 45 two-row spring barley lines with adult
plant resistance.
Line Pedigree
Stripe Rust Reaction
Night/day temp
65/70F 54/60F
OPS 1 BCD12xD3-6/B61(Ops) 7 7
OPS 2 BCD12xD3-6/B61(Ops) 6 2
OPS 3 BCD12xD3-6/B61(Ops) 1 1
OPS 4 BCD12xD3-6/B61(Ops) 0 1n
OPS 5 BCD12xD3-6/B61(Ops) 5 5
OPS 6 BCD12xD3-6/B61(Ops) 1 1
OPS 7 BCD12xD3-6/B61(Ops) 0 1
OPS 8 BCD12xD3-6/B61(Ops) 1 1
OPS 9 BCD12xD3-6/B61(Ops) 8 8
OPS 10 BCD12xD3-6/B61(Ops) 9 9
OPS 11 BCD12xD3-6/B61(Ops) 0 0
OPS 12 BCD12xD3-6/B61(Ops) 8 8
OPS 13 BCD12xD3-6/B61(Ops) 1 1
OPS 14 BCD12xD3-6/B61(Ops) 1 1
OPS 15 BCD12xD3-6/B61(Ops) 2 1
OPS 16 BCD12xD3-6/B61(Ops) 1 1
OPS 17 BCD12xD3-6/B61(Ops) 5 5
OPS 18 BCD12xD3-6/B61(Ops) 4 4
OPS 19 BCD12xD3-6/B61(Ops) 1 1
OPS 20 BCD12xD3-6/B61(Ops) 0 1
OPS 21 BCD12xD3-6/B61(Ops) 1 1
OPS 22 BCD12xD3-6/B61(Ops) 6 7
OPS 23 BCD12xD3-6/B61(Ops) 5 7
OPS 24 BCD12xD3-6/B61(Ops) 1 1
OPS 25 BCD12xD3-6/B61(Ops) 1 1
OPS 26 BCD12xD3-6/B61(Ops) 1 1
OPS 27 BCD12xD3-6/B61(Ops) 7 7
OPS 28 BCD12xD3-6/B61(Ops) 1 2
OPS 29 BCD12xD3-6/B61(Ops) 9 9
OPS 30 BCD12xD3-6/B61(Ops) 1 1n
OPS 31 BCD12xD3-6/B61(Ops) 8 8
OPS 32 BCD12xD3-6/B61(Ops) 8 8
OPS 33 BCD12xD3-6/B61(Ops) 5 5
OPS 34 BCD12xD3-6/B61(Ops) 1n 1
OPS 39 BCD12xD3-6/B61(Ops) 1n 1
OPS 40 BCD12xD3-6/B61(Ops) 2 1
OPS 50 BCD12xD3-6/B61(Ops) 1n 4
OPS 66 BCD12xD3-6/B61(Ops) 2n 2n
OPS 69 BCD12xD3-6/B61(Ops) 4 4
OPS 78 BCD12xD3-6/B61(Ops) 2 2
OPS 84 BCD12xD3-6/B61(Ops) 1n 1
BCD 47 5 5
BCD 12 5 4
D3-6/B23 1 1
D3-6/B61 1 1

Table 3. Seedling reaction to stripe rust of 44 spring and winter wheat breeding lines.
69
Variety/Line Pedigree
Stripe Rust Reaction
Night/day temp.
18/22C 12/14
Kold 1285/Astrix 1n 1n
Strider OR 1860164/Steptoe 1 4
Kab 37 Kold/88Ab536 7 7
Kab 47 Kold/88Ab536 2 6-7
Kab 51 Kold/88Ab536 1n 1
Stab 7 Strider/88Ab536 6 9
Stab 47 Strider/88Ab536 0 3
Stab 113 Strider/88Ab536 7(3plts), 3(12plts) 7(2plts), 3(15plts)
00AB 547 98AbKab/BC49 1 1
88 Ab 536 Ne 76129/Morex//Morex 9 9
Harrington 8 8
BU 16 BCD47xD3-6/B23(BU) 1n 1n
BU 27 BCD47xD3-6/B23(BU) 2n 1n
BU 28 BCD47xD3-6/B23(BU) 3 8
BU 37 BCD47xD3-6/B23(BU) 1 1
BU 38 BCD47xD3-6/B23(BU) 2n 1
BU 45 BCD47xD3-6/B23(BU) 2n 1
AJO 12 BCD47xD3-6/B61(Ajo) 2n 1n
AJO 44 BCD47xD3-6/B61(Ajo) 3 5
AJO 59 BCD47xD3-6/B61(Ajo) 2n 1n
AJO 61 BCD47xD3-6/B61(Ajo) 2n 1n
OPS 19 BCD12xD3-6/B61(Ops) 2n 1n
OPS 66 BCD12xD3-6/B61(Ops) 2n 1n
OPS 78 BCD12xD3-6/B61(Ops) 1n 1n
BCD 47 4 6
BCD 12 5 7
Icaro/Acuario 34 Icaro/Acuario 34 1n 7
OPS/He 6890 38-3 OPS/He 6890 38-3 3n 3
Bu/He 6890 39-1 Bu/He 6890 39-1 2n 2n
Ajo/He 6890 18-3 Ajo/He 6890 18-3 1 6
Bu/He 6890 9-3 Bu/He 6890 9-3 1 1
Bu/He 6890 36-3 Bu/He 6890 36-3 1n 1
Sal/He 6890 4-1 Sal/He 6890 4-1 2n 3n
OPS/He 6890 36-1 OPS/He 6890 36-1 1n 3n
Bu/He 6890 9-2 Bu/He 6890 9-2 2n 3n
Bu/BCD 47 16-3 Bu/BCD 47 16-3 4 6
Sal/He 6890 37-1 Sal/He 6890 37-1 2n 1
Sal/He 6890 24-2 Sal/He 6890 24-2 2n 4-5
Mex 18-Robust ped. Mex 18-Robust ped. 5 6
Ajo/BCD 47 7-2 Ajo/BCD 47 7-2 4 7
Oxbow/He6890 11 Oxbow/He6890 11 7 9
OPS/BCD 47 51-3 OPS/BCD 47 51-3 2n 2n
OPS/BCD 47 13-2 OPS/BCD 47 13-2 2n 1n
Baronesse Baronesse 8 8

70
RELATED PROJECTS
Breeding materials and barley lines to be released in Montana have been screened for
disease resistance in cooperation with the Montana Foundation Seed Program.
Scald and net blotch populations are being monitored. No new virulence types have
occurred over the last few years. Drought conditions have not been conducive to
pathogen development in many parts of the state.
Assistance was provides in regard to disease screening programs for barley stripe rust and
other leaf diseases.
We continue to maintain numerous leaf pathogens of barley.
PUBLICATIONS
Castro A., X. Chen, A. Corey, T. Fillichkin, P.M. Hayes, M. Johnston, S. Sandoval-Islas,
and H. Vivar, 2002. Stripe rust resistance QTL pyramids in barley. In: Plant and Animal
Genome X, San Diego, January 12-16 2002. Abstracts, p 179.
Castro A., X. Chen, A. Corey, T. Fillichkin, P.M. Hayes, M. Johnston, S. Sandoval-Islas,
and H. Vivar. Pyramiding of seedling barley stripe resistance QTL. In preparation for
Crop Science

71
TWO-ROWED BARLEY IMPROVEMENT PROJECT
Jerome D. Franckowiak
Department of Plant Sciences
North Dakota State University
Fargo, ND 58105
Barley Breeding Objectives
The primary objective of the barley breeding programs in the Agricultural Experiment
Station, North Dakota State University (NDSU) is to develop and release improved
barley cultivars for production in North Dakota (ND) and adjacent states. New cultivars
should be acceptable to ND growers and those who use or process barley. Basic and
applied research is conducted on barley to provide information that will aid achievement
of the barley improvement goals, improve cultural practices, and enhance the
understanding of barley genetics. This report emphasizes research on improvement of
two-rowed barley for ND. The research activities of the two-rowed barley program are
coordinated with the activities of other barley research projects at Fargo: the six-rowed
barley improvement program, the barley quality program in the Department of Cereal
Science, the barley pathology program in the Department of Plant Pathology, and the
barley genetics program at the USDA-ARS Northern Crops Science Laboratory.
General Seasonal Information
Two-rowed barley cultivars and experimental lines were evaluated in yield trials grown at
six ND locations during 2001. In addition the Carrington, Dickinson, Hettinger,
Langdon, Minot, and Williston Research Extension Centers conducted yield trials at offstation
sites. Pure seed increase plots of 34 entries in variety and advanced yield trials
were grown in large drill strips at Casselton and Fargo. Pure seed increases of 17
historical cultivars and 66 experimental lines in intermediate yield trials were grown in
small drill strips at Fargo. The barley yield trials were sown between 26 April and 15
May. The Western Spring, the Western Spring Dryland, and the Canadian Western Tworow
cooperative barley nurseries were each grown at two locations.
The yield trials were planted relatively early in 2001 and stand establishment was good at
all locations. At Carrington, the herbicide Puma applied prior to several days of cold
temperatures and caused differential damage to plots. Standing water in late June
damaged plants in the Casselton, Fargo, and Prosper nurseries. Plots at Hettinger were
destroyed by hail. Yield plots at Langdon were not harvested because of severe lodging
and subsequent leaf diseases. Spot blotch, incited by Cochliobolus sativus, was severe in
the nurseries at Casselton and Prosper. Spot blotch was also severe in several fields of
‘Harrington’ barley in the Minot area. The incidence of Fusarium head blight (FHB),
caused mainly by Fusarium graminearum, was high in nurseries at Carrington and
Langdon sites. Septoria leaf blotch, incited primarily by Septoria passerinii, were
observed after heading in nurseries throughout ND. Net blotch, incited by Pyrenophora
teres f. teres, were found on a few lines at Carrington and Minot. Notes on taken on
lodging at Williston and grain protein values were estimated. Yields averaged about 75
bu/a from field plots at Fargo, 80 bu/a at Minot, and 90 bu/a at Williston.

72
Varieties and Variety Tests
Conlon was released in 1996 by NDSU for production in western ND and was
recommended as a two-rowed malting barley by AMBA in May 2000. In 2001, Conlon
was the second most popular barley cultivar in ND, sown on 5.7% of the 1.8 million
acres of barley. Conlon heads more than one day earlier than Bowman and retains test
weight well compared to other cultivars. It has shown some resistance to FHB and the
accumulation of the toxin deoxynivalenol (DON). Conlon is resistant to net blotch and
has the genes Mlg and Mlk for resistance to powdery mildew. It is susceptible to spot
blotch and septoria leaf blotch. As a result, it is better adapted for production in western
ND. The yields of Conlon are about 10 to 15% lower than the best six-rowed cultivars in
most ND yield trials. Several growers reported that in 2001 Conlon grain frequently had
much lower DON readings than that of Robust.
Advanced and Intermediate Yield Trials
2ND18365 (2B91-4947/ND15403) was most promising line in advanced yield trials.
2ND19012 (Logan//ND16463/ND15409) and 2ND19121 (ND15468/ND16092//
ND16461) were the most promising lines in intermediate yield trials. The number of
promising lines is low because few lines had low grain protein values combined with
high yields. Differential stunting of plants in the Carrington trial by the herbicide Puma
further reduced the number of promising lines. ND16092 (ND13297/ND14701) and
some of its derivatives showed a level of tolerance to Puma similar to that observed of
six-rowed barley cultivars. ND15403 (Logan sib/ND13297) and ND16461 (ND13296/
ND14760) are the low grain protein parents of several promising lines.
Lines entered in advanced and intermediate yield trials were screened for field response
to spot blotch and grown in the Fusarium head blight nursery at Osnabrock. The best
lines in 2001 yield trials were tested for seedling responses to net blotch. Researchers in
the Department of Plant Pathology conducted field and seedling tests for disease
reactions. Seed lots of the best lines in the advanced and intermediate trials were
submitted to the USDA-ARS Cereal Crops Research Unit at Madison, WI for malt
quality tests.
Yield and agronomic comparisons of 2ND18365 with Conlon indicate that it yields more,
has lower lodging readings, and is more resistant to leaf spot diseases (Table 1). It is
slightly later and taller than Conlon. Malt quality tests indicate that 2ND18365 has
higher values for malt extract, soluble protein, and alpha-amylase activity than Conlon
(Table 2). It also has slightly lower grain protein values. Its diastatic power values are
lower than those of Conlon and its kernels were not quite as plump.
Line 2ND19119 (ND15403/ND15368//ND16453) was identified as a high yielding line
with large, plump kernels in 2000. It had high extract values and very low grain protein
values. Data from 2001 trials indicate that 2ND19119 has high yield potential and
adequate resistance to leaf spot diseases. However, 2ND19119 was very susceptible to
Puma injury in 2001 and its kernels showed poor hull adherence. This brief history of
2ND19119 demonstrates that low grain protein and large seed size can be combined in
Midwest two-rowed barley, but also that numerous other agronomic traits are difficult to
control in the breeding program.

Table 1. Agronomic trait comparisons for 2ND18365 and check cultivars using data
from nurseries grown in North Dakota from 1999 to 2001.
Entry
Yield (bu/a)
Heading date
(after 5/31)
73
Plant height
(inches)
Leaf
spot
(1-9) 1
Lodging (1-
9) 2
Test
weight
(lbs/bu)
Trials 8 10 11 3 5 5
2ND18365 83.7 26.7 31.3 3.8 2.5 50.3
Conlon 76.1 25.9 30.3 5.4 4.5 50.1
Drummond 81.4 27.9 32.3 3.3 2.0 48.9
Stander 82.2 28.7 31.4 3.6 3.2 49.1
1 A leaf spot score of 1 = no leaf disease symptoms and 9 = nearly complete defoliation.
2 A lodging score of 1 = no lodging, 9 = nearly flat.
Table 2. Malt quality comparisons for 2ND18365 and check cultivars using data
obtained from analyses of barley samples grown in North Dakota during 1999 and 2000.
Entry*
Plump
kernels
(%)
Kernel
weight
(mg)
Barley
protein
(%)
Malt
extract
(%)
Betaglucans
(ppm)
Wort
protein
(%)
Wort N/
total N
(%)
Diastatic
power
(ΕL)
Alpha-
amylase
(20Ε)
Trials 3 3 3 3 3 3 3 3 3
2ND18365 78.7 38.0 13.6 80.7 293 6.40 47.1 98 87.8
Conlon 81.3 36.9 14.5 78.5 288 5.21 35.9 139 67.5
Drummond 78.1 32.9 14.2 78.2 154 6.16 43.1 196 72.5
Stander 80.2 33.2 14.3 79.1 207 6.79 47.8 167 83.8
* Data courtesy of the USDA-ARS, Cereal Crops Research Unit, Madison, WI.
Preliminary Yield Trials
Four hundred five new selections were planted in the 2001 preliminary yield trials at
Fargo and Langdon using seed from the 2000-2001 Yuma, AZ nursery. Based on
heading dates, yield and grain protein data taken from the Fargo nursery and FHB
readings from the scab screening nursery at Osnabrock, seed samples of 200 selections
and 36 checks from the Fargo nursery were submitted to the USDA-ARS Cereal Crops
Research Unit, Madison, WI, for malt quality testing. Seed samples of 209 selections
plus checks were submitted to the Department of Plant Pathology for testing of seedling
reactions to net blotch. The most promising lines in the 2001 preliminary yield trials had
high yields at Fargo, resistance to net blotch, and low grain protein values. The best lines
include 2ND19888 (ND16523//ND15468/ND16092), 2ND19929 (TR258/ND17437),
2ND20040 (ND15403//ND16453/A64), and 2ND20121 (ND17383//ND16680/
ND17490).
Early Generation Material
Head rows (each from a single F2 spike) representing the F3 progenies of 128 crosses

74
were planted at two locations. Half of the 15,000 head rows were planted at Casselton on
19 May and the other half at Prosper on 29 May. Stand establishment was good, but
standing water damaged the head rows at both locations. Head rows were selected for
harvest based on spot blotch reaction, plant height, and estimated plant vigor. After
harvest and threshing, nearly half of the seed lots were discarded based on thin kernels,
poor seed color, or undesirable threshing characteristics. Seed lots from 1310 head rows
and 36 check rows were submitted to the Department of Cereal Science for determination
of grain protein values. Based on the results, two spikes from each of 693 head rows
were planted in a winter nursery near Yuma, AZ to increase seed for the 2002
preliminary yield trials. Diastatic power values will be estimated using remnant seed
before rows are selected for harvest. Relative straw strength, early maturity, and general
appearance will also be used as criteria to identify rows for harvest.
The crosses planted in the 2001 head rows were made to combine leaf spot resistance
with low grain protein and high malt extract. However, a severe spot blotch epidemic
was observed in nurseries at both Casselton and Prosper. Only a few head rows were
found to have low leaf spot incidence. This was unexpected because most parents were
moderately resistant to spot blotch. Isolates were made from infected leaves of
2ND16461 by Mr. Yongliang Sun, Department of Plant Pathology, NDSU. The causal
agent was identified as Cochliobolus sativus. Seedling disease tests conducted by Mr.
Sun and Dr. Stephen Neate showed that many two-rowed lines and several six-rowed
cultivars are susceptible to this new isolate, but resistant to older isolates.
The F2 progenies of 119 crosses were planted at Fargo and Prosper on 26 May and 29
May, respectively. During late June and early July standing water damaged plants in both
F2 nurseries. Spikes were selected from individual plants in the best progenies at both
locations. Also, F2 progenies of 99 promising crosses were sent to Christchurch, New
Zealand or Yuma, AZ for rapid generation advance in winter nurseries. Many of these
crosses were made to incorporate FHB resistance and early maturity genes into elite tworowed
breeding materials.
Over 350 crosses were made in 2001 spring and fall greenhouse nurseries. The primary
breeding goal was to combine large seed size and low grain protein with better FHB
resistance. Crosses designed to add resistance to the new spot blotch isolate were a
priority in the fall greenhouse nursery. Because the Eam6.h of Midwest barley cultivars
is located in the repulsion phase with QTLs for FHB resistance in chromosome 2H,
several crosses were made to evaluate the Eam5.x, eam9.l, or eam10.m genes for early
maturity in locally adapted material. A few crosses were made to incorporate the Rph15
gene for leaf rust resistance. The F1 generation of some crosses will be grown in the
2002 spring greenhouse, others will be grown at Langdon this summer.
Special Projects
1. Development of Morphological Marker Stocks in Bowman Barley. Backcrossing
individual morphological marker genes into the Bowman genetic background was
continued in 2001. Selected Bowman backcross-derived lines were grown near
Christchurch, New Zealand to collect agronomic data. Many morphological

75
mutants reduce plant vigor and grain yield; however, a few dense spike and
semidwarf mutants appeared to cause little yield reduction and improved lodging
tolerance. The agronomic value of several mutants should be reevaluated at other
locations and in better genetic backgrounds.
2. Recurrent Selection for Fusarium Head Blight Resistance. The male-sterile
facilitated recurrent selection population for FHB resistance in two-rowed barley
has undergone two cycles of selection for FHB resistance. Agronomically
desirable plants were selected at Langdon from rows derived male sterile plants
harvested from the Yuma nursery in 2001. Rows from these 259 seed lots were
planted in the 2001-2002 winter nursery at Yuma to facilitate random mating for
the next cycle of selection.
3. Resistance to Septoria passerinii. Ms. Manju Kathikeyan completed her studies
on the inheritance of resistance to septoria leaf blotch in barley. Inheritance tests
indicate that dominant genes control seedling reactions to S. passerinii in several
resistant cultivars. The genes for resistance to S. passerinii appeared to have
additive interactions. In highly resistant wild barley, Hordeum vulgare subsp.
spontaneum, accessions, two or more dominant genes controlled seedling
reactions to septoria leaf blotch. Incorporating resistance into elite breeding
material is complicated by related pathogens and by the lack of reliable field or
greenhouse tests.
4. Resistance to Fusarium Head Blight and Plant Height Genes. Recent studies have
mapped at least three QTLs for FHB resistance in chromosome 2HL. One near
the Vrs1 locus, one near the Eam6 locus, and one distal from the Vrs1 locus.
Besides the vrs1 and Eam6 loci, several other agronomically important genes are
located in this region of chromosome 2H. The hcm1 reduces plant height, the lin1
gene is associated with fewer rachis nodes per spike and increased kernel
plumpness, and the Hcm2 appears to increase peduncle length. The assumed
sequence of these genes in 2HL of Midwest six-rowed barleys is vrs1.a - lin1.a -
hcm1.a - hcm2.b - Gth1 - Eam6.h. The Gth1 (toothed or barbed lemma veins)
gene is often associated with low malt extract values in two-rowed barley. Highly
FHB resistant accessions appear to have the genes Vrs1.d - Lin1 - Hcm1 - Hcm2 -
Gth1 - eam6 at these loci. Ms. Nadejda Krasheninnik is examining the height
genes in several progenies in order to confirm that the above analysis in correct.
She is also studying the maturity mutant, mat-c, located near the Gth1 locus in
chromosome 2HL. In crosses heterozygous for alleles at both loci, segregates
having a recombination between the vrs1 and mat-c loci can be identified
visually. If an FHB resistance gene is located between these markers, desirable
recombinants could be easy to identify.
5. Resistance to Fusarium Head Blight and Early Maturity Genes. Several
introduced cultivars from China and Japan have moderately resistant to FHB in
tests conducted at Hangzhou, China. However, they head very early and low
yielding in ND. When several of these lines were crossed to Midwest two-rowed
lines, very few FHB resistant selections were recovered. Genes controlling
photoperiod response caused the difficulties in evaluation and utilization of FHB

76
resistance from Chinese and Japanese barley cultivars. Mr. Guotai Yu is studying
photoperiod response genes and FHB resistance genes in this material. Progenies
will be grown in Hangzhou, China; Fargo, ND; and Yuma, AZ to evaluate
photoperiod responses and to select desirable lines. Selected progenies and lines
will be screened for FHB reactions in China and at Osnabrock, ND. They will
also be evaluated for agronomic traits and malt quality parameters.
6. Early Maturity Genes in Midwest Barley. If recombination between the Eam6.h
allele and QTLs for FHB resistance cannot be identified in Midwest malting
barley, other maturity genes must be used. Initial experiments with other maturity
genes suggest that genes controlling plant height should be revisited as barley
lines adapted to the Upper Midwest are developed. The most promising maturity
genes are Eam5.x from India via CIMMYT lines, eam9.l from China via Japan,
and eam10.m from Russia via CIMMYT lines. In 2001 the eam9.l was found to
interact with sdw1 dwarfing gene to produce relatively early semidwarf plants.
The Eam5.x may show a similar interaction with the sdw1 gene. If these
observations can be verified, utilization of the sdw1 gene in development barley
cultivars for ND may be possible.
Personnel in the Department of Plant Sciences
Jerome D. Franckowiak, Professor and two-rowed barley breeder.
Roger C. Genoch, Research Technician III.
Manju Kathikeyan, an M.S. degree candidate from India.
Nadejda Krasheninnik, a Ph.D. degree candidate from Russia.
Guotai Yu, a Ph.D. degree candidate from China.
Publications
Franckowiak, J.D. 2001. Accumulating genes for disease resistance in two-rowed barley
for North Dakota. p. 39-46. In H.E. Vivar and A. McNab (eds.). Breeding Barley in the
New Millennium: Proceedings of an International Symposium. CIMMYT, Mexico., D.F.
Franckowiak, J.D. 2001. Coordinator’s report: Chromosome 2H (2). Barley Genet.
Newsl. 31: http://wheat.pw.usda.gov/ggpages/bgn/31/ul1txt.htm#2H.
Franckowiak, J.D. 2001. Coordinator’s report: Semidwarf genes. Barley Genet. Newsl.
31: http://wheat.pw.usda.gov/ggpages/bgn/31/ul1txt.htm#semidwarf.
Franckowiak, J.D., and R.D. Horsley. 2001. Development of scab resistant barley
varieties for North Dakota. p. 14-20. In Proc. 33 rd . Barley Improvement Conference.
American Malting Barley Assoc., Milwaukee, WI.

77
SIX-ROWED BARLEY IMPROVEMENT PROJECT
Richard D. Horsley
Department of Plant Sciences
North Dakota State University
Objectives
The six-rowed barley improvement research program at the Agricultural Experiment
Station, North Dakota State University, Fargo, is a cooperative effort among the
Departments of Plant Sciences, Cereal and Food Sciences, and Plant Pathology. The
fundamental objective of the program is to develop and release improved six-rowed
barley cultivars acceptable to barley producers in North Dakota and adjacent areas in the
United States, and to those who use or process this barley. Basic and applied research is
conducted at NDSU on barley to provide information that will facilitate achievement of
the barley improvement goals, improve cultural practices, and enhance our understanding
of barley.
Barley Breeding
Advanced Testing Program - General
Important commercial barley cultivars, new cultivars, and promising advanced selections
were evaluated in ten trials at seven experimental locations in North Dakota during 2001.
In addition, "off-station" trials with new and check cultivars were conducted by the
Carrington, Dickinson, Hettinger, Langdon, North Central (Minot), and Williston
Research Extension Centers. Pure seed increase plots of experimental lines (26) in
Varietal and Advanced Yield Trials were grown at Fargo and Casselton. Pure seed
increase plots of experimental lines (44) in the Intermediate Malting Barley Yield Trial
were grown at Fargo. Barley yield trials at all locations were sown between 26 April and
17 May. Sowing at all locations was about seven to ten days later than average due to
wet soil conditions. Regional trials grown were the Mississippi Valley Barley Nursery
and the Canadian Western Cooperative Six-row Trial.
Plant emergence in yield trials was fairly uniform at all locations. As in recent years,
growing conditions during the two halves of the growing season varied drastically.
Precipitation from sowing up to mid-June was below average. These conditions limited
the yield potential of the crop, especially in northeastern North Dakota. From mid-June
to mid-July, precipitation was at levels that favored development of Fusarium head blight
(FHB). Several back-to-back rain storms in July with high winds caused extensive
lodging in all small grain crops grown in northeast North Dakota.
The major disease problem occurring on barley produced in northeastern and northcentral
North Dakota was FHB, incited primarily by Fusarium graminearum. Severity of
FHB was minimal at Fargo, and moderate to high at Carrington, Osnabrock, and Minot.
Levels of FHB at Minot were the greatest observed since the epidemic began in 1993.
Overall, the percent of FHB found in our yield trial nurseries in 2001 was slightly less
than that in 2000. The most disturbing aspect of this disease is that it continues to move
west in the state into areas where it was not a problem previously. All cultivars grown in

78
North Dakota are moderately susceptible to the pathogens inciting FHB. Foliar diseases
were most severe at Osnabrock. The predominant foliar disease at this site was septoria
speckled leaf blotch (SSLB), incited by Septoria passerinii. All six-rowed cultivars
currently grown in North Dakota are susceptible to SSLB. High levels of SSLB along
with successive rainstorms resulted in severe lodging in our Osnabrock yield trials. The
only trials harvested at this location were those that included lines with SSLB resistance.
The North Dakota Agricultural Experiment Station (NDAES) released Drummond, tested
as ND15477, in June 2000. Drummond yields greater than Robust and slightly less than
that of Lacey (Table 1). Drummond has a heading date similar to Robust, and has
stronger straw and lower FHB severity ratings than two other new varieties, Lacey
(University of Minnesota) and Legacy (Busch Ag. Resources Inc.) (Table 1). However,
the lower FHB severity in Drummond does not translate into lower DON accumulation.
In 2001, Drummond was grown for production of Registered Seed. In 2002, only seed of
the classes Foundation and Registered are available.
Miller Brewing completed two years of plant scale evaluation of Drummond and found it
satisfactory. Based on their testing, Drummond was added to the list of recommended
varieties by AMBA. Anheuser-Busch completed one year of plant scale evaluation of
Drummond and found it satisfactory. A second year of evaluation using seed produced in
2001 will begin soon.
Table 1. Agronomic comparisons of six-row varieties grown in North Dakota yield trials,
1996-2001.
Days to Plant
FHB
Yield heading height Lodging severity‡ DON§
Variety (bu/ac) (days after 5/31) (inches) (1-9)† (%) (ppm)
Station years 11 11 11 3 4 5
Robust 77 25 35 5.3 6.0 2.2
Stander 78 26 33 3.9 7.3 3.1
Foster 80 25 34 5.1 7.9 2.8
Lacey 79 25 32 3.8 5.6 1.9
Legacy 78 27 34 4.0 5.0 2.6
Drummond 78 25 34 2.4 4.3 2.9
†Lodging score of 1 equals no lodging, 9 equals severe lodging.
‡FHB = Fusarium head blight severity.
§DON = Deoxynivalenol.
Foster, released by the North Dakota Agricultural Experiment Station (NDAES) in 1995,
was added to the list of varieties recommended for malting by AMBA in July 1996.
Foster has yielded slightly greater than all currently released varieties; yet, has weaker
straw than all but Robust (Table 1). Foster appears best adapted to all malting barley
growing regions of North Dakota except the northern Red River Valley because of its
weaker straw. Foster has lower grain protein than currently grown varieties; thus, this
may allow growers in the central and western malting barley growing region of North
Dakota to produce barley with acceptable grain protein more consistently. Foster was
grown on about 10,000 acres (

79
this variety has decreased over the last four years because many growers are sensitive to
the “itchiness” caused by its long rachilla hairs.
Twenty-one experimental lines were grown at four North Dakota locations (Fargo,
Carrington, Minot, and Langdon) in their third or fourth year of yield trials. The USDA-
ARS Cereal Crops Research Unit, Madison, WI, evaluated selected lines for malt quality
and Dr. Stephen Neate evaluated lines for resistance to spot and net blotch in greenhouse
tests. Three lines currently are being evaluated in the AMBA Pilot Scale Evaluation
Program. The lines are ND16301 (Foster//ND12200/6B88-3213), ND16903
(Foster//ND12200/BT428), and ND16922 (ND14161/ND14296). Comparisons of these
lines to currently grown varieties are presented in Tables 2-7.
Table 2. Agronomic comparisons of ND16301, Robust, Stander, and Foster based on
North Dakota yield trials, 1996-2001.
Grain Days to Plant Stem FHB§
Variety yield heading height Lodging breakage severity DON
(bu/ac) (days after 5/31) (inches) (1-9)† (1-5)‡ (%) (ppm)
Station years 30 29 26 10 17 9 13
ND16301 77 25 32 3.3 2.9 6.0 3.4
Robust 72 26 34 4.9 4.3 6.8 2.8
Stander 76 27 32 3.4 3.0 7.7 3.9
Foster 76 26 33 4.8 4.0 7.8 3.5
†Lodging of 1 = no lodging and 9 = severe lodging.
‡Stem breakage of 1 = no breakage at harvest and 5 = severe breakage at harvest.
§ FHB = Fusarium head blight.
DON = Deoxynivalenol.
Table 3. Malt quality comparisons of ND16301 and other six-rowed barley cultivars grown in North
Dakota yield trials, 1996-2001†.
Barley Plump Malt Wort Diastatic Alpha-
protein kernels extract protein S/T‡ power Amylase
Entry (%) (%) (%) (%) (%) ( o L) (20 o DU)
Station years 26 26 26 26 26 26 26
ND16301 13.3 71 78.5 5.78 44.5 183 62.9
Morex 14.1 53 77.2 5.87 43.5 168 64.9
Robust 13.8 64 77.6 5.68 42.3 166 50.9
†Data courtesy of the USDA-ARS Cereal Crops Research Unit, Madison, WI.
‡Soluble protein to total protein ratio.

Table 4. Agronomic comparisons of ND16903, Robust, Stander, and Foster based on
North Dakota yield trials, 1998-2001.
Grain Days to Plant Stem FHB§
Variety yield heading height Lodging breakage severity DON
(bu/ac) (days after 5/31) (inches) (1-9)† (1-5)‡ (%) (ppm)
Station years 11 11 11 5 8 5 7
ND16903 79 27 33 4.0 3.0 5.5 2.8
Robust 75 26 36 4.9 4.1 8.6 3.1
Stander 78 26 34 4.4 3.2 9.7 3.3
Foster 82 25 34 4.6 3.7 8.7 3.5
†Lodging of 1 = no lodging and 9 = severe lodging.
‡Stem breakage of 1 = no breakage at harvest and 5 = severe breakage at harvest.
§ FHB = Fusarium head.
DON = Deoxynivalenol.
Table 5. Malt quality comparisons of ND16903 and other six-rowed barley cultivars
grown in North Dakota yield trials, 1998-2001†.
Barley Plump Malt Wort Diastatic Alphaprotein
kernels extract protein S/T‡ power Amylase
Entry (%) (%) (%) (%) (%) ( o L) (20 o DU)
Station years 8 8 8 8 8 8 8
ND16903 13.7 70 77.4 6.01 44.6 168 70.2
Morex 14.3 50 77.1 6.09 43.8 174 72.1
Robust 14.4 57 77.8 6.02 43.0 181 56.7
†Data courtesy of the USDA-ARS Cereal Crops Research Unit, Madison, WI.
‡Soluble protein to total protein ratio.
Table 6. Agronomic comparisons of ND16922, Robust, Stander, and Foster based on
North Dakota yield trials, 1998-2001.
Grain Days to Plant Stem FHB§
Variety yield heading height Lodging breakage severity DON
(bu/ac) (days after 5/31) (inches) (0-9)† (0-5)‡ (%) (ppm)
Station years 11 11 11 4 7 4 6
ND16922 82 25 34 4.3 3.2 6.4 3.5
Robust 75 26 36 4.9 4.1 8.6 3.1
Stander 78 26 34 4.4 3.2 9.7 3.3
Foster 82 25 34 4.6 3.7 8.7 3.5
†Lodging of 0 = no lodging and 9 = severe lodging.
‡Stem breakage of 0 = no breakage at harvest and 5 = severe breakage at harvest.
§ FHB = Fusarium head blight.
DON = Deoxynivalenol.
80

Table 7. Malt quality comparisons of ND16922 and other six-rowed barley cultivars
grown in North Dakota yield trials, 1998-2001†.
Barley Plump Malt Wort Diastatic Alpha-
protein kernels extract protein S/T ‡ power Amylase
Entry (%) (%) (%) (%) (%) ( o L) (20 o DU)
Station years 7 7 7 7 7 7 7
ND16922 14.0 73 78.1 6.04 44.7 169 79.0
Morex 14.3 49 77.1 6.04 43.5 167 72.0
Robust 14.3 59 77.8 5.96 42.9 175 55.8
†Data courtesy of the USDA-ARS Cereal Crops Research Unit, Madison, WI.
‡Soluble protein to total protein ratio.
81
Intermediate and Preliminary Yield Trials
Forty-five experimental lines were grown in the Intermediate Malting Barley Yield Trial
at four locations (Fargo, Carrington, Langdon, and Minot) for their second year of
evaluation. Selected lines were evaluated for malt quality by the USDA-ARS Cereal
Crops Research Unit at Madison, WI and for spot blotch and net blotch reactions in
greenhouse tests conducted by Dr. Neate. Four hundred ninety eight experimental lines
were grown in Preliminary Yield Trials at Osnabrock and Fargo.
Early Generation Selections
Single spikes from the F3 and F4 generations were sown at Fargo and Osnabrock in head
rows. About 17,100 head rows representing material from 124 crosses were sown.
Selection of head rows in the field was based on maturity, plant height, straw strength,
kernel plumpness, kernel color, FHB resistance, awn type, spike length, spike erectness,
and spike density. About 1,600 selections were made and submitted to Dr. Paul
Schwarz’s laboratory (Dept. of Cereal Science) for malt quality prediction tests.
Selections were evaluated for barley kernel assortment, barley grain protein, test weight,
barley kernel color, and barley diastatic power. Spikes from the selected rows were sown
in Yuma, AZ to increase seed for 2002 yield trials. Rows from the selections with the
best malt quality estimates will be harvested and advanced to the Preliminary Yield
Trials.
Sixty-seven F2 populations were grown at Fargo and Osnabrock. Selection among and
within populations was based on the same criteria used in the F3 and F4 progeny rows.
Nine F1 populations from crosses made in the 2001-spring greenhouse were grown in the
field at Osnabrock. All F1 populations had at least one parent with partial FHB
resistance. One hundred forty two crosses were made during fall of 2001 in the
greenhouse to combine favorable agronomic characteristics, disease resistance, and malt
quality traits. Over 85% of the crosses made used parents that showed resistance to at
least one of the following diseases: FHB, spot blotch, net blotch, stem rust, leaf rust,
speckled leaf blotch, and leaf scald.

82
Breeding for Fusarium Head Blight Resistance
A mist-irrigated FHB epidemic nursery was established near Osnabrock, ND in 1998.
Plants in the mist-irrigated nursery are inoculated by spreading F. graminearum infected
corn and barley grain throughout the nursery. Remnant seed from approximately 8,000
F3 and F4 progeny rows grown in an adjacent uninoculated nursery was used to sow hill
plots in the mist-irrigated nursery. Selection of progeny rows for harvest in the
uninoculated nursery was based, in part, on the level of FHB resistance of the
corresponding hill plot in the mist-irrigated nursery. Harvested grain from the selected
progeny rows were evaluated for DON accumulation. The most resistant plants in the
corresponding hill plots were individually harvested and were evaluated for FHB
resistance in the greenhouse during fall 2001 and winter 2001-2002. Based on
information obtained from the greenhouse screening, seed from the most resistant plants
will be harvested and used for sowing parents for crossing in the 2002-fall greenhouse
and will be advanced for additional field screening during summer 2002.
Western North Dakota Malting Barley Program
The 2001 North Dakota Legislature recommended that the North Dakota Agricultural
Experiment Station spend up to $288,000 on research and development of malting barley
for western North Dakota. Representatives Frank Wald of Dickinson, ND and Bob
Skarphol of Tioga, ND championed this program through the legislative process. These
gentlemen along with other state legislatures recognized the growing importance of
western North Dakota for malting barley production due to the Fusarium head blight
epidemic in eastern North Dakota the last nine years. The expansion of our effort in
western North Dakota will not result in a reduction of our current malting barley research
effort for eastern North Dakota.
A limitation in producing malting barley in western North Dakota is that all current
varieties and production practices were developed for conditions in eastern North Dakota.
When current varieties are grown under dryland conditions in the west, levels of kernel
plumpness and grain protein are typically unacceptable for malting. When these same
varieties are grown under irrigation, they typically lodge because their straw is too tall
and weak. Thus, new malting barley varieties specifically developed for irrigated and
dryland production in western North Dakota must be developed. Also, production
practices and recommendations for these new varieties must be developed concurrently.
Significant expansions for the new research program are planned for the Dickinson and
Williston Research Extension Centers. Research to be conducted includes:
• Development of six- and two-rowed malting barley varieties for dryland production.
New barley varieties for dryland production will have at least 70% plump kernels,
grain protein below 13%, and acceptable malting and brewing quality.
• Development of six- and two-rowed malting barley varieties for irrigated
production. New barley varieties for irrigated production will have high yield
potential (≥ 150 bu/ac), strong straw, multiple disease resistance, and acceptable
malting and brewing quality.

83
• Management studies for malt barley production under irrigated and dryland
conditions. Factors limiting success of consistent malt barley production in western
North Dakota are low levels of plump kernels and excessive levels of grain protein
under dryland conditions, and weak straw and diseases under irrigated conditions.
Research on the effects of nitrogen fertilization and stored spring soil moisture on
kernel plumpness, grain protein, lodging, and diseases will provide information that
growers can utilize to increase their likelihood of developing acceptable malting
barley.
Special Projects
1. Gioconda Garcia, a Ph.D. candidate from Ecuador, mapped the low-protein
characteristic from Karl barley, using the cross Azure/ND5377, to the
centromeric region of chromosome 6H. Mapping to the same region were a
QTL for kernel color and diastatic power. Gioconda could not determine if the
associations between grain protein, kernel color, and diastatic power were due to
linkage or pleiotropy.
2. Ken Lamb, a Ph.D. candidate from Minnesota joined the project in June 2000.
His research will be to identify molecular markers linked to genes conferring
FHB resistance in a doubled-haploid (DH) population derived from the cross
Foster/C93-3230-24. The line C93-3230-24 (B2912/Heitpas 5) is a six-rowed
line developed by BARI that does not derive its FHB resistance from Chevron.
The mapping population will be evaluated for FHB infection, deoxynivalenol
(DON) content, days to heading and maturity, plant height, spike nodding angle,
and molecular markers. Preliminary work has found chromosomal regions
associated with FHB resistance in chromosomes 2H, 5H, and 7H. These
associations were significant at all environments where the population was
grown. The region in chromosome 2H associated with FHB resistance has the
largest effect and is also associated with tall plant height and late maturity. The
region in chromosome 5H associated with FHB resistance is located in the
middle of the long arm, while the region in chromosome 7H is located on the
short arm. These regions also were found to be associated with FHB resistance
in a previous mapping study using Chevron as the resistant parent.
3. In a continuation of our work on the genetics of malt modification, a second
mapping population was developed from the cross Beacon/Hazen. Hazen has
poor modification and Beacon good malt modification. Development of an
RFLP and SSR map for this population is continuing. The population was
grown at two ND locations in 1998, 1999, 2000, and 2001. Entries from the
1998, 1999, and 2000 experiments were sent to Dr. Berne Jones’s laboratory for
malt analyses. Loci controlling malt modification will be mapped. We want to
determine if loci controlling malt modification in the SM population map to
similar loci in the Beacon/Hazen population.

84
Plant Sciences Personnel
R.D. Horsley, Associate Professor and six-rowed barley breeder
Marci Green, Research Specialist II
Jeremy Pederson, Research Specialist II and M.S. Candidate
Jason Faller, Ag. Research Technician III
Gioconda Garcia, Ph.D. Candidate
Ken Lamb, Ph.D. Candidate
Publications
Bolin, P., P. Schwarz, B. Jones, R. Horsley. 2001. Effect of malting parameters on the
development of soluble protein in 6-rowed barley. ASBC Newsl. 61:PB24.
Horsley, R.D., J.D. Franckowiak, P.B. Schwarz, and B.J. Steffenson. 2002. Registration
of Drummond barley. Crop Sci. (accepted).
Horsley, R.D., M.J. Wentz, and P.B. Schwarz. 2001. Relationships between common
malt modification measurements. ASBC Newsl. 61:PB24.
Rudd, J.C., R.D. Horsley, A.L. McKendry, and E. Elias. 2001. Host plant resistance
genes for Fusarium head blight: sources, mechanisms, and utility in conventional
breeding systems. Crop Sci. 41:620-627.
Schwarz, P., J. Barr, M. Joyce, J. Power, and R. Horsley. 2002. Analysis of malt grist by
manual sieve Test. J. Am. Soc. Brew. Chem. 60(1):10-13.
Urrea, C.A., R.D. Horsley, and B.J. Steffenson. 2002. Heritability of Fusarium head
blight resistance and deoxynivalenol accumulation from barley accession CIho 4196.
Crop Sci. (accepted).
Urrea, C.A., R.D. Horsley, B.J. Steffenson, and J.D. Franckowiak. 2002. Registration of
6NDRFG-1 six-rowed barley germplasm line with partial Fusarium head blight
resistance. Crop Sci. (accepted).

85
STUDIES ON BARLEY DISEASES AND THEIR CONTROL
Stephen Neate
Department of Plant Pathology
North Dakota State University
Fargo, ND 58105
OBJECTIVES
Diseases are important in limiting the yield and quality of malting barley production in
the upper mid-west of the US. The objectives of the Barley Pathology Project at North
Dakota State University is to investigate barley diseases and develop timely, practical
methods for disease control to ensure that the quantity and quality of barley are not
limited by disease. We aim to achieve this goal through the development of cultivars with
genetic resistance, as well as development of cultural and chemical management
strategies. To most efficiently achieve this goal it is also essential to investigate the
population biology and basic ecology of pathogens that cause disease on barley. We
have an ongoing program of monitoring and research to accomplish this goal. We also
work closely with breeders to develop barley cultivars with multiple disease resistance.
1. Evaluate elite barley germplasm and segregating breeding populations for
resistance to Fusarium Head blight (FHB). Working together with the
breeders, Dr. J. Franckowiak, and Dr R. Horsley, NDSU, the barley pathology
project has collaborated in identifying FHB resistance in approximately 2500
irrigated plots of barley accessions including Minndak, Foster/CIho4196,
Foster/Bari, Elite, and transgenic populations. These accessions, as well as 9000
segregating breeding lines in plots of NDSU barley breeders were evaluated for
FHB resistance. The resistant lines were subsequently evaluated for their FHB
resistance in the greenhouse and the DON level of the inoculated kernels were
determined. A summary of the results can be viewed on the US Wheat and
Barley Scab Inititative web site.
2. Screen barley cultivars for resistance to spot blotch. A total of 600 barley
entries was screened for spot blotch resistance to isolate SB85 of Cochliobolus
sativus at the Fargo field site last year. Both the infection types and the
percentage (0-100%) of leaf area affected by disease were evaluated. Among the
tested 150 breeder’s lines, most of 6-rowed and 50% of 2-rowed lines were found
to be resistant (with severity level

86
3. Screen breeders lines for resistance to net blotch. 400 breeder’s lines were
screened for net blotch (Pyrenophora teres f. teres) resistance in the greenhouse
last year. Ninety eight out of 219 tested 2-rowed lines were found to have good to
high levels of resistance (with infection types 4. 3, 2, or lower). Among the 6rowed
lines, half of them only exhibited a moderate level of resistance to net
blotch, only lines ND19711, ND 19742, and ND19289 exhibited low infection
phenotypes to isolate 89-19 of P. teres f. teres. These results confirmed previous
screening which clearly showed that in lines originating from the the breeding
programs at NDSU, 2 row barley has greater resistance than 6 row.
4. Screen barley for resistance to crown rust. A further study was conducted for
the genetics of resistance to barley crown rust (Puccinia coronata var. hordei) in
wheat.. F2 populations, derived from Chris monosomic wheat lines crossed to a
spring type susceptible selection of PI350005, were inoculated with crown rust
isolate 91-36sp1. The disease reactions of the F2 progenies fit closely to the
expected single gene ratio and the phenotype data was provided for the on-going
molecular mapping of the resistance gene in wheat.
Several barley lines were evaluated for their resistance to crown rust in the field.
Logan, PC249a, and Hor2596 were found resistant to crown rust with reaction
types of MR or R. Aim and Azure are susceptible to crown rust in the field.
5. Molecular mapping of the barley stem rust resistance gene rpg4. In a
collaborative effort on physical mapping of the barley stem rust resistance gene
rpg4 , barley pathology project of NDSU has screened 100 OS6 x Harrington F2
families and SQ501 derivatives to pathotype QCC-2 of Puccinia graminis f. sp.
tritici at both low and high temperatures. In addition, 102 recombinant barley
families for rpg4 resistance were also tested against pathotype QCC-2. Analysis
is ongoing.
6. Verifying the existence of new spot blotch races. With Dr J Frankowiak we
have screened 46 diverse genetic barely lines in the glasshouse against 3 putative
new races of Cochliobolus sativus that caused much higher levels of disease in the
field in 2001. Preliminary results indicate that these are new races that are able to
cause increased disease.
OTHER FUNDS AND FUTURE DIRECTIONS
As one of the principal investigators on the SBARE Malting Barley for Western North
Dakota project I have developed collaborations with Dr Marcia McMullen and Mr Roger
Ashley to substantially increase the numbers of barley fields sampled for leaf, stem head
and root diseases in Western North Dakota. Currently about 190 barley fields are
sampled annually with patchy distributions in some parts of the state. We aim to increase
the sampling to 500 fields. This sampling will not only provide spatial data that will be
available to farmers and researchers about actual problems in the field but will provide
the project with a bank of isolates that can be used to monitor pathotype distributions.
The money allocated from SBARE does not fully cover the anticipated project
expenditure.

87
With funding from the US Wheat and Barley Scab Initiative, obtained by Dr L Dahleen
USDA-ARS Fargo, we are screening in the glasshouse 60 lines of transgenic Conlon
barley with anti toxin gene TRI101 and yeast metabolite transporter gene PDR5 for
resistance to Fusarium head blight. Preliminary results indicate that lines containing
either gene can be effective at reducing disease. Promising lines will be tested in the
field in 2002.
Another future direction will be to look at manipulating inoculum levels in the stubble of
previous crops to reduce the disease pressure on barley. To date most disease control
methods are aimed at reducing disease levels once the infection has begun. Manipulation
of stubble to reduce inoculum will take place by a combination of mechanical, chemical
and microbiological methods. Collaborative research projects are under negotiation with
Dr D Schisler USDA-ARS Peoria Il to test biological control strains of Cryptococcus
nodaensis yeast and with Dr J. Kloepper Auburn University, AL to test a three
component Bacillus mix for reduction of inoculum potential and disease due to Fusarium
graminearum.
With Dr P Bregitzer USDA-ARS Aberdeen ID and Peggy Lemaux UC Berkley, we have
negotiated a collaborative project in which we test transgenic Drummond barley with
antifungal genes TLP1 and TLP4 and trichothecene pathway genes TRI101 and TRI102
against a range of barley diseases. Germplasm is not expected to become available for
this project for 12 months at which time funding will be sought to support this component
of the project.
With Dr B Steffenson UM and Dr D Garvin USDA-ARS Minneapolis I have negotiated a
collaborative project to screen 120 H. vulgare ssp spontaneum introgression lines for
resistance to disease caused by a range barley pathogens.
Personnel:
Stephen Neate, Associate Professor / Project Leader (since October 8 th 2001)
Glen Statler, Prof / Dept Chair / Project Leader (until October 8 th 2001)
Yongliang Sun, Research Specialist I
Cooperators:
Mr Roger Ashley, Dickinson Research Extension Center, ND
Dr. Lynn Dahleen, USDA-ARS, Fargo, ND
Dr. Jerome Franckowiak, NDSU, Fargo, ND
Dr. Richard Horsley, NDSU, Fargo, ND
Dr. Shahryar Kianian, NDSU, Fargo, ND
Dr. Andy Kleinhofs, WSU, Pullman, WA
Dr Joseph Kloepper, Auburn University, AL
Dr Marcia McMullen, NDSU, Fargo, ND
Dr David Schisler, USDA-ARS Peoria Il

88
Publications: (generated by previous project members while supported by AMBA)
Edwards, MC; Fetch, TG; Schwarz, PB; Steffenson, BJ 2001 Effect of Barley yellow
dwarf virus infection on yield and malting quality of barley. PLANT-DISEASE. 85: 202-
207.
Schwarz, PB; Schwarz. JG; Zhou, A; Prom, LK; Steffenson, BJ 2001 Effect of Fusarium
graminearum and F. poae infection on barley and malt quality. MONATSSCHRIFT-
FUR-BRAUWISSENSCHAFT. 54: 55-63.
Zhong, SB; Steffenson, BJ 2001 Genetic and molecular characterization of mating type
genes in Cochliobolus sativus. MYCOLOGIA. 93: 852-863.
Zhong, SB; Steffenson, BJ 2001 Virulence and molecular diversity in Cochliobolus
sativus. PHYTOPATHOLOGY 91: 469-476.

89
MALTING AND BREWING QUALITY OF BARLEY
Paul Schwarz
Department of Cereal Science
North Dakota State University
Fargo, ND 58105
OBJECTIVES AND JUSTIFICATION
A primary objective of the barley quality program at North Dakota State University is to
provide individuals in the barley improvement program with barley and malt quality data
that is required for the development and release of acceptable malting varieties.
Prediction tests are performed on early generation lines to aid in selection for improved
malting quality. Micro-malting and malt quality analyses are performed on materials
from barley genetics studies and other special cooperative projects. Basic research
efforts are directed toward the biochemical components of barley and malt, in order to aid
in the understanding and definition of malt quality. A survey of the regional barley crop
is conducted on an annual basis. This information is provided to the sponsors in the form
of weekly and annual reports.
BARLEY VARIETY DEVELOPMENT
1. Early Generation Quality Testing Program. Approximately 3,125 early generation
two- and six-rowed lines and cultivars, grown in 2001, were submitted for preliminary
screening by near infrared reflectance (NIR) for protein. The potential diastatic power of
1,135 of the breeder’s lines were analyzed. An additional 1,300-1,750 six-rowed samples
were screened for kernel assortment, test weight and/or color. Lines and varieties (120)
from the 2001 NDSU variety plot trials were micro-malted and are awaiting analysis for
standard quality parameters.
2. Deoxynivalenol Testing Program. The overall goal of the DON testing program in
Cereal Science is to expedite the development of scab resistant malting barley cultivars
for the upper Midwest. DON analysis services are provided to barley breeders and
pathologists at NDSU, University of Minnesota, and Busch Agricultural Resources, Inc.
The service is provided to all cooperators at a minimal charge, which includes only the
cost of expendable supplies, chemicals, and sample grinding labor.
Approximately 7,500 barley samples from the 2001 crop were analyzed for DON by gas
chromatography with electron capture detection (GC-ECD) (Table I). Samples included
breeder’s lines, crop survey samples, and samples from research studies. This is 2,500
more samples than analyzed in 2000, and does not include the analysis of 1,350
standards. The 2001 crop samples were analyzed beginning September 1, 2001 and are
to complete in March, 2002.
A barley DON check sample service is operated as a service to 13 academic and industry
labs. Two samples are shipped to each participant on a monthly basis. Results of this
check service have indicated that precision of DON analysis in our laboratory improved
during 2001. While our results average slightly below the grand mean, the standard
deviation was the lowest of any of the 13 participating laboratories.

90
Table I
2001 Crop Samples Analyzed for DON in the Department of Cereal Science
Sample Type Sample Number
Breeder Samples Completed
(BARI, Dr Dahleen, Dr Griffey, Dr. Horsley, Dr Smith)
6,189
Breeder Samples to be analyzed (March 2002) 900
2002 Regional Crop Survey 297
Collaborative Check Services 48
Cereal Science Research (Dr Schwarz) 188
AMBA Member Companies 15
Other 49
Total 7,686
3. AMBA Pilot-Malting Studies. The laboratory participated in pilot-malting studies
coordinated by AMBA. This included the prescreening of 22 six-rowed barley samples,
the 2001 Harrington-Robust collaborative, and the 2001 AMBA crop plot evaluations.
4. Cooperative Research with NDSU Plant Science. Micro-malting and malt quality
evaluations were conducted as part of collaborative studies with Dr Horsley, NDSU
Plant Sciences. Approximately 680 samples were malted an analyzed as part of a studies
on glyphosate, and genetics of low-protein.
RESEARCH PROJECTS
1. Arabinoxylans of Barley, Malt and Wort. The importance of β-glucans in malting
and brewing has long been recognized, and associated problems include the reduced
recovery of malt extract, diminished rates of lautering, shortened filter life, and the
formation of gels and haze. However, arabinoxylans (pentosans), which are another
major component of barley cell walls, have received considerably less attention. Cereal
arabinoxylans are partially water-soluble, high-molecular weight polysaccharides, with
the potential to form viscous solutions and gels, and as such, may be, at least partially,
associated with problems previously attributed to β-glucans. Research results have
indicated that both high molecular weight β-glucans and arabinoxylans are more
troublesome in terms of membrane filtration.
The study of arabinoxylans has been hindered by the fact that there is no standardized
procedure for their determination in barley, malt , wort or beer. The value of total
arabinoxylan data on whole ground barley or malt is questionable. This follows as the
husk of the barley grain is composed largely of insoluble arabinoxylan and cellulose.
Determination of the soluble and insoluble arabinoxylan present in the aleurone and
endosperm cell walls should be of greater interest to the maltster and brewer, but may be
masked by the greater levels in the husk. As is generally accepted to be the case for the

91
β-glucans, it might be most instructive to determine the level of arabinoxylan in wort or
beer.
The overall objectives of this research area project are to (1) develop methodology for the
determination of arabinoxylans in wort in beer., and (2) to characterize the MW profile
of arabinoxylans present in wort and beer and to evaluate the influence of processing
parameters on these profiles. Both are Ph.D. thesis projects, and limited progress is
reported as the candidates have just begun research.
Method development for determination of arabinoxylans has focused on refinement of
the GC method originally described by Schwarz and Han (J. Am. Soc. Brew. Chem.
53:157, 1995). This method involves acid hydrolysis of freeze-dried beer solids and the
determination of xylose and arabinose as alditol acetates. A limited amount of
ruggedness testing has been conducted to date.
In terms of MW profile, work to this point has focused on development of isolation
procedures that will allow analysis of the entire amount of each polysaccharide without
interference from other polysaccharides or other materials. Experiments have been
conducted to evaluate selective enzymatic digestion of dextrins (with amyloglucosidase),
β-glucans (with lichenase), and arabinoxylan (with endo-xylanase). The very low
molecular weight carbohydrates (mono- to oligosaccharides) are removed by
ultrafiltration, and the remaining high molecular weight fraction will be analyzed by high
performance size exclusion chromatography (HPSEC) with light-scattering/viscometric
detection. Results to date have shown that the enzymes do not appear to exhibit side
activities, and thus are suitable for selective digestion. However, the ultrafiltration
method has not yielded acceptable reproducibility.
2. Fusarium Head Blight (FHB) and Malt Quality. Fusarium-related research
focuses on the control of Fusarium growth during malting, and assessment the
relationship between Fusarium infection levels and damage to the quality of the grain.
The use of a Geotricum starter culture in steeping has been reported to reduce Fusarium
growth and DON (Boivin, P. and Malanda, M. 1997 MBAA Tech. Quart. 34:96). We are
currently evaluating the use of a commercial Geotricum culture on samples from the
2001 crop in cooperation with Degussa BioActives (Waukesha, WI) and Institut Français
Des Boissons De La Brasserie Malterie (IFBM, Nancy, France). Several pilot-maltings
have been completed, but data for general release is not yet available. Cooperative
research on the use of physical treatments for control of Fusarium growth during malting
is being conducted with Dr Charlene Wolf-Hall (PI, Cereal and Food Science, NDSU). A
recently completed M.S. thesis project evaluated the effect of hot water sterilization,
UV/electron beam-, and gamma-irradiation on barley germination and FHB levels. This
preliminary study indicated that hot water sterilization (45 o C) may be effective in
reducing levels of viable Fusarium, without significant damage to germination.
Additional work in this area has been funded through a USDA-NRI grant.
A cooperative study with Dr Brian Steffenson (Plant Pathology, UM) and Dr Richard
Horsley (Plant Sciences, NDSU) on the relationship between Fusarium infection levels
and damage to the barley/malt quality has been ongoing for several years. Samples

92
(Robust), with varying levels of DON were collected in eastern North Dakota as part of
the 1995-2000 regional crop surveys The samples were micro-malted (150, N=2) and
standard quality parameters determined. Plate count (% infected kernels), DON,
xylanase, protease and ergosterol were determined as markers of FHB infection. All
malting and analytical work for this project was completed in 2001. Statistical analysis
of the relationship(s) between the various FHB markers and grain quality is ongoing.
Preliminary results indicate, that while significant correlations exist between several
quality parameters and FHB markers, they will likely be of limited predictive value.
Ongoing work focuses on the characterization of endo-xylanase(s) produced by Fusarium
graminearum.
REGIONAL BARLEY CROP QUALITY
The 2001 survey of the regional barley crop was prepared in cooperation with the
American Malting Barley Association. Periodic summaries of the crop quality were
made available to AMBA members throughout the harvest of 2001. A total of 275 sixrowed
and 21 two-rowed samples were collected in North Dakota and the primary barley
producing regions of Minnesota. Protein and kernel plumpness of the 2001 regional sixrowed
malting barley crop was slightly improved over that seen in 2000. However, color
scores and test weight were comparable to slightly poorer than those observed in 2000
(Table II).
Two hundred and ninety-seven samples collected throughout North and South Dakota
and Minnesota were tested for DON. The average level of DON observed in six-rowed
samples from the 2001 crop (2.6 ppm) was slightly higher than of 2000, but was still
lower than the eight year average (Figure 1). Approximately 39% of the regional sixrowed
crop tested exhibited less than 0.5 ppm DON, which compares to 25% below 0.5
ppm in 2000 (Figure 2).
Table II.
Traditional Six-Rowed Malting Barley Regional Averages for Various Quality
Parameters, 1990-2001.
Year Samples Moisture Color Score % Plump Test Wt (lbs/bu) Protein %
2001 206 12.7 8 75 45.7 13.2
2000 307 12.8 7 69 46.1 13.7
1999 433 13.1 7 69 44.9 12.8
1998 465 12.7 5 68 46.4 12.9
1997 485 13.4 7 74 44.7 12.8
1996 512 13.6 7 83 47.4 12.5
1995 496 13.4 7 76 45.8 12.7
1994 520 13.0 7 76 46.2 12.6
1993 540 12.5 8 68 43.8 12.8
1992 552 13.3 7 82 47.6 12.3
1991 507 12.7 7 68 44.9 13.1

10
9
8
7
6
5
4
3
2
1
0
93
Figure 1. Average DON levels (ppm) in the samples tested for the 1993-2001 regional
barley crop quality surveys.
>1.0
48.7%
1993 1994 1995 1996 1997 1998 1999 2000 2001
2001
0.6-1.0
12.0%
>1.0
30.9%
1.0
51.7%

Dr Paul Schwarz
CEREAL SCIENCE PERSONNEL
Associate Professor
Mr. John Barr Chemist II
Mr. James Gillespie Chemist II
Ms Patricia Stanley Chemist I (departed March , 2002)
Ms Kristin Pavlish Chemist I (departed August 2001)
Dr Adela Cruz Tuhkanen Research Associate (departed August 2001)
Ms Jun Ying Yan Laboratory Assistant
Mr Mark Haztenbeller Graduate Research Assistant
Mr. Genaro Ordon Graduate Research Assistant
Mr. Paul Sadosky Graduate Research Assistant
94
Dr Lynn Dahleen
COOPERATORS
USDA-ARS NCSL
Dr Jerry Franckowiak Dept. of Plant Sciences, NDSU.
Dr Richard Horsley Dept. of Plant Sciences, NDSU.
Dr Berne Jones USDA-ARS CCRU
Dr Brian Steffenson Dept. of Plant Pathology, University of Minnesota
Dr Charlene Wolf-Hall Dept. of Cereal and Food Science, NDSU
PUBLICATIONS
Schwarz, P., Stanley, P. and Solberg, S. 2002. Activity of lipase during mashing. J. Am.
Soc. Brew. Chem. (60), in-press
Schwarz, P.B., Jones, B. L., and Steffenson, B.J. 2002. Enzymes associated with
Fusarium infection of barley. J. Am. Soc. Brew. Chem. (60), in-press.
Schwarz, P., Barr, J., Joyce, M., Power, J., and Horsley, R. 2002. Analysis of malt grist
by manual sieve Test. J. Am. Soc. Brew. Chem. 60(1): 10-13.
Schwarz, P.B., Schwarz, J. G., Zhou, A., Prom, L.K., and Steffenson, B. J. 2001. Effect
of Fusarium graminearum and F. poae infection on barley and malt quality.
Monatsschrift für Brauwissenschaft 54(3/4):55-63.
Edwards, M.D., Fetch, Jr., T.G., Schwarz, P.B., and Steffenson, B.J. 2001. Effect of
Barley yellow dwarf virus Infection on Yield and Malting Quality of Barley. Plant
Disease 85(2):202-207
Sadosky, P. and Schwarz, P. 2001 ABSTRACT: Effect of arabinoxylans, beta-glucans,
and dextrins on the viscosity and membrane filterability of beer. MBAA Communicator
52(2):14.
Schwarz, P.B. 2001. ABSTRACT: Relationship between Fusarium Head Blight infection
and the malting quality of barley. MBAA Communicator 52(2):11-12.

95
Bolin, P., Schwarz, P., Jones, B., and Horsley, R. 2001. ABSTRACT: Effect of malting
parameters on the development of soluble protein in six-rowed malting barley. Brewers
Digest. 76 (3):40.
Horsley, R., Wentz, M., and Schwarz, P. 2001. ABSTRACT: Relationships between
common malt modification measurements. Brewers Digest 76(3):40.
Smith, K., Schwarz, P., and Barr, J. 2001. ABSTRACT: Impact of Fusarium Head Blight
disease management on malting barley. Brewers Digest 76(3):41.
Stanley, P., Schwarz, P., and Solberg, S. 2001. ABSTRACT: Activity of lipase during
malting and mashing. Brewers Digest 76(3):41.

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ATTEMPTS TO TRANSFORM NEWER CULTIVARS WITH GENES AFFECTING
TRICHOTHECENE TOXIN AND FHB LEVELS
Lynn S. Dahleen and M. Manoharan
Cereal Crops Research Unit, USDA-ARS and
Dept. of Plant Sciences, NDSU
Objective
To produce transgenic commercial barley cultivars that express anti-toxin genes which
reduce DON levels and Fusarium infection.
Introduction
Fusarium head blight (FHB), caused primarily by Fusarium graminearum, has been one of
the most destructive diseases of barley since the early 1990s, resulting in huge economic
losses for growers (McMullen et al.1997). Of particular concern is the production of
deoxynivalenol (DON), a potential pathogen virulence factor (Desjardins et al. 1996,
Proctor et al. 1995, 1997) that is harmful to humans and livestock (Busby and Wogan 1981).
Chemically modifying DON or transporting it out of cells could improve resistance to FHB
while at the same time reducing DON accumulation in grain. FsTri101 (TriR), isolated from
F. sporotrichioides, encodes a 3-OH trichothecene acetyltransferase that converts DON to a
less toxic acetylated form. PDR5, an ATP-binding cassette transporter isolated from
Saccharomyces cerevisiae, acts as an efflux transporter, shunting DON across the plasma
membrane from the interior of the cell. We have transformed the commercial malting barley
cultivar Conlon with the Tri101 and PDR5 genes with the aim of reducing DON level in
infected grain.
Methodology
Development of the transgenic lines was described in last year’s report. Experiments
have resulted in seven transgenic events for Tri101 and six events for PDR5. A
transformation event consists of all the plants regenerated from one callus clump that was
bombarded with the genes. Four of the events for each gene resulted in tetraploid plants.
Lines from the three diploid Tri101 events and the two diploid PDR5 events were
advanced two generations by self-pollination and seed used for field trials in 2001. Ten
lines each of the five diploid transformation events were planted in short rows in two
replicates. One replicate was planted in the misted, inoculated nursery at Langdon, ND
and one replicate was planted just outside the inoculated nursery, so each would have
different levels of FHB. Non-transgenic Conlon was planted every fifth row. Traits
measured included height, % FHB, seed weight per 100 spikes, and DON concentration.
Greenhouse tests are underway. Plants are spray inoculated, held in a mist chamber for
24 hours, and then scored for % FHB 14 days later. The two lines from each
transformation event that had the lowest FHB in the field have been tested and additional
lines are being grown for further testing. After harvest, seed will be sent to the NDSU
Barley Malt Quality Lab for DON measurements.

97
Results
Results from field and greenhouse tests of the diploid transgenics are presented in Table
1. Acetylase activity tests indicated that Tri101 was expressed in all 30 lines. FHB
infections in lines with Tri101 were similar to or somewhat less than the Conlon nontransgenic
controls in both the field and greenhouse trials. However, DON levels in the
Tri101 lines from the field were equal to or higher than Conlon, sometimes substantially
higher. DON tests on the greenhouse-grown plants will be conducted when seed is
mature.
Lines with PDR5 showed more promising results. Northern analysis showed that PDR5
was transcribed in the transgenic lines. In the field and greenhouse, all PDR5 lines had
FHB levels equal or less than the Conlon control rows. Unlike lines with Tri101, only
five PDR5 lines showed increased DON. Eleven of the lines had a DON reduction of at
least 1 ppm, with DON in three of the lines reduced more than 5 ppm. DON tests on the
greenhouse-grown plants will be conducted when seed is mature.
Most transgenic lines were shorter than Conlon, with the shortest lines almost 30%
shorter than the nontransgenic control. Transgenic lines also had reduced seed weights
per 100 spikes, with the poorest lines showing a 60% reduction in seed weight. The
reduction in seed weight appears to be related to DON levels. PDR5 lines with the lowest
DON generally had the smallest reduction in seed weight.
Greenhouse screening of the transgenic lines will continue until spring planting. Lines
will be planted in replicated field plots this spring for further FHB evaluation and
additional controls will be included. Plants will be scored for height, heading date, %
FHB, and yield. After harvest, cleaned seed will be used for DON analysis.
Additional transgenic line development is underway. Various combinations of the genes
Tri101, PDR5, a rice chitinase and a rice thaumatin-like protein are being inserted into
Conlon and Drummond. Conlon lines carrying Tri101 and PDR5 are being crossed to put
both genes into the same plant. We also plan to cross the Conlon transgenics with other
elite 2- and 6-rowed cultivars to transfer the anti-toxin genes into other backgrounds. As
these various lines are developed, they will be tested in the lab for the presence and
expression of the transgenes and in the field for the effects of the genes on FHB and
DON levels.
Personnel
Mrs. Luming Brewer – part-time laboratory technician
Mrs. Mary Wentz - part-time laboratory technician
Recent Publications
Dahleen, L.S., Okubara, P.A. and Blechl, A.E. Transgenic approaches to combat
Fusarium head blight in wheat and barley. Crop Sci. 40:628-637. 2001.
Dahleen, L.S. and McCormick, S.E. Trichothecene toxin effects on barley callus and
seedling growth. Cer. Res. Comm. 29:115-120. 2001

98
Manoharan, M., L. Dahleen and T. Hohn. 2001. Transformation of a commercial barley
cultivar with genes for resistance to Fusarium head blight. American Society of
Agronomy Abstracts.
Manoharan, M., L. Dahleen and T. Hohn. 2001. Transformation of a commercial barley
cultivar with genes for resistance to Fusarium head blight. National Fusarium Head
Blight Proceedings. p. 21.
Table 1. Field and greenhouse tests of transgenic barley containing Tri101 (1-1, 1-2, 1-3) or
PDR5 (2-1, 2-2).
Average FHB Average Average Average Seed
Event Line Location Infection Height DON Weight
Conlon Average Field 13% 93 cm 14 ppm 83 grams
Conlon Average Greenhouse-1 19 - - -
Greenhouse-2 22 - - -
1-1 Average Field 14 86 16 59
1-1R1 Field 11 90 11 59
1-1R1 Greenhouse-2 5 - - -
1-1R2 Field 13 93 13 62
1-1R2 Greenhouse-2 9 - - -
1-2 Average Field 10 88 30 58
1-2R1 Field 13 86 22 59
1-2R1 Greenhouse-2 9 - - -
1-2R2 Field 11 91 17 69
1-2R2 Greenhouse-2 9 - - -
1-3 Average Field 9 88 34 49
1-3R1 Field 5 83 27 45
1-3R1 Greenhouse-2 9 - - -
1-3R2 Field 11 90 19 61
1-3R2 Greenhouse-2 7 - - -
2-1 Average Field 7 88 14 63
2-1R1 Field 7 82 10 71
2-1R1 Greenhouse-1 2 - - -
2-1R2 Field 9 89 14 61
2-1R2 Greenhouse-1 5 - - -
2-2 Average Field 7 90 13 63
2-2R1 Field 5 88 9 72
2-2R1 Greenhouse-1 5 - - -
2-2R2 Field 5 88 7 77
2-2R2 Greenhouse-1 8 - - -
Field averages are from both reps of 10 lines/event; Greenhouse data from 10 pots with two plants/pot. FHB was rated
on 10 spikes/row in the field and 3 spikes/pot in the greenhouse. Seed weight was measured on 100 spikes/row.

99
DEVELOPING BARLEY TISSUE CULTURE SYSTEMS TO IMPROVE PLANT
REGENERATION AND REDUCE SOMACLONAL VARIATION
Lynn S. Dahleen and Phil Bregitzer
USDA-ARS, Fargo, ND and Aberdeen, ID
A concerted effort to optimize tissue culture conditions for Harrington and Morex
has resulted in significant improvements in plant regeneration from tissues of these
important cultivars (see previous AMBA reports; Dahleen and Bregitzer, 2002). In
addition, these media improvements are applicable to many other tested cultivars of
diverse pedigree, with plant regeneration increases averaging 15-fold that obtained on the
original media. Vigorous regeneration is critical to successful transformation of barley
cultivars, and the improved techniques have been used to recover transgenic plants from
several difficult-to-transform cultivars, including Morex (Bregitzer et al., 2000).
Previous systems were very genotype-dependent (Wan and Lemaux, 1994), and the use
of unimproved culture systems has confined many transformation efforts to amenable
cultivars such as Golden Promise. Major efforts to introduce antifungal protein genes into
elite malting cultivars are underway (Bregitzer, Dahleen, Lemaux, research in progress).
It is expected that direct transformation of six-rowed cultivars will assist in screening for
FHB resistance.
In addition to facilitating the transformation of elite cultivars, improvements in
plant regeneration may be associated with reductions in somaclonal variation (SCV). In a
study of three different types of barley tissues, it was found that plants regenerated from
highly differentiated, meristematic tissues (which have superior regeneration
characteristics) were agronomically superior to plants regenerated from relatively
undifferentiated, embryogenic cultures (Bregitzer et al., 2002). Preliminary information
regarding this study can also be found in our 1999 report to AMBA. These studies did
not, however, address the relative importance of increased differentiation, versus the
changes in media formulations that are critical to creating and maintaining the
meristematic tissues. Interestingly, although 6-benzylamino purine (BAP) is of critical
importance to the successful production and maintenance of meristematic tissues, it can
be used also in the less differentiated, embryogenic tissues that have been used to develop
our improved medium. Thus, it was of interest to determine if the inclusion of BAP at
low levels could have a role in reducing SCV,
Although we are pleased with our progress towards a genotype-independent, nonmutagenic
tissue culture and transformation system for barley, we wished to further
investigate potentially useful media modifications and their role in reducing SCV. Two
objectives were pursued in the last year:
1) Investigate the influence of silicon on plant regeneration. Briefly, the justification
for these experiments is that silicon is not considered an essential nutrient and is
routinely omitted from common media formulations despite evidence that silicon
has a major role in plant growth and development (Epstein, 1999).
2) Determine if plants regenerated from our improved tissue culture media were
agronomically superior to those regenerated from the original media.

100
Methodology
1) Investigating the influence of silicon on plant regeneration
Identical experiments were conducted at Fargo, ND, and Aberdeen, ID. Following the
procedures described in Bregitzer et al. (2000), the plant regeneration response of callus
derived from immature embryos of three cultivars--Morex, Harrington, and Golden
Promise-- were measured in response to various levels of silicon supplementation (as
SiO2; control (0), 10, 100, 500, 1000, and 5000 :M Si for initial experiments). Data were
analyzed using the SAS procedure CATMOD.
2) Measuring somaclonal variation in plants derived from the original and improved
media
The experiments conducted last year (The effects of media improvements on newer
cultivars and advanced breeding lines, AMBA Prog. Rep. 2000) compared the
regeneration responses on our original and improved media. Four regenerated plantstwo
from the original media, two from the improved media--were grown to maturity from
eight cultivars or breeding lines: Colter, Crystal, Conlon, Baronesse, Morex, 90Ab321,
Drummond, and Golden Promise. Seed from these plants, and their respective single
plant-derived parental controls, was increased in New Zealand during their 2000-2001
growing season. The agronomic performance of these lines were evaluated in replicated
plots at two locations, Aberdeen (irrigated) and Tetonia, (dryland) ID. Standard smallplot
yield trials were conducted: 4 replicates at each location; 40 sq. ft. per plot; traits to
be measured include heading date, plant height, lodging percentage, grain yield, test
weight, and percentage plump kernels.
Results
Influence of silicon on plant regeneration
One of two planned experiments has been completed at each location, and the second
experiment is nearing completion. The Aberdeen data do not include Harrington because
of plant health problems in the growth chamber. Significant differences in plant
regeneration were found among cultivars, locations and silicon concentrations, but the
interaction between silicon concentration and cultivars was not significant.
The addition of silicon to the media increased green plant regeneration for all cultivars at
both locations. Overall, regeneration rates were highest when 500 :M Si was added to the
media, which induced, on average, more than double the number of regenerated plants
induced on medium lacking silicon. The amount of silicon in the media appeared to have
no effect on the number of albino plants regenerated.
A second set of experiments is underway at both locations, dropping the 10 and 5000 :M
Si concentrations. These experiments will confirm whether the 500 :M Si concentration
results in increased green plant regeneration.
Agronomic performance of regenerated plants
Two important differences between the "original" and "improved" media formulations
are the increased copper concentration and the addition of BAP. Previous agronomic

101
trials of plants regenerated from media containing high or low concentrations of copper
did not indicate any relationship between copper levels and agronomic performance
(Bregitzer, unpublished). Given the critical role of BAP for the induction and
maintenance of the highly-differentiated, meristematic tissues that have been found to
reduce SCV, we were interested in whether SCV could be reduced by the inclusion of
BAP in the relatively undifferentiated, embryogenic culture system that was the subject
of these investigations, and which is by far the most common type of tissue culture
system used for in vitro manipulations of barley.
Table 2 shows the agronomic performance of plant derived from non-tissue-cultured
controls, plants regenerated from the original media, and plants derived from the
improved media. The results were consistent with previous data in that there was a
tendency for reduced performance from tissue culture-derived plants. However, no
consistent differences were noted between plants derived from the two media systems,
and the average performances of all lines revealed no relationship of agronomic
performance and the media system used. Given the average 15-fold increase in
regenerability provided by the improved media system, these data also did not show any
relationship between regenerability and agronomic performance. Thus, although the
improved media system should facilitate transformation of elite cultivars, meaningful
reductions in SCV will require also the establishment and maintenance of the more
differentiated, meristematic tissues described by Cho et al. (1998) and Zhang et al. (1999)
Personnel
Mrs. Luming Brewer – laboratory technician – tissue culture
Mrs. Mary Wentz - laboratory technician – tissue culture
Mr. Vince Edwards--Biological Science Technician
References
Bregitzer, P., S. Zhang, M-J. Cho, and P. G. Lemaux. 2002. Reduced somaclonal
variation in barley is associated with culturing highly differentiated, meristematic tissues.
Crop Sci. 42: (in press).
Bregitzer, P., R.D. Campbell, L.S. Dahleen, P.G. Lemaux, and M-J. Cho. 2000.
Development of transformation systems for elite barley cultivars. Barley Genetics
Newsletter 30:10-12.
Bregitzer, P., L. Dahleen, and P.G. Lemaux. 1999. Development and implementation of
improved tissue culture media for the in vitro manipulation of malting barley. Ann. Prog.
<strong>Report</strong> on Malting Barley Res., pp. 1-8.
Cho, M-J., W. Zhang, and P.G. Lemaux. 1998. Transformation of recalcitrant barley
cultivars through improvement of regenerability and decreased albinism. Plant Sci.
138:229-244.
Dahleen, L.S., and P. Bregitzer. An improved media system for high regeneration rates
from barley immature embryo-derived callus cultures of commercial cultivars. Crop Sci
42: (in press).

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Epstein, E. 1999. Silicon. Ann. Rev. Plant Mol. Biol. 50:641-664.
Wan, Y., and P.G. Lemaux. 1994. Generation of large numbers of independently
transformed fertile barley plants. Plant Physiol. 104:37-48.
Zhang, S., M-J. Cho, T. Koprek, R. Yun, P. Bregitzer, and P.G. Lemaux. 1999. Genetic
transformation of commercial germplasm of oat (Avena sativa L.) and barley (Hordeum
vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings.
Plant Cell Rep. 18:959-966.
Table 1. Comparative genotype regeneration responses to different concentrations of
silicon in tissue culture media. All control means were set at one. Data are reported as
number of green plants per petri dish as compared to the control response.
Si Concentration (:M)
Genotype Location 0 10 100 500 1000 5000
GP combined 1.0 1.9 1.1 2.5 1.0 0.7
Morex combined 1.0 0.8 1.4 1.5 1.2 1.3
Harrington Fargo 1.0 0.9 2.0 2.2 1.5 2.0
GP + Morex combined 1.0 1.5 1.2 2.2 1.0 0.9

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Table 2. Agronomic performance of non-tissue cultured controls and plants regenerated
from original and improved media.
Parental line Media
Heading
date
(julian)
Height
(cm)
Lodging
(%)
Yield
(bu/ac)
Test
weight
(#/bu)
% Plump
Kernels
90Ab321 Improved 167 56.1 24.4 86.9 53.2 86.1
90Ab321 Original 165 59.0 9.7 89.1 53.5 88.9
90Ab321 control 165 58.0 14.4 90.7 53.1 87.8
Baronesse Improved 174 65.8 34.7 78.7 51.8 77.6
Baronesse Original 174 65.6 34.1 87.0 52.3 82.5
Baronesse control 173 63.4 31.3 88.0 52.4 83.1
Colter Improved 168 69.3 17.2 108.9 51.3 67.8
Colter Original 168 65.3 16.9 101.7 51.0 69.9
Colter control 167 67.4 5.6 110.7 52.2 78.0
Conlon Improved 168 68.8 6.9 99.9 53.9 96.2
Conlon Original 168 66.6 3.4 98.8 54.0 97.6
Conlon control 170 70.3 4.2 103.2 54.8 98.0
Crystal Improved 175 69.9 25.6 101.2 53.4 81.2
Crystal Original 175 70.6 16.3 100.9 53.9 83.2
Crystal control 173 72.3 29.4 101.9 53.4 78.3
Golden Promise Improved 175 57.5 30.0 78.0 51.2 60.9
Golden Promise Original 175 58.1 37.2 77.7 50.6 52.9
Golden Promise control 175 54.6 32.5 73.5 50.9 55.3
Morex Improved 169 70.5 26.6 84.8 50.1 69.5
Morex Original 169 72.9 30.6 79.7 49.6 71.3
Morex control 168 72.5 9.4 92.6 51.2 82.9
ND15477 Improved 169 70.3 6.3 98.7 52.0 81.7
ND15477 Original 170 71.6 3.1 100.0 52.6 82.0
ND15477 control 169 71.3 7.5 107.6 52.5 86.7
mean of all lines Improved 171 66.0 21.4 92.0 52.1 77.7
mean of all lines Original 170 66.2 18.9 91.8 52.2 78.4
mean of all lines control 170 65.9 17.2 95.6 52.4 80.1

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BARLEY VIRUS DISEASES
Michael C. Edwards
USDA-ARS Cereal Crops Research Unit, Red River Valley Agricultural Research
Center, and Department of Plant Pathology, North Dakota State University, Fargo, North
Dakota.
Objectives/Rationale
The ultimate goal of this research program remains unchanged from previous years. That
goal is to reduce barley losses that occur as a result of virus diseases. Our objectives and
rationale therefore remain unchanged. Virus diseases continue to result in significant
yield losses in barley and other cereals. Because significant improvement in our ability
to control virus diseases depends to a great extent upon significant improvement in our
understanding of how viruses function and cause disease, this research program
emphasizes molecular genetic studies of host-virus interactions. The goal of these studies
is to identify the factors involved in viral pathogenicity and host resistance. Our studies
primarily involve barley stripe mosaic virus (BSMV) and oat blue dwarf virus (OBDV).
Because we have evidence that resistance to BSMV is targeted to basic processes such as
virus replication and movement, just as is the case with other viruses, we feel there is
much to gain from studying this virus as a model. Like barley yellow dwarf virus,
OBDV is present in barley fields every year, albeit to a much lesser extent. Also like
BYDV, OBDV is limited to the phloem of infected plants. Thus, certain aspects of
OBDV pathogenesis may be similar to that of BYDV.
Another goal of our project is to eliminate BSMV infections from the barley in the
National Small Grains Germplasm Collection (in cooperation with ARS personnel in
Idaho [Drs. Wesenberg & Bockelman]). The NSGGC serves as a valuable source of new
germplasm for the improvement of malting barley varieties as well as others. Many of
the barley entries are infected with BSMV, a seed-borne pathogen. Since clean seed
stocks are vital for effective control of BSMV, it is extremely important to maintain the
collection in a BSMV-free condition. Thus, we will attempt to identify all entries
infected with BSMV and eliminate BSMV from seed stocks of these entries. Sources of
tolerance to BSMV ultimately may be identified as well.
Another goal of our program is to facilitate the development of new malting barleys
adapted to the upper Midwest through the coordination of the Mississippi Valley Uniform
Regional Barley Nursery.
Methods
For evaluation of the NSGGC, procedures involve the evaluation of accessions (or
entries) for the presence of BSMV, rogueing of infected plants, and production of
BSMV-free seed. In most past years, 2000 new entries were grown in field plots at the
USDA-ARS National Small Grains Germplasm Research Facility in Aberdeen, Idaho.
We had originally planned to attempt the production of virus-free seed from known
infected accessions using methods similar to those used last year and first used in 1997.
This normally involves planting each accession in hills to minimize the possibility of

105
BSMV spread between entries. However, it was decided to re-examine about 2000
previously-tested entries using row planting to increase the amount of seed produced
from entries testing virus-free. All of the seed lots tested had been previously found to be
infected with low levels of BSMV or had been designated as ‘possibly infected.’ Both
visual observations and ELISA testing of fresh plant samples were used to determine the
presence of BSMV. One leaf piece was removed from each plant in a row and samples
were pooled for ELISA testing. Rows with visibly infected plants or with plants that
tested positive were rogued. If ELISA results were negative, the rows were harvested
and the seed used by the curator of the small grains collection to replace contaminated
seed. If ELISA results were positive, the seed was not harvested.
Results
Eradication of BSMV from barley in the National Small Grains Collection.
Although for the last few years we have moved beyond bulk testing of entries to the more
difficult task of eradicating BSMV from entries already known to be infected, we decided
to again bulk test some of the entries that were considered to have low rates of BSMV
infection. This provided a better chance of producing BSMV-free seed without losing the
genetic diversity in many of the entries.
A high rate of infection (approximately 15%) was observed. Of 1978 entries tested, 302
were infected with BSMV. Another 8.5% tested beyond the normal ‘healthy’ range, and
will need to be re-evaluated. Testing will continue in 2002 with a similar number of
entries.
We have produced BSMV-free seed for over 17,000 barley entries in the National Small
Grains Collection since the inception of this collaborative project with ARS personnel in
Aberdeen. Elimination of BSMV from seed of the entries will greatly enhance the value
of the collection and should assist in prevention of BSMV contamination of breeding
programs.
The Mississippi Valley Uniform Regional Barley Nursery was again coordinated.
Specific data on this nursery can be obtained in the 2001 Mississippi Valley Barley
Nursery <strong>Report</strong>. These reports are available as ‘pdf’ files by email or will be available
on-line at our website when it is updated (http://www.fargo.ars.usda.gov/cer/cer_home.htm).
Cooperators:
D. Wesenberg and H. Bockelman, USDA-ARS, National Small Grains
Germplasm Research Facility, Aberdeen, Idaho
Additional Personnel Engaged in Barley Virus Research:
Renee McClean, Biological Laboratory Technician
Adi Santoso, new postdoctoral research associate

106
GERMPLASM ENHANCEMENT FOR RWA RESISTANCE
D.W. Mornhinweg, D.R. Porter, J.A. Webster
USDA-ARS Plant Science and Water Conservation Research Laboratory
Stillwater, Oklahoma
Prebreeding for adapted germplasm lines
The prebreeding program is designed to bring resistance genes from unadapted
germplasm lines into adapted malting and feed barley backgrounds for all barley growing
regions in the U.S. It involves repeated backcrossing with intermittent screening with a
time commitment of approximately seven years from the first cross until homozygous
resistant BC3F3 lines can first be evaluated as observation lines in the field. This is an
ongoing process involving various resistant lines and adapted cultivars currently in all
phases of the program. Field testing was preformed in cooperation with Phil Bregitzer
(spring barley) and Darrell Wesenberg/Charles Ericksen (winter barley) all scientists with
the USDA-ARS in Aberdeen, Idaho; Bob Hammon and Frank Piears, Colorado State
University; and David Baltensberger, University of Nebraska. In the summer of 2001,
ten advanced generation RWA-resistant spring barley lines were evaluated in replicated
yield trials at 4 locations in Idaho, 2 locations in Nebraska and 2 locations in Colorado.
Five of these lines involved six, 6-row backrounds and five lines involved five, 2-row
backgrounds with RWA resistance in all ten lines derived from STARS-9301B.
Preliminary Yield Trials were conducted at one location in Idaho on a total of 33 adapted
RWA-resistant winter barley germplasm lines involving three susceptible winter barley
backgrounds and 9 RWA-resistant sources. Preliminary yield trials were conducted at 4
locations in Idaho for 187 advanced generation adapted spring barley germplasm lines
involving 14 susceptible backgrounds and 23 RWA-resistant sources. A preliminary
yield trial was conducted in Stoneham, Colorado on 114 adapted advanced generation
RWA-resistant spring barley germplasm lines in an Otis background involving 3 different
sources of RWA resistance. A 30 entry subset of these lines was also evaluated in a
Preliminary yield trial in Aberdeen, Idaho. Five hundred and sixty advanced generation
adapted winter RWA-resistant barley germplasm lines were evaluated in headrows at
Aberdeen, Idaho and rows selected for future yield testing in 2002. These lines
involved one susceptible background and 9 RWA-resistant sources. 1,000 adapted
BC3F3/4 winter barley germplasm lines were evaluated in observation rows in Western
Colorado involving 3 susceptible backgrounds and 7 RWA-resistant backgrounds and
600 adapted BC3F3/4 winter barley germplasm lines were evaluated in observation rows
in Aberdeen, Idaho involving 2 susceptible background and 7 RWA-resistant
backgrounds. Rows were selected for preliminary yield testing in 2002 and heads were
selected for evaluation in headrows in 2002 in both Idaho and Colorado. 1,000 BC3F3/4
adapted spring RWA -resistant barley lines were evaluated in observation rows in Idaho.
Selections were made for preliminary yield trials in 2002. Numerous adapted RWAresistant
spring barley germplasm lines were increased for pure seed in the field in Idaho
and in the greenhouse in Stillwater, OK. Eight spring BC3F2 populations involving 6
susceptible backgrounds and 2 RWA-resistant sources and 11 winter BC3F1 populations

107
involving two susceptible backgrounds and 11 RWA-resistant sources were increased
towards evaluation as BC3F3 observation rows in 2002 and 2003 for future germplasm
development. 43,593 seedlings were screened towards germplasm development and
future germplasm release of winter, spring, feed and malt barleys. Six BC2F1 crosses
were made towards germplasm development involving 6 susceptible spring barleys and
one RWA-resistant source.
Genetic Studies
Complete genetic analysis has been performed on 9 out of a total of 108 RWA -resistant
lines developed by the USDA-ARS in Stillwater. All 9 lines showed multiple gene
control for RWA resistance. Genetic diversity studies are planned for all resistant lines to
determine if these lines carry different genes for resistance to RWA. This type of
analysis requires 3 years for development of seed necessary for each test. Complete
inheritance studies were conducted for 2 unadapted RWA-resistant germplasm lines.
Four spring F1 populations were increased to F2 for future use in inheritance studies.
Ten spring barley BC1F1 crosses, 9 spring barley F1/RF1 crosses, and 5 spring barley F1
crosses were make for future genetic studies. Eight TC1F1:F2 winter barley populations
and 14 TC1F1:F2 spring barley populations were increased for future genetic diversity
studies. Two spring barley and 11 winter barley TC1F1 populations were increased to
TC1F2 for future genetic diversity studies. Six spring barley testcrosses of resistant x
resistant F1 on susceptible barleys and, 5 spring barley R X R F1, and 30 winter barley R
X R F1 crosses were made in the spring of 2001 for future genetic diversity studies.
Cooperative screening
Six hundred advanced lines were screened for resistance for 3 barley breeders.
Other aphids
A small scale replicated study was conducted in the greenhouse on 10 barley lines
previously identified in the literature as either susceptible or resistant to Bird cherry oat
aphid (BCO) or resistant to Barley yellow dwarf mosaic virus. Seedlings were infested
with BCO at the 1 leaf stage and measurements on shoots and roots were made in an
attempt to determine the effect of Bird cherry oat aphid feeding on these lines as
seedlings. Identical measurments were made on an identical planting which was grown
in the same greenhouse at the same time but kept aphid free by repeated insecticide
applications. Analysis of data is ongoing.
Personnel
Germplasm Enhancement
Dolores W. Mornhinweg, Geneticist
David R. Porter, Research Geneticist

108
Cooperators
Darrell Wesenberg, Research Agronomist, USDA-ARS, Aberdeen, ID
Phil Bregitzer, Research Geneticist, USDA-ARS, Aberdeen, ID
Charles Erickson, Agronomist, USDA-ARS, Aberdeen, ID
Berne Jones, Research Biochemist, USDA-ARS, Madison, WI
Frank Piears, Professor, Colorado State University
Bob Hammond, Professor, Colorado State University
Dave Blatsenberger, Professor, University of Nebraska
Recent Publications
Mornhinweg, D.W., D.R. Porter and J.A. Webster. 1998. Registration of Stars-9577B
Russian Wheat Aphid Resistant Barley Germplasm. Crop Sci. 39: 882-883.
Mornhinweg, D.W., P.P. Bregitzer, B.L. Jones, F.B. Peairs, T.L. Randolph, D.R. Porter,
and J.A. Webster. 1999. Effect of Early and Late RWA Infestation on Agronomics and
Malting Quality of Adapted Barley Germplasm Lines. Proceedings 16 th American
Barley Researchers Workshop p. 69.
Mornhinweg, D.W., and D.R. Porter. (submitted to Crop Science) Inheritance of RWA-
resistance in spring barley germplasm line, STARS-9577B.

109
THE OREGON BARLEY IMPROVEMENT PROGRAM
Patrick Hayes
Dept. of Crop and Soil Science
Oregon State University
Corvallis, OR 97331
541-737-5878
patrick.m.hayes@orst.edu
Summary
The primary focus of the OSU breeding program, with AMBA funding, is malting quality
winter 6-row. A secondary focus is malting quality spring 6-row. We are attempting to
integrate the two efforts by developing facultative varieties that can be either fall or
spring sown. We have devoted considerable effort over the past ten years to transferring
quantitative resistance to stripe rust to our germplasm. We have incorporated stripe rust
resistance in all types of germplasm, both winter and spring and 2-row and 6-row, but
the focus of our mapping and molecular selection efforts has been spring 2-row.
<strong>Progress</strong> in all areas of endeavor is summarized in this report.
2001 was not a good year for Oregon barley production. Oregon barley acreage was
reduced by water disputes in the Klamath Basin. From a research perspective, however,
prospects are encouraging. We have three winter barley selections in the AMBA Pilot
program. STAB 113 is in a first year of testing; STAB 7 and 47 were advanced to a
second year; and STAB 7 is in accelerated increase for plant scale testing. We have
developed some promising 6-row spring malting germplasm. And, most encouraging, our
pyramids of stripe rust resistance genes are working. We have shown that quantitative
resistance to stripe rust can be mapped, introgressed, and transferred to elite agronomic
and malting quality backgrounds using molecular marker assisted selection.
Furthermore, these pyramids of resistance genes were resistant in the face of the reported
new virulence in Peru. Another encouraging development was the performance of
Recombinant Substitution Lines based on introgressing pieces of the Hordeum vulgare
subsp. spontaneum genome into Harrington.
Winter barley improvement
2001 was a dry year in much of the Pacific Northwest and yields were lower than usual
even at irrigated locations, such as Aberdeen. Agronomic data from trials at Pendleton
(dryland), Pullman (dryland) and Aberdeen are shown in Tables 1 – 4. The winter
malting selections are still not as high yielding as the winter feed varieties, but if
performance is compared to spring malting types at the same locations, the comparisons
are more favorable. Stripe rust resistance data are presented in Table 5. The field
epidemic intensities at Toluca and at Davis are high; accordingly, while we would like to
see lower levels of rust on some of these selections, the level of protection should be
sufficient for most production environments in the Pacific Northwest. These data support
the contention that seedling resistance is not necessarily a predictor of adult plant

110
resistance. Some of the lines show good resistance to the apparent new stripe rust
virulence at Huancayo, Peru.
We are focusing our winter malting barley efforts on genotypes that do not require
vernalization. Results for our mapping lead us to hypothesize that in the development of
88ab536 there was either a crossover event leading to the separation of the genes that
determine cold tolerance and vernalization on chromosome 7 (5H) or that an allelic
architecture was created that leads to an epistatic down-regulation of the vernalization
requirement. These hypotheses will be elaborated on in our proposal for continued
funding. The immediate practical implication is that most of our winter malting barley
selections can be spring-sown. This would offer a safety net to growers, in the event of
extensive winter injury, and it could lead to the development of varieties with very broad
adaptation. The yield of some of the winter selections under spring-sown conditions is
competitive (Table 7). We hypothesize that the agronomic potential of these lines may
be limited by the malting quality donor (Morex, via 88Ab536). Accordingly, as will be
elaborated on in our proposal for continued funding, we propose to transfer winter
hardiness QTL alleles to more recent Midwestern malting quality varieties and thus raise
the bar of agronomic performance. Quality samples of the spring-sown winter malting
selections were submitted, but data have not been received, so the consistency of quality
expression across sowing dates is not known at this point.
The malting quality of winter selections grown under fall-sown conditions at Pendleton,
Pullman, and Aberdeen is shown in Table 6. For comparative purposes, we include the
malting data from analyses of the same sample by different labs. Of these selections,
STAB 7 is in accelerated seed increase in preparation for Plant Scale testing; STAB 7 and
STAB 47 are currently both in a second year of the AMBA Pilot program; and STAB 113
is in a second year of first level testing in the AMBA Pilot program. KAB 47 and KAB
51 drill strips were planted at Pendleton and Aberdeen in Fall, 2001 in preparation for
submission to the AMBA Pilot program.
A notable feature of many of the winter malting selections is low grain protein, and this
leads to lower than desired specs for other malt parameters. So, while much of the rest of
the barley research community struggles with ways to lower grain protein, we are
working on ways to increase it. In cooperation with S. Petrie and K. Rhinhart
(Pendleton) we initiated a series of management trials in the fall of 2001. We are
continuing with expanded versions of these trials this year and after this season, we will
be able to present a comprehensive data analysis and a management package. At this
point, it appears that we will be able to manage nitrogen (via split fall/spring
applications) in order to achieve target levels of grain protein and enzymatic properties.
However, the timing of the split applications will be critical, and there may be specific
requirements for specific genotypes.
Winter 6-row: 2001-2002 season
Germplasm development
Crosses are made at Corvallis and lines advanced to homozygosity through doubled
haploid production, and/or single seed descent. With the emphasis on facultative growth
habit, we are using SSD. With SSD, molecular markers can be used to indirectly select

111
for traits that are difficult/expensive to assess under field conditions. This selection
during generation advance will increase the likelihood of developing lines with target
profiles. With doubled haploids, one produces many lines without target profiles leading
to a waste of agronomic and quality testing resources.
Crosses: Based on performance of current winter lines under spring seeding we
hypothesize that agronomic performance is limited by the spring parent (88ab536
– Morex). Accordingly, we reasoned that we should focus on converting spring
types with better agronomic performance than Morex – e.g. Excel and Stander.
We made crosses of STAB 7, 47, 113, Kab 47, Kab 51 with Excel and Stander.
The advance of this germplasm will be the subject of our proposal for continued
winter malting barley development.
Germplasm advance: Progeny from the following cross combinations are
currently being advanced via SSD in the greenhouse: STAB 7/ STAB 113;
STAB113/STAB 47, KAB 43, and KAB 50; STAB 47/ KAB43, and KAB 51;
STAB 7/KAB 43 and KAB 51.
Cycle 1: Head rows
The following SSD populations were planted in the fall of 2001 at Corvallis as F4 head
rows:
• STAB 113/Kab 43; Stab 113/Kab 50;Stab 113/Stab47; Stab 47/Kab 51; Stab
7/Kab 51; Stab 7/Kab 43; Stab 7/Stab 113; Stab 113/Stab 47
• Strider/Orca six-row lines (F7) selected for BYDV resistance
• A set of winter barley top crosses
Cycle 2. Single replication yield trials
• All STAB white aleurone mapping population lines and parents; 140 entries.
Cycle 3. Replicated yield trials
• Selected STAB and KAB lines, plus checks at Pendleton, Oregon; Pullman,
Washington; Aberdeen, Idaho.
Cycle 4: Pre-release
• AMBA Pilot scale drill strips: STAB 47, STAB 7, STAB 133, KAB 47; KAB
51 at Pendleton and Aberdeen
• Management trials: Nitrogen, Chloride, Zinc, and date of planting at
Pendleton and Moro.
• On farm one acre trials:
o STAB 113: Paul Williams at Davenport, WA
o Stab 47: Bill Jepsen at Heppner, OR
o Kab 37: Melvin Stonebrink at Wallowa, OR

112
Tri-state Extension testing : Oregon - STAB 7, STAB 47, STAB 113, KAB 37; Idaho
Idaho - STAB 7, STAB 47, STAB 113
Upcoming Releases
• No winter varieties were proposed for release.
• We have planted Breeder’s Seed head rows of all selections the
AMBA Pilot program.
• One acre of STAB 7 will be increased in a spring seeding in cooperation with the
Washington Crop Improvement Association in order to have sufficient seed for a
plant scale test.
Variety performance updates
There was commercial production of Kold and Strider.
Spring Barley Improvement
6-row: The consensus of discussions with western spring 6-row malting barley users was
that the two most promising varieties for western production are Excel and Stander.
Drawbacks to these varieties, in western production, are thin seed and susceptibility to
stripe rust. Accordingly, we implemented a marker-assisted selection program to transfer
stripe rust resistance QTLs from the varieties Orca (2-row) and Tango (6-row). 1,200 F5
SSD lines were evaluated for stripe rust resistance in Mexico in the summer of 2001.
One hundred forty selections had rust severities < 20%. We proceeded with one cycle of
greenhouse seed increase and are currently genotyping these lines to determine their
stripe rust resistance allele architecture. The resistant lines are currently planted at Davis,
California and Ciudad Obregon, Mexico; we will proceed with F7 head row evaluations
at Tule Lake, California and (in a cooperative effort with BARI) at Fort Collins,
Colorado. We will confirm stripe rust resistance this summer at Toluca, Mexico.
Samples from agronomically promising lines at Tule Lake and Ciudad Obregon will be
submitted for malting quality assessment, laying the groundwork for improvement of
spring western 6-row. As mentioned in the preceding section on winter malting barley
improvement, we are hoping to integrate our 6-row malting barley efforts so the same
varieties will be suitable for fall and spring sowing.
2-row: The goal of our 2-row spring barley improvement efforts has been a public sector
variety better than Baronesse. Ideally, we would like a Baronesse with malting quality.
All of our spring barley improvement efforts have been conducted within a framework of
disease resistance. Broad-spectrum resistance to stripe rust, and other diseases, will be an
excellent insurance policy. These resistance genes will help to ensure the stability of
barley production by preventing expensive and unpleasant surprises. Our strategy for
spring 2-row improvement has been to pyramid resistance QTLs using marker-assisted
selection. These efforts have paid off. As shown in Table 9, genotypes with multiple
resistance QTL alleles have lower levels of stripe rust than lines with one or no QTL.
Some of these resistance gene pyramid selections were also resistant to the reported new
virulence detected at Huancayo, Peru in 2001 (Table 8). We are confirming these
preliminary data with more extensive tests in Ecuador and Peru.

Germplasm Development:
113
Crosses: Due to resource constraints faced by the Tri-State Barley Commissions,
and the fact that there are strong two-row breeding programs in the Pacific
Northwest, we decided to de-accelerate our 2-row breeding efforts for at least one
cycle. We had focused our crossing efforts on transferring disease resistance QTL
alleles to Merit. In order to keep the germplasm moving through the pipeline, we
sent seed of these crosses to the BARI program for advance and selection.
Germplasm advance: Per crossing, we curtailed germplasm advance, with the
exception of 6-row populations involving Lacey/Tango and Sara/Tango.
After one cycle of greenhouse increase, and a cycle of stripe rust assessment in
Mexico in the summer of 2001, we increased the BCD47/Baronesse population of
422 doubled haploid lines in New Zealand. All lines with BCD 47 levels of stripe
rust resistance that are of Baronesse height and maturity will be planted in single
rep yield trials at Pendleton.
Cycle 1: Head rows
2001
• Due to drought conditions and the resulting water wars we were not
able to grow plots in the Klamath Basin in 2001.
2002
• 105 lines BSR resistant 6-rows from the Orca/Stander/Tango/Excel
SSD populations will be planted at Tule Lake.
Cycle 2: Single replication yield trials
2001
• An Augmented PYT consisting of 205 lines, including checks, was
grown at Pendleton. This nursery consisted primarily of double
haploids derived from crosses of the stripe rust resistance gene
pyramids (Ops, Ajo, Bu, and Sal) with a Czech selection (He6890).
The drought stress at Pendleton, coupled with selection against short
statue, allowed us to select lines for replicated trials in 2002. Malting
quality data on these selections has not been received from the CCRU.
• RCSLYT - 52 entries from the Hordeum vulgare subsp. spontaneum
introgressions with Harrington were grown at Pendleton, Aberdeen,
Pullman, Saskatoon, and Bozeman. These data, shown in Table 10,
are quite exciting: the spontaneum accession clearly contributed
positive alleles for seed size and weight. In some cases, this led to
higher yield. Malting quality data on these selections has not been
received.

2002
114
• An Augmented PYT of approximately 100 lines selected from the
BCD47/Baronesse population will be grown at Pendleton.
Cycle 3: Replicated yield trials
2001
• 6RYT - Facultative winter barley lines, selections from the
introgression of stripe rust resistance genes into Colter, and selections
form the ICARDA/CIMMY T program with malting barley parentage
were grown at Pendleton, Pullman, and Aberdeen in replicated trials
and at Klamath Falls and Moscow as single rep trials. Data are
presented in Table 7. Malting quality data on these selections has not
been received.
• 2RYT - 56 entries from the Bu, Ajo, Ops, and Sal stripe rust
resistance pyramids were grown at Pendleton, Pullman, and Aberdeen
in replicated trials and at Klamath Falls and Moscow as single rep
trials. Data are presented in Table 8. Malting quality data on these
selections has not been received.
2002
• 2RYT – Selections from the Bu, Ajo, Ops, and Sal germplasm and
selections from the crosses of BU, Ajo, Ops, and Sal with He6890 or
BCD47 will be grown at Pendleton, Pullman, and Aberdeen in
replicated trials and at Klamath Falls and Moscow as single rep trials.
• RCSLYT - 20 entries from Hordeum vulgare subsp.
spontaneum/Harrington project will grown at Pendleton, Aberdeen,
Pullman, and Fort Collins.
• 6RYT - 15 entries including winter facultative lines, CR/SR lines and
Mexico selections will be grown at Pendleton, Pullman, and Aberdeen
in replicated trials and at Klamath Falls and Moscow as single rep
trials
Cycle 4: Pre-release
BCD 47 was dropped from the AMBA program due to high enzyme activity. This short
stature, late season variety is not suitable for dryland production. Accordingly it cannot
compete with Baronesse under dryland conditions. We had intended to assess its
commercial potential under irrigation in the Klamath Basin, but we were not able to do so
due to the lack of irrigation. These trials will be conducted in 2002.
Other experiments:
2001

115
• All lines in spring yield trials, except the Hordeum vulgare subsp.
spontaneum/Harrington project were grown at Toluca, Mexico (in cooperation
with H. Vivar and F. Capettini) and at Mt. Vernon (in cooperation with X.
Chen) for stripe rust assessment.
• The experiments were planted in the Fall of 2001 at UC Davis in cooperation
with L. Jackson and L. Gallagher. The multiple resistance gene pyramids were
assessed for seedling resistance by M. Johnson (Montana State University).
Upcoming variety releases
BCD47
• If the on-farm trials in the Klamath Basin are promising, we will pursue
release as a feed variety.
Sara
• Sara is a 6-row, stripe rust-resistant hooded barley for forage production.
Variety performance updates
There was commercial production of Orca and Tango.
Project Profile
Project Personnel
Patrick Hayes, Professor
Jennifer Kling, Research Professor
Isabel Vales, Research Assistant Professor
Isabel Vales, Research Assistant Professor
Luis Marquez-Cedillo, post-doc
Lol Cooper, post-doc
Ann Corey, Senior Research Assistant
Tanya Filichkin, Research Assistant
Ariel Castro, Graduate Research Assistant
Ivan Matus, Graduate Research Assistant
Jari VonZitzewitz, Graduate Research Assistant
Kelley Richardson, Graduate Research Assistant
Publications
Sato, K. T. Inukai, and P. M. Hayes. 2001. QTL analysis of resistance to the rice blast
pathogen in barley (Hordeum vulgare). Theor. Appl. Genet. 102:916-920.
Van Sanford, D., J. Anderson, K.. Campbell, J. Costa, P. Cregan,, C. Griffey, P. Hayes,
and R. Ward. 2001. Discovery and development of molecular markers linked to Fusarium
Head Blight resistance: an integrated system for wheat and barley. Crop Sci. 638-644.

116
Karsai, I., K. Meszaros, L. Lang, P.M. Hayes, and Z. Bedo. 2001. Multivariate analysis
of traits determining adaptation in cultivated barley. Plant Breeding 120:217-222.
Marquez-Cedillo, L.A., P.M. Hayes, A. Kleinhofs, W.G. Legge, B.G. Rossnagel, K.
Sato, S.E. Ullrich, D.M. Wesenberg, and the NABGMP. 2001. QTL analysis of
agronomic traits in barley based on the doubled haploid progeny of two elite North
American varieties representing different germplasm groups. Theor. Appl. Genet.
103:625-637.
Costa, J.M., S. Kramer, C. Jobet, R. Wolfe, A. Kleinhofs, D. Kudrna, A. Corey, S.
McCoy, O. Riera-Lizarazu, K. Sato, T. Toojinda, I. Vales, P. Szucs, and P. M. Hayes.
2001. Molecular mapping of the Oregon Wolfe Barleys: an exceptionally polymorphic
doubled-haploid population. Theor. Appl. Genet.103:415-424.
Germplasm registrations
Wesenberg, D.M.., D.E. Burrup, W.M. Brown, V.R. Velasco, J.P. Hill, J.C. Whitmore,
R.S. Karow, P.M. Hayes, S.E. Ullrich, and C.T. Liu. 2001. Registration of Bancroft
barley. Crop Sci. 41:265-266.
Book Chapters
P.M. Hayes, A. Castro, L. Marquez-Cedillo, A. Corey, C. Henson, B.L. Jones, J. Kling,
D. Mather, I. Matus, C. Rossi, and K. Sato. Genetic Diversity for Quantitatively Inherited
Agronomic and Malting Quality Traits. In R. Von Bothmer, H. Knupffer, T. van Hintum,
and K. Sato (ed.). Diversity in Barley. Elsevier Science Publishers, Amsterdam. in press
Table 1. Agronomic data for Oregon winter barley selections compared to check varieties,
averaged across three locations (Pendleton, Pullman, and Aberdeen; 2001).
Yield Test weight Plump seed
Variety/selection (lbs/acre) (lbs/bu) (on 6/64)
88Ab536 5366 51 78
Strider 6912 50 82
Hundred 6132 48 49
812 6474 50 74
STAB 7 5439 51 68
STAB 47 5053 51 83
STAB 113 5752 52 80
KAB37 6068 51 68
KAB47 4988 50 67
KAB51 5473 52 85

117
Table 2. Agronomic data for Oregon winter barley selections compared to check varieties,
averaged across three locations (Pendleton, Pullman, and Aberdeen; 2000 - 2001).
Yield Test weight Plump seed
Variety/selection (lbs/acre) (lbs/bu) (on 6/64)
88Ab536 5278 52 82
Strider 7408 51 88
Hundred 6666 50 61
812 6756 51 80
STAB 7 5920 52 76
STAB 47 5187 52 87
STAB 113 6419 53 79
KAB37 6668 53 76
KAB47 5532 52 74
KAB51 5686 53 89
Table 3. Agronomic data for Oregon winter barley selections compared to check varieties (2001).
Yield Test weight Plump seed
Location /Year Variety/selection (lbs/acre) (lbs/bu) (on 6/64)
Pendleton 2001 88Ab536 4483 49 61
Strider 6811 48 78
Hundred 6341 49 35
812 5785 49 61
STAB 7 5532 49 56
STAB 47 5384 52 81
STAB 113 5453 52 75
KAB37 6007 51 61
KAB47 5728 49 50
KAB51 4998 52 86
Pullman 2001 88Ab536 4761 50 83
Strider 6638 48 86
Hundred 5353 43 51
812 6040 48 78
STAB 7 5136 49 75
STAB 47 4342 49 87
STAB 113 5756 50 85
KAB37 5454 49 70
KAB47 5261 50 78
KAB51 5190 49 91
Aberdeen 2001 88Ab536 6854 54 91
Strider 7286 54 83
Hundred 6701 52 61
812 7598 53 82
STAB 7 5650 54 74
STAB 47 5424 53 82
STAB 113 6048 54 79
KAB37 6744 54 73
KAB47 3947 51 73
KAB51 6230 54 77

118
Table 4. Agronomic data for Oregon winter barley selections compared to check varieties (2000- 2001).
Yield Test weight Plump seed
Location /Year Variety/selection (lbs/acre) (lbs/bu) (on 6/64)
Pendleton 88Ab536 4622 51 72
Strider 7132 50 87
Hundred 6271 49 53
812 5792 51 71
STAB 7 6252 51 70
STAB 47 5761 52 87
STAB 113 5805 52 70
KAB37 6161 52 74
KAB47 5725 50 65
KAB51 5459 53 91
Pullman 88Ab536 4218 53 83
Strider 6772 51 86
Hundred 5532 47 51
812 5839 51 78
STAB 7 4998 52 75
STAB 47 4027 52 87
STAB 113 5836 53 85
KAB37 5757 52 70
KAB47 5323 52 78
KAB51 4816 52 91
Aberdeen 88Ab536 6993 53 91
Strider 8321 54 90
Hundred 8194 52 74
812 8637 53 90
STAB 7 6511 54 82
STAB 47 5772 53 88
STAB 113 7615 55 86
KAB37 8088 55 82
KAB47 5549 53 82
KAB51 6782 55 88
Table 5. Adult plant stripe rust disease severity of winter barley varieties and checks at Toluca, Mexico; Davis,
California; Huancayo, Peru (data provided by H .Vivar, L. Jackson, and B. Brown) and reaction under controlled
environment conditions when inoculated with an isolate corresponding to race 24 (data courtesy of M. Johnston).
Stripe rust Stripe rust Davis, Stripe rust Stripe rust reaction
Variety/selection Toluca, Mexico CA Huancayo, Peru (seedling)
% severity % severity % severity 1 – 9 scale
88 Ab 536 90 100 100 9
Kold 20 0 0 1
Strider 0 0 0 1
Hundred - 30 70 -
Eight-Twelve - 60 70 -
Scio - 40 80 -
Kab 37 30 20 60 7
Kab 47 0 5 80 2
Kab 51 0 50 10 1
Stab 7 40 20 40 6
Stab 47 30 10 60 0
Stab 113 20 40 50 3

119
Table 6. Malting quality of winter barley selections compared to 88AB536. Pendleton, Pullman, and
Aberdeen 1999- 2001.
Malt Barley Wort Diastatic Alpha
Variety or
extract protein protein power amylase
Selection Data source Location Year % % %
0
ASBC 20 0 Beta
glucan
Units ppm
88 Ab536 ConAgra Pendleton 1999 80.3 10.1 4.4 143 54.0 84.0
88 Ab536 CCRU Pendleton 1999 78.7 11.3 4.3 143 53.5 128.0
88 Ab536 BARI Pendleton 2000 78.8 10.9 3.1 170 55.2 348.4
88 Ab536 ConAgra Pendleton 2000 81.7 8.7 4.3 119 51.0 301.0
88 Ab536 BARI Aberdeen 2000 80.0 11.4 5.0 166 56.6 323.2
88 Ab536 BARI Pullman 2000 82.1 11.3 4.3 155 45.1 626.9
88 Ab536 BARI Pendleton 2001 77.2 12.2 4.8 153 49.3 312.0
88 Ab536 ConAgra Pendleton 2001 78.4 12.3 4.9 199 61.0 188.0
88 Ab536 ConAgra Pullman 2001 79.4 12.6 5.6 189 61.0 220.0
Average 79.3 11.5 4.7 159.6 51.6 288.8
Stab 7 ConAgra Pendleton 1999 82.2 9.4 4.2 129 76.0 53.0
Stab 7 CCRU Pendleton 1999 81.1 10.1 3.8 115 60.9 67.0
Stab 7 ConAgra Pendleton 2000 82.2 9.9 4.6 135 60.0 296.0
Stab 7 BARI Pendleton 2000 79.9 10.2 2.8 145 49.7 538.6
Stab 7 BARI Aberdeen 2000 78.9 13.4 5.2 155 51.2 471.3
Stab 7 BARI Pullman 2000 81.5 10.3 3.6 103 46.4 540.6
Stab 7 ConAgra Pendleton 2001 80.1 11.4 4.8 164 78.0 189.0
Stab 7 BARI Pendleton 2001 78.0 11.3 4.2 134 42.1 337.9
Stab 7 BARI Aberdeen 2001 78.9 12.1 5.1 132 48.4 280.9
Stab 7 ConAgra Pullman 2001 81.4 10.9 4.8 172 76.0 161.0
Stab 7 BARI Pullman 2001 78.6 11.0 4.4 118 39.4 392.3
Average 80.3 10.9 4.3 136.5 57.1 302.5
Stab 47 ConAgra Pendleton 1999 78.0 11.8 5.0 150 61.0 56.0
Stab 47 CCRU Pendleton 1999 76.8 12.9 4.4 149 61.1 64.0
Stab 47 ConAgra Pendleton 2000 79.6 10.2 4.4 117 59.0 229.0
Stab 47 BARI Pendleton 2000 77.8 10.9 3.0 140 51.7 458.7
Stab 47 BARI Aberdeen 2000 77.8 12.3 5.5 148 63.7 207.9
Stab 47 BARI Pullman 2000 81.9 11.0 4.5 134 50.5 592.3
Stab 47 ConAgra Pendleton 2001 80.0 11.7 4.8 145 72.0 75.0
Stab 47 BARI Pendleton 2001 78.3 11.1 4.4 130 45.9 325.7
Stab 47 BARI Aberdeen 2001 76.4 13.7 5.3 135 54.3 220.0
Stab 47 ConAgra Pullman 2001 78.6 13.4 6.1 219 77.0 108.0
Stab 47 BARI Pullman 2001 76.4 13.6 5.6 138 44.1 274.5
Average 78.4 11.9 4.9 143.6 58.2 238.7

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Table 6 (cont.). Malting quality of winter barley selections compared to 88AB536. Pendleton, Pullman, and
Aberdeen 1999- 2001.
Malt Barley Wort Diastatic Alpha
Variety or
extract protein protein power amylase
Selection Data source Location Year % % %
0
ASBC 20 0 Beta
glucan
Units ppm
Stab 113 ConAgra Pendleton 1999 80.8 9.0 4.4 147 52.0 45.0
Stab 113 CCRU Pendleton 1999 79.9 10.1 3.8 132 41.2 71.0
Stab 113 ConAgra Pendleton 2000 81.6 9.4 4.4 125 58.0 88.0
Stab 113 BARI Pendleton 2000 79.4 9.9 3.0 152 51.9 244.2
Stab 113 BARI Aberdeen 2000 81.5 10.0 5.3 122 52.0 248.4
Stab 113 BARI Pullman 2000 82.7 8.6 4.0 102 40.7 460.9
Stab 113 BARI Pendleton 2001 79.1 10.3 4.7 114 48.0 117.9
Stab 113 ConAgra Pendleton 2001 80.3 10.8 4.5 135 57.0 31.0
Stab 113 BARI Aberdeen 2001 77.6 12.2 4.8 130 43.2 311.7
Stab 113 ConAgra Pullman 2001 80.3 11.0 5.2 151 59.0 65.0
Stab 113 BARI Pullman 2001 78.5 11.1 4.8 111 41.0 236.3
Average 80.5 10.0 4.6 126.5 49.9 151.5
Kab 47 ConAgra Pendleton 1999 80.7 10.0 4.7 126 80.0 19.0
Kab 47 ConAgra Pendleton 2000 82.2 8.4 4.2 86 62.0 169.0
Kab 47 BARI Pendleton 2000 81.6 8.4 3.0 90 69.7 234.8
Kab 47 BARI Aberdeen 2000 79.4 12.6 5.8 140 66.5 358.6
Kab 47 BARI Pullman 2000 82.3 11.4 5.3 141 62.8 352.2
Kab 47 ConAgra Pendleton 2001 79.3 11.7 5.2 169 80.0 33.0
Kab 47 BARI Pendleton 2001 78.2 11.8 4.9 113 38.1 225.0
Kab 47 BARI Aberdeen 2001 76.9 13.2 5.6 113 45.7 228.1
Kab 47 ConAgra Pullman 2001 79.8 12.4 5.7 152 70.0 151.0
Kab 47 BARI Pullman 2001 78.0 12.7 5.1 117 40.9 347.5
Average 80.2 11.0 4.9 126.0 63.3 210.0
Kab 51 ConAgra Pendleton 1999 80.6 8.8 4.2 107 56.0 45.0
Kab 51 ConAgra Pendleton 2000 82.8 8.3 4.0 100 59.0 119.0
Kab 51 BARI Pendleton 2000 79.0 11.3 2.7 149 53.1 613.8
Kab 51 BARI Aberdeen 2000 79.2 11.7 5.3 139 53.8 553.5
Kab 51 BARI Pullman 2000 81.4 10.8 4.1 125 48.0 825.2
Kab 51 ConAgra Pendleton 2001 78.4 12.8 4.8 183 68.0 140.0
Kab 51 BARI Pendleton 2001 76.9 12.6 4.1 115 55.8 386.6
Kab 51 BARI Aberdeen 2001 76.7 12.6 4.6 111 38.2 399.4
Kab 51 ConAgra Pullman 2001 79.3 12.8 4.9 157 59.0 208.0
Kab 51 BARI Pullman 2001 77.8 12.6 4.6 123 36.8 493.0
Average 79.5 11.3 4.3 133.1 54.4 376.0

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Table 7. Across location summary of the six-row spring barley yield trial, 2001.
Yield lbs/acre Heading Lodging † Test wt. Plump
Entry Name Aberdeen Boseman Pendleton Pullman Avg date % lbs/bu %
1 Baronesse 7471 6179 3572 5915 5784 177 43 52.4 89.2
3 Stander 8058 5336 3329 5108 5458 174 60 52.4 89.8
14 CR/SR 16 6763 5555 2886 4169 4843 176 97 51.5 75.4
15 CR/SR 66 6020 5414 2690 5227 4838 176 80 47.8 60.5
17 Mex 2 6768 4875 2764 4563 4743 175 60 48.3 74.7
9 Stab 113 6588 5280 2098 4858 4706 176 37 51.1 73.9
4 Stab 7 6221 5144 2513 4826 4676 182 100 50.0 57.8
16 CR/SR 102 6624 4830 2595 4514 4641 176 100 49.2 71.0
2 Tango 6495 4875 2485 4642 4625 172 73 48.5 69.8
6 Stab 34 6494 5438 2300 4197 4607 177 60 49.4 45.0
11 Kab 47 6573 4166 2532 4972 4561 181 57 49.4 43.2
12 Kab 50 6363 4294 2353 4315 4332 176 83 50.5 64.3
18 Morex 6298 4222 2889 3906 4329 175 100 50.9 78.5
7 Stab 47 6082 4486 2536 4195 4325 174 73 50.2 73.1
5 Stab 33 6254 4075 2521 4334 4296 181 97 48.1 66.9
8 Stab 64 5665 3739 2336 4543 4071 175 97 49.0 39.7
10 Kab 43 5809 4251 2285 3703 4012 172 37 51.1 76.6
13 Kab 51 5976 4688 1749 3621 4009 178 33 51.1 84.0
Mean 6474 4825 2580 4534 4603 176 71 50.1 68.5
se 398 240 142 365 817 1 13 0.6 6.9
Prob.>F 0.0135

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Table 9. Stripe rust disease severity, area under the disease progress curve (AUDPC), and
infection rate in BCD lines (based on 7 environments) classified according to the presence or
absence of resistance alleles at QTL regions on chromosomes 4, 5 and 7.
QTL location Resistance phenotype
Chr. 4 Chr. 5 Chr. 7 Disease severity AUDPC Infection rate
- - - 50.8 850 0.0424
+
- - 25.0 438 0.0248
-
+
- 31.7 587 0.0303
- -
+
40.2 648 0.0345
+ +
- 15.8 241 0.0171
+
-
+
7.7 160 0.0169
-
+ +
21.1 341 0.0224
+ + +
8.9 226 0.0092
r² of the model (%) 46.0 44.6 59.6
Table 10. Agronomic data for Harrington and 36 RCSL derived lines from Harrington /
Caesarea 26-24 in five environments in 2001.
Yield (lb/acre) Kernel Plumpness (%) Test Weight (lbs/bu) Thousand Kernel Weight (g)
H # RCSL H RCSL H RCSL H RCSL
Environment 0 0 max min 0 0 max min 0 0 max min 0 0 max min
Aberdeen, ID 6600 6160 7482 4327 80 81** 94 58 50 51 ** 54 47 41 45** 54 36
Pullman, WA 5517 5206 6440 3889 97 96** 99 90 53 52** 54 50 44 46** 51 40
Bozeman, MO 5196 5305 6051 3208 74 78* 97 49 51 53 * 55 50 41 46 ** 51 40
Pendleton, OR 2893 2805** 3376 1639 86 83** 95 39 54 53** 54 52 43 45 55 39
Saskatchewan, CA 1891 1924 2400 1043 92 86** 97 64 54 52* 54 49 44 46* 53 40
# H = Harrington; * significant at p

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MOLECULAR MARKER ASSISTED MODIFICATION OF TRADITIONAL HIGH
QUALITY MALTING BARLEYS
A. Kleinhofs, D. von Wettstein & S. E. Ullrich
Dept. Crop and Soil Sciences
Washington State University
Pullman, WA. 99164-6420
The major emphasis during the reporting period was on the Harrington x Baronesse cross.
Harrigton x Baronesse
Yield Analysis
In summer 2001, 8 BC2 lines and 20 BC3 lines were planted in the regional yield trials at
three locations: Pullman, Fairfield, WA and Royal Slope, WA. These lines yielded
similar to Baronesse in 2000 and were selected to represent the highest yielding lines
from all locations and years with the exception of K11-3, which was included as a low
yielding check. 26 lines grown in Pullman, 12 lines from Fairfield, and 26 lines from
Royal Slope yielded statistically similar to Baronesse. 14 of the 26 lines from Pullman, 8
of 12 lines from Fairfield, and 19 of the 26 lines from Royal Slope yielded higher than
Harrington, although differences were not significant (Table 1).
Malting Analysis
Malting analysis was performed at Madison, WI in 2001 on selected lines grown at
Pullman in summer 2000. Analysis was done using an experimental Joe White malting
system. 62 lines were malted, with two replications when possible. From this data, 13
lines produced malting characteristics similar to Harrington as well as yields similar to
Baronesse.
In fall 2001, 28 lines grown at Royal Slope in summer 2001 were malted again at
Madison, WI. Standard malting analysis was used on three replications for all lines.
Based on the combined yield and malting data, 6 lines have consistently yielded similar
to Baronesse with malting quality similar to Harrington (Table 2). A subset of these lines
exhibiting the best yield and malting data will be grown in the state uniform nurseries in
summer 2002.
Genetic Analysis
When comparing the combined yield data of the BC2 and BC3 populations to the
genotype data of the NILs, two regions on 2HL and one region on 3HL are suggested as
containing yield related QTL. The proximal region of 2HL is bracketed by Bmy2 -
MWG699 while the distal region is bracketed by ABG072 - ksuD22. On 3HL, the QTL
region is bracketed by MWG571A - MWG961. Each high yielding NIL contains at least
one of these regions (Fig. 1). One interesting note is that line 7 does not contain any
Baronesse alleles in these target regions, yet yielded significantly greater than Harrington
at Pullman in 1999 and 2000, and statistically similar to Baronesse, although not
significantly greater than Harrington, in all locations in 2001. This suggested that other
yield QTLs from Baronesse exist that have not been identified. Also, line K11-3 contains

124
Baronesse alleles in the 3HL target region, but yields significantly less than Baronesse.
This could be explained by the possible presence of other genes in the genome that could
negatively interact with the Baronesse yield genes in an epistatic manner. Experiments to
determine the amount, location in the genome, and action of these genes are underway.
Morex x Steptoe
Morex derived lines F13 and A23. These lines are Morex with chr3 (F13) and chr3 and
chr5 (A23) fragments from Steptoe.
Yield improvement in these lines is being tested by introgression of the Steptoe large
seed QTL; crosses made summer 2000. Backcrosses and reselection were conducted in
summer 2001.
Yield improvement by increasing the head length is in progress. We crossed selected
lines with selected lax head lines in summer 2000. Backcrosses and reselection were
conducted summer 2001.

125

126
BARLEY IMPROVEMENT IN WASHINGTON
S.E. ULLRICH, V.A. JITKOV, J.A. CLANCY, M.C. DUGGER, A. KLEINHOFS, D.V. WETTSTEIN
Department of Crop and Soil Sciences
Washington State University
Objectives and Rationale
The WSU barley breeding program's primary objective is to release new varieties that are
higher yielding, better adapted and/or with higher quality than the comparable varieties
currently being grown in the state. Agronomic traits, malting and brewing quality, and
nutritional quality are evaluated during selection. The majority of breeding lines are 2row,
and 6-row spring types as these are used most commonly in rotations. There has
been a shift to more emphasis on 2-rows vs 6-rows in the last few years.
Barley is an important agricultural commodity in Washington State and has had an
average rank of 16 th over the last 5 years among the top 40 commodities produced in the
state. Barley is the 3 rd most important agronomic field crop behind wheat and hay.
Washington ranked 4th among states in barley production 1997-2001. Barley production
in Washington was over 21 million bushels from 430,000 acres seeded in 2001. The
average statewide yield for 2001 was 50 bu/a primarily from dryland production.
Baronesse, planted on nearly 73% of the state's barley acreage was the leading cultivar in
2001. Malting types were planted on about 10% of the acreage (about 43,000 acres), and
winter types on about 1%. The recommended malting types grown in rank order were
Harrington, Morex, and Garnet. There is much greater potential for malting barley
production in Washington than is realized. The gap between feed and malting types has
primarily been due to the level of industry activity in the state and the dominance of one
feed cultivar (currently Baronesse, previously Steptoe). The major drive in recent years
has been to replace Baronesse with public and hopefully a malting type varieties.
Methodology
1. Germplasm Development
Modified pedigree-bulk, single seed descent, and doubled haploid breeding techniques
are employed along with molecular marker assisted selection techniques in collaboration
with A. Kleinhofs. Parent building, as well as line development, occur utilizing
Midwestern varieties for the 6-row quality base, western U.S. and Canadian varieties for
the 2-row quality base and European varieties for diversity in combinations with adapted
western genotypes and for export quality base. Although most European varieties are
inferior to North American varieties in malting quality by U.S. standards, there is
potential to derive U.S. export types. The adaptation of some European 2-row cultivars to
western U.S. conditions has been very good in recent years as well. Adaptation factors
primarily include yield, lodging resistance, and barley stripe rust resistance.
Cooperation particularly among the Pacific Northwest (Idaho, Oregon and Washington)
breeders has been occurring through annual research review meetings and germplasm

127
exchanges. Spring and winter barley germplasm exchanges also occur with several major
Canadian, Brasilian, and European breeding programs.
2. Agronomic Testing
Yield trials were conducted at 15 major nursery sites in 2001, but two nurseries were not
harvested due to drought conditions. More advanced lines were also tested in USDA
winter and spring regional nurseries for assessment of environmental adaptability. In
addition four on-farm tests of six varieties coordinated by Great Western Malting Co. was
conducted in 2001 making it the 12th year of on-farm variety testing in Washington.
Cumulative precipitation in eastern Washington in 2001 was well below average, with
planting and harvest of spring varieties about average. Yields and quality were about
average due to the favorable distribution of rainfall during the growing season in the
Palouse, but well below average in the central part of the state. In spite of a relatively
mild winter, barley stripe rust was at a relatively low level for the third year in a row
throughout the inland PNW. Winter and spring barley volunteers survived well in
Washington due to the mild winter.
3. Malt Quality Evaluations
Selections that appear to have good agronomic potential are included in replicated yield
trials with grain from these trials malted and analyzed by the USDA-ARS Cereal Crops
Research Unit at Madison and in industry laboratories.
4. Barley Quality Evaluations
Barley from lines and cultivars in replicated yield trials and other studies (i.e., fertilizer,
cultural practice, nutrition trials, etc.) as well as early generation breeding lines are often
tested for total protein, beta-glucan content, moisture, plumpness and kernel and test
weight in our laboratory.
Results
1. Spring Barley
Farmington (WA9504-94) was released in 2001 jointly with Oregon, Idaho, and the
USDA-ARS. This 2-row has displayed good agronomic and malting quality potential
(Tables 1-3), as well as partial resistance to stripe rust. Its yield is equal or greater than
that of Baronesse in higher rainfall and irrigated areas of eastern Washington. It
significantly outyields all other currently grown cultivars in Washington. Grain of
WA9504-94 from the 1999 harvest was entered into the AMBA Pilot Scale Testing
Program, but was unacceptable for further testing. However, it was submitted again in
2001. Adaptation is particularly good in the higher production zones of eastern
Washington, Oregon, and Idaho.
Another 2-row, WA8682-96 (no name yet) will be released in 2002. It has wider
adaptation than Farmington, and is expected to compete well with Baronesse over most
of eastern Washington (Tables 1and 2). Over 26 site-years, 2000-2001, WA8682-96 had
greater or equal yields compared with Baronesse at 13 of the 15 nursery sites. It has high
test weight and kernel plumpness compared to other commercial cultivars. It also has

128
partial resistance to barley stripe rust. Micromalt data indicate that WA8682-96 has
malting quality potential (Table 3). Grain of WA8682-96 from the 2000 harvest was
entered into the AMBA Pilot Scale Testing Program, but was rated unacceptable.
However, it will be submitted again in 2002.
Year
Table 1a. WA 8682-96 Relative yields (% Baronesse).
Locations
Pomeroy Fairfield Pullman Farm. R. Slope Walla2 Ritzville
1998 98
1999 118* 99 95
2000 108* 94 89* 105 107 108*
2001 84 99 91* 96 100 105
Avg. 96 99 93 101 103 105 103
Farmington # 96/99 99/91* 96/95* 104/105 100/111* 100
# - Farmington data 5yr/2 yr means
* - Significantly different from Baronesse @ p = 0.10.
Year
Table 1b. WA 8682-96 Relative yields (%Baronesse).
Locations
Dusty Reardan Almira Bick. Lamont Dayton Anatone St. John
2000 102 95 123 100 86* 105 100
2001 89 108 97 73 90 93 99
Avg. 95 100 97 123 94 87 100 100
Farmington # 95*/98 114* 92*/100
# - Farmington data 5yr/2 yr means
* - Significantly different from Baronesse @ p = 0.10.

129
Table 2. 2000-2001 Agronomic Performance of Selected Barley Varieties.
Overall (26 location/years)
Plant Ht Plump Lodg.* TW Yield Yield
Varieties in. % % lb/bu lb/a %Bar.
WA 8682-96 27 87 39 51.8 4585 98
Baronesse 25 83 14 50.9 4690 100
Farmington 23 80 11 50.5 4505 96
Harrington 27 87 15 49.6 4344 93
Bancroft 28 81 27 50.2 4337 92
Gallatin 28 81 21 51.9 4282 91
Orca 29 94 7 51.1 4067 87
Mean 28 85 20 50.9 4384 93
* Averaged over 3 location/years in which lodging occurred.
Table 3. Malting Performance of Farmington and WA 8682-96, 1998-2001*.
Variety/ Malt Protein, % DP α-amylase β-glucan
Line Extract, % Total Soluble S/T
o
ASBC
o
20 DU ppm
WA 8682-96 78.4 12.5 4.3 36 91 55 386
Farmington 78.6 12.8 4.2 35 116 41 442
Harrington 79.6 12.6 5.3 42 113 69 301
* Courtesy of the CCRU, USDA-ARS, Madison, WI.
The newer advanced 2-row lines WA8608-97, WA8601-97, WA10144-96, and WA8792-
96 have performed particularly well in agronomic terms (Table 4), and WA10144-96
appears to have good export malting quality, while the other lines appear to have little
malting quality potential (Table 5). WA8674-96 and WA7908-96 were entered into the
2001 AMBA Pilot Scale Testing Program (Table 6), but were put on hold in the program
because of overall performance until the pilot malt results are known. A major objective
in the 2-row program, as well as the 6-row program, which has been met with
considerable success, is to increase kernel size (% plump). This is particularly
advantageous in dry years, as most of Washington barley production is dryland. Another
objective is to reduce plant height and increase lodging resistance to effectively extend
adaptation to irrigated production. Incorporating barley stripe rust resistance into
breeding lines is a major objective as well.

Lines
Lines
130
Table 4. Advanced Lines Agronomic Traits 2001
State Uniform Nursery (13 locations).
Plant Ht Plump TW Yield Yield
in. % lb/bu lb/a %Bar.
WA 8608-97 26 92 50 4041 98
WA 8601-97 25 85 49 4030 98
WA 8792-96 27 90 51 3989 97
WA 10144-96 25 87 49 3955 96
Harrington 26 92 49 3943 96
Baronesse 24 86 50 4111 100
Table 5. Malting Quality of Selected 2-row Lines, 1998-2001*.
Malt Protein % DP α-amylase β-glucan
Extract % Total Soluble S/T o ASBC 20 o DU ppm
WA 8608-97 77.5 13.1 3.8 30 73 42 403
WA 8601-97 77.1 12.8 3.7 30 65 42 365
WA 10144-96 77.2 12.0 4.2 37 82 43 228
WA 8792-96 77.1 13.3 4.1 32 84 49 369
Harrington 79.1 12.7 5.0 41 122 68 228
* Courtesy of USDA-ARS, CCRU, Madison, WI
Table 6. Spring 2-Row Barley Malting Data*, 1998-2001.
Variety/ Malt Protein, % DP α-amylase β-glucan
Line Extract, % Total Soluble S/T
o
ASBC
o
20 DU ppm
WA8674-96 78.6 12.6 3.9 31.5 101 46 488
WA7908-96 78.7 13.1 5.4 42.2 134 61 529
Harrington 80.1 12.5 5.0 41.4 113 71 298
* Courtesy of the CCRU, USDA-ARS, Madison, WI.
The newest crop of 2-row and 6-row selections have also displayed improved agronomic
characteristics including competitive yield levels compared with Baronesse (Table 7) and
barley stripe rust resistance. Several of these lines appear to have good malting quality as
well (Table 8).

131
Table 7. Spring 2- and 6-Row # Barley Agronomic Data 2000, 2 Locations.
HD PH Plump TW Yield %
Variety Julian In. % lb/bu lb/a Baron.
WA 9263-98 # 182 36 94 50.5 6919 100
WA 8481-97 178 34 92 52.1 6826 98
WA 9262-98 # 181 35 91 50.3 6755 97
WA 7194-98 178 33 97 53.7 6691 96
WA 7153-98 180 33 98 53.2 6606 95
WA 10497-97 177 32 96 54.0 6592 95
Harrington 178 35 96 52.5 6474 93
Morex # 174 39 81 51.0 5288 76
Baronesse 178 33 94 53.7 6938 100
Table 8. Spring 2- and 6-Row # Barley Malting Data*, 1999-2001.
Variety/ Malt Protein, % DP α-amylase β-glucan
Line Extract, % Total Soluble S/T
o
ASBC
o
20 DU ppm
WA 9263-98 # 81.4 11.5 5.0 45.6 123 76 150
WA 8481-97 79.0 12.0 4.0 34.6 94 49 230
WA 9262-98 # 80.2 12.2 5.3 45.0 119 79 134
WA 7194-98 78.7 13.2 4.2 33.7 97 50 575
WA 10497-97 78.6 12.7 4.3 34.9 103 49 390
Harrington 79.4 12.9 4.7 38.2 111 67 306
Morex # 79.4 13.3 5.0 39.3 163 56 185
* Courtesy of the CCRU, USDA-ARS, Madison, WI.
We are in a rebuilding phase for 6-row types. Much of our material was discarded
after the 1998 season due to barley stripe rust susceptibility. Virtually all the 6-row
malting cultivars from the Midwest and West are very susceptible to barley stripe rust,
and of course these cultivars have been used heavily in crossing. Therefore, we are
trying to rebuild with rust resistance as well as quality. Still the most challenging aspect
of our 6-row program is to combine Midwestern quality with western adaptation into
selections acceptable to growers, maltsters, and brewers. And now adding stripe rust
resistance is a further challenge. Simple and complex cross combinations are made to
build the desired new and rare genotypes. There are also several 6-rows and 2-rows in a
stripe rust resistance backcross program.

132
2. Molecular Breeding
Molecular marker assisted backcross breeding projects are underway in collaboration
with A. Kleinhofs to incorporate Baronesse yield genes into Harrington to make
Harrington more agronomically fit under western dryland conditions. These molecular
breeding projects are designed to improve the competitiveness of malting barley grown in
the Pacific Northwest. Five of the Harrington/Baronesse backcross lines have been
entered into the Extension State Uniform Nursery in 2002. See Kleinhofs’ contribution to
this report. In addition, we have 12 stripe rust resistance molecular marker assisted
backcross populations in process. Field screening of four of the populations has revealed
a high proportion of resistant types.
3. Winter Barley
Winter barley production is limited in Washington primarily due to direct competition
with winter wheat-- the state's principal crop--and a lack of sufficient winterhardiness. A
major breakthrough will be required to substantially improve the winterhardiness of
barley. This has been a worldwide problem. The winter program has been deemphasized
due to low grower interest, budget constraints and greater reliance on the
Oregon State and USDA-ARS-Aberdeen programs for winter germplasm development.
Our program evaluates germplasm only.
Other Projects
1. North American Barley Genome Mapping Project (NABGMP)
The breeding program collaborates in the NABGMP and has grown the doubled haploid
mapping populations from the Steptoe/Morex, Harrington/TR306, and Harrington/Morex
crosses for agronomic and quality evaluation, quantitative trait loci (QTL) analysis and
economic trait gene mapping. A major project has provided greater understanding of the
genetics of dormancy/preharvest sprouting in barley grain. QTL analysis has revealed
two major loci on chromosome 7 and minor genes on chromosomes 1 and 4. Current
dormancy research focuses on fine structure mapping of the chromosome 7 QTLs and
single locus inheritance patterns, as well as, mapping dormancy QTLs in a Triumph
/Morex cross through collaboration with I. Romagosa in Spain. QTL validation, fine
mapping, and QTL marker assisted selection experiments have been completed or are in
progress for dormancy, major malting quality traits, and yield on chromosomes 1 and 4.
Efforts are underway to develop and incorporate molecular marker assisted selection
(MMAS) procedures into the breeding program to apply information from NABGMP and
improve breeding efficiency. Collaboration with A. Kleinhofs, P.M. Hayes and D.
Wesenberg has been instrumental. Funding has been through NABGMP and USDA-
CSREES-NRICGP grant programs.

133
2. Direct Seeding Cropping Systems
In collaboration with R.J. Cook, and others, several research projects are underway to
evaluate barley adaptation to direct-seeded production in dryland cropping systems.
Specific projects address barley variety soil borne root pathogen reactions, variety
performance under direct-seeded vs conventional tillage in continuous cropping systems
in intermediate and low rainfall areas of eastern Washington, and barley as a rotation
crop in continuous small grains cropping systems in low rainfall areas. Thus far,
considerable differences in barley variety reaction to high soil borne disease conditions
have been demonstrated with some genotypes showing some resistance or tolerance.
Also thus far, barley yields have been less under direct seeding compared with
conventional seeding, but little genotype x variety interaction for yield has been
indicated.
3. Proanthocyanidin-free Barley
In collaboration with D. von Wettstein, proanthocyanidin-free barley lines are being
developed to eliminate the chill haze problems in beer and possibly enhance the
nutritional value of barley. Sodium azide mutagenesis has provided new
proanthocyanidin-free mutants for biochemical, agronomic, disease, and malting quality
evaluations. The mutants are used as parents in crosses with high quality genotypes.
Several newer 6- and 2-row lines appear to have considerably improved agronomic and
quality characteristics especially the recently selected pigmented proanthocyanidin-free
(PANT) mutants. Two lines have been approved for prerelease seed increase in 2002. An
additional project involves the incorporation of a bacterial engineered heat stable, betaglucanase
gene into proanthocyanidin-free barley via transformation. Feeding as well as
malting evaluations are underway with advanced lines.
4. Hulless/Waxy Barley
Crosses between waxy and hulless and standard varieties have been made. A
collaborative effort with I. Romagosa provides doubled haploid breeding lines of waxy
hulless types to the WSU program. We continue to evaluate progeny for improved
food/feed and industrial use type barleys. Our first hulless barley, Bear, was released in
1997. Feed quality is excellent compared with covered cultivars and the hulless cultivars,
Condor and Falcon. New collaboration is evolving with B.-K. Baik in the WSU Food
Science and Human Nutrition Department for the evaluation and development of barley
food products. A first effort is looking at genotype effects on color and discoloration of
raw and cooked barley.
5. Forage Barley
Work has been ongoing for several years to replace the standard forage or hay barley,
Belford, which has been around since the 1940’s. The new hooded six-row barley
Washford was released in 1997. Washford has out-yielded Belford by 15% in hay dry
weight and 22% in grain, and it has greater lodging resistance with about the same quality
profile. New lines are being developed to further improve upon Washford.

134
6. Breeding for Pest Resistance
Increased efforts are underway to breed for resistance to relatively new or potential pest
problems in the Pacific Northwest. The priority order of effort is barley stripe rust
(collaborating with X. Chen and D. Wesenberg), soil borne root diseases (collaborating
with R.J.Cook), Hessian fly (collaborating with R. Ratcliffe), and Russian wheat aphid
(collaborating with D. Mornhinweg). Breeding lines resistant to these pests are emerging
in the breeding program.
Future Directions
Future directions of the barley improvement program at WSU include the development of
improved germplasm both at the pre- and post-cultivar release stages. Barley malting,
brewing, and nutritional quality will continue to be emphasized along with yield and
adaptation. New selection procedures will be employed to improve the efficiency,
effectiveness, and acceleration of the cultivar development program. The barley genome
mapping project will contribute to breeding efficiency and effectiveness through
improved genetic knowledge of economically important traits and molecular markerassisted
selection procedures. This is a particular emphasis currently. A gradual
movement toward transformation of barley as techniques become available is ongoing
with cooperative studies. Associated basic genetic and biochemical characterization
studies of malting and nutritional quality traits will continue.
WSU Barley Program Personnel
S. E. Ullrich, Professor and Scientist, Project Leader.
A. Kleinhofs, Professor/Scientist, Collaborator, molecular genetics and breeding, WSU.
D. von Wettstein, R.A. Nilan Distinguished Barley Professor, Collaborator,
proanthocyanidin free barley, transformation, WSU.
V. A. Jitkov, Breeding Field Research Associate, in charge of field/greenhouse/seedhouse
operations.
J. A. Clancy, Research Technologist III, in charge of the barley quality and molecular
genetics lab with partial support from AMBA.
M.C. Dugger, Research Technologist I, breeding field/greenhouse/seedhouse assistant .
C. Kruger, M.S. Graduate Research Assistant working on evaluation of barley traits
associated with direct-drilled cropping systems.
W. Hotchkin, M.S. Graduate Research Assistant.
ADDITIONAL COOPERATIVE EFFORTS OCCUR WITH THE FOLLOWING SCIENTISTS:
X. Chen - barley stripe rust, USDA-ARS, WSU.
R.J. Cook - root diseases, WSU.
B.-K. Baik – barley food quality, WSU.

135
D.M. Wesenberg – spring and winter regional nurseries, barley stripe rust, QTL
validation, germplasm exchange, USDA-ARS, Aberdeen, ID.
P.M. Hayes - QTL validation, germplasm exchange, OSU, Corvallis, OR.
B.L. Jones - malting quality analyses, USDA-ARS, Madison,WI.
D.W. Mornhinweg - Russian wheat aphid, USDA-ARS, Stillwater, OK.
R.H. Ratcliffe – Hessian fly, USDA-ARS, West Lafayette, IN.
I. Romagosa- genetic studies of barley adaptation and quality and breeding hulless barley,
University of Lleida, Lleida, Spain.
G. Arias- breeding and malting quality, EMBRAPA, Passo Fundo, R.S., Brazil.
SELECTED 2001 PUBLICATIONS:
Horvath, H., L.G. Jensen, O.T. Wong, E. Kohl, S.E. Ullrich, J. Cochran, C.G.
Kannangara, and D. von Wettstein. 2001. Stability of transgene expression, field
performance and recombination breeding of transformed barley lines. Theor. Appl.
Genet. 102:1-11.
Wesenberg, D.M., D.E. Burrup, W.M. Brown, Jr., V.R. Velasco, J.P. Hill, J.C.
Whitmore, R.S. Karow, P.M. Hayes, S.E. Ullrich, and C.T. Liu. 2001. Registration of
‘Bancroft’ barley. Crop Sci. 41:265-266.
Marquez-Cedillo, L.A., P.M. Hayes, A. Kleinhofs, W.G. Legge, B.G. Rossnagel, K. Sato,
S.E. Ullrich, D.M. Wesenberg, and the NABGMP. 2001. QTL analysis of agronomic
traits in barley based on the doubled haploid progeny of two elite North American
varieties representing different germplasm groups. Theor. Appl. Genet. 103:625-637.
Ullrich, S.E., C.E. Muir, and J.A. Froseth. 2001. Registration of ‘Bear’ barley. Crop Sci.
41:1994-1995.
Ullrich, S.E., C.E. Muir, M.L. Nelson, and J.A. Froseth. 2001. Registration of
‘Washford’ barley. Crop Sci. 41:1995.
Ullrich, S.E., J.W. Burns, P.E. Reisenauer, V.A. Jitkov, D. von Wettstein, and A.
Kleinhofs. 2001. Spring barley performance in 2000. Wheat Life 44(1):46-48.
Ullrich, S.E. and M. Palmer Sullivan. 2001. WSU releases Farmington, a new two-row
spring barley variety. Wheat Life 44(5):20.
Dofing, S.M., S.S. Jones, S.E. Ullrich, K.K. Kidwell, K.G. Campbell, and D. Boze. 2001.
2000 cereal variety evaluation results. Dept. of Crop and Soil Sci. Tech. <strong>Report</strong> 01-1.
WSU. 120 pp.
Ullrich, S. E., V.A. Jitkov, J. A. Clancy, M.C. Dugger, and C.I. Kruger. 2001. Barley
improvement research. pp. 33-36. In J. Burns and R. Veseth (ed.). 2001 field day
proceedings: Highlights of research progress. Dept. of Crop and Soil Sci. Tech <strong>Report</strong>
01-4. WSU.
Cook, R.J., S.E. Ullrich, W.F. Schillinger. 2001. Performance of advanced lines and
varieties of spring barley seeded directly into wheat or barley stubble. pp. 87-88. In J.

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Burns and R. Veseth (ed.) 2001 field day proceedings: Highlights of research progress.
Dept. of Crop and Soil Sci. Tech. <strong>Report</strong> 01-4. WSU.
Ullrich, S.E. 2001 Barley production and improvement in Washington State. Barley
Newslttr. 44: http://wheat.pw.usda.gov/ggpages/BarleyNewsletter/44/Wash<strong>Report</strong>1.html.
Schmierer, D., N. Kandemir, D. Kudrna, D. Wesenberg, S. Ullrich, and A. Kleinhofs.
2001. Molecular marker-assisted selection for increased yield of traditional malting
barley cultivars. Barley Genetic Newslttr. 31:6-11.
http://wheat.pw.usda.gov/ggpages/bgn/31/schmierer.htm.
Ullrich, S.E., D. von Wettstein, J.W. Burns, and A.Kleinhofs. 2001. Spring barley
performance in 2001. Wheat Life 44 (11): 50-54.
Schmierer, D.*, N. Kandemir, D. Kudrna, D. Wesenberg, S.E. Ullrich, and A. Kleinhofs.
2001. Molecular marker-assisted selection for improved yield in traditional malting
barley cultivars. Plant & Animal Genome IX International Conference (Jan. 13-17, San
Diego). Final Abstracts Guide, p 163. Poster presentation.
Kruger, C.I.*, R.J. Cook, W. Schillinger, V.A. Jitkov, R. Sloot, and S.E. Ullrich. 2001.
Emergence characteristics of barley genotypes in a controlled environment with siolborne
disease pressure. ASA-CSSA-SSSA <strong>Annual</strong> Meetings Abstracts CD-ROM. Oral
Paper presented at the 2001 Western Society of Crop Science <strong>Annual</strong> Meetings, Tucson,
AZ, 6/11-13/01.
Hotchkin, W. S.*, S.C. Spaeth, V.A. Jikov, and S.E. Ullrich. 2001. Relationship between
embryo morphology and stand establishment in hulless barley: breeding implications.
ASA-CSSA-SSSA <strong>Annual</strong> Meetings Abstracts CD-ROM. Oral Paper presented at the
2001 Western Society of Crop Science <strong>Annual</strong> Meetings, Tucson, AZ, 6/11-13/01.
Kruger, C.I.*, R.J. Cook, W. Schillinger, V. Jitkov, R. Sloot, and S.E. Ullrich. 2001.
Stand establishment and performance characteristics of direct seeded barley genotypes.
2001 ASA-CSSA-SSSA <strong>Annual</strong> Meeting Abstracts CD-ROM.
http://www.annualmeeting2001.com/Abstracts/c01-kruger143324-P.PDF. Poster
presented at the 2001 ASA-CSSA-SSSA <strong>Annual</strong> Meetings, Charlotte, NC, 10/21-25/01.
Voltas, J*., I. Romagosa, S.E. Ullrich, Van Eeuwijk, F.A. 2001. Identification of adaptive
patterns in the ‘Steptoe’ X ‘Morex’ barley mapping population integrating genetic,
phenotypic, and environmental information. 7 th International Quantitative Trait Locus
Mapping and Marker-Assisted Selection Workshop. Valencia, Spain, 10/19-20/01.
available at http://www.ivia.es/qtlmas/listados/modificar.php3?indice=26.

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A STUDY OF THE MALTING QUALITY OF NEW BARLEY SELECTIONS
Allen D. Budde, Berne L. Jones, Eddie Goplin and David M. Peterson
Cereal Crops Research Unit, USDA, ARS and
Department of Agronomy, University of Wisconsin - Madison
Objectives. This project is an on going research collaboration with several state and
federal malting barley breeding projects. Breeders' selections are micro-malted, analyzed
and evaluated for malting quality. We can thereby provide accurate data and quality
evaluations of new selections and thus facilitate the development of improved malting
barleys. We also have carried out analyses that involve measuring the malting quality of
experimental lines. These will lead to a better understanding of the biochemistry behind
malting quality and/or will allow breeders to more effectively produce barley lines that
have improved malting quality.
Materials and methods. The methods used for the physical and chemical analyses of
micro-malts are, for the most part, ASBC procedures. Some automated versions of the
standard methods are used, in which case they have been standardized against the
recommended procedures.
Micro-malts are prepared with 170 g (db) or, when seed was limited, with 120 g samples.
The samples are steeped to 45% moisture at 16ºC, germinated for 5 days at 17ºC, with
turning for 3 min each half hour, and kilned over a period of 24 hours to a final
temperature of 85ºC. These conditions are used to obtain malts having quality
characteristics similar to those prepared commercially.
All analytical data and evaluations of the micro-malts are summarized in individual
reports that are returned to the breeders who had submitted the samples. At the end of the
analysis year, the results are compiled into four annual regional reports that are posted at
http://www.dfrc.ars.usda.gov/ccru/cc00001.html on our website. Data from the pilot
scale analyses are submitted to AMBA for collaborative evaluation.
Results.
Barley samples brewed. We have discontinued the pilot brewing of lines that showed
commercial potential, but are maintaining our brewing equipment in operating condition
and sometimes use it for experimental studies.
Malting and analyzing research samples. During the 2000 crop year, we malted and
analyzed the quality of 993 samples (listed above, under ‘experimental’) for various
USDA and non-USDA barley researchers, who needed malting quality information to
carry out their research projects. These analyses were carried out in addition to analyzing
samples that were submitted by breeders as part of their yearly breeding line quotas.
Some of the research projects for which we analyzed samples were: 1) barleys that
received various rates of nitrogen application; 2) lines that were developed and used to
map the locations of QTLs on chromosomes; 3) proanthocyanidin-free barley lines; and
4) various projects being carried out at the CCRU.

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Barley samples malted and subjected to malting quality analysis.
2000 Crop Year Summary
4,674 samples were received for analysis:
Breeders’ line micro-malts (170 g) 2,673
Breeders’ line micro-malts (120 g) 340
Western Spring Nursery 102
Mississippi Valley Nursery 130
AMBA Pilot malting samples 22
Breeders’ experimental studies 993
‘In House’ experimental studies 414
Total 4,674
2001 Crop Year Summary
3,523 samples have been received (as of 2-20-02) for analysis:
Breeders’ line micro-malts (170 g) 2,611
Breeders’ line micro-malts (120 g) 142
Western Spring Nursery 100
Mississippi Valley Nursery 116
AMBA Pilot malting samples 22
Malts for experimental studies 382
Malts for ‘in house’ experiments 150
Total 3,523
New equipment. During the year, we replaced our LECO 428 Nitrogen Analyzer with
an updated LECO 528 Analyzer. The analytical principles used by the instruments are
identical and the results from the two are interchangeable. We also purchased and have
received three new steep tanks this year. These will be installed and tested as soon as the
current crop year samples have all been analyzed.
Studies with the Joe White Malting Systems
Christopher Martens, Allen Budde and Berne Jones
Cereal Crops Research Unit, USDA, ARS
Objectives. Our Joe White (JW) Micromalting systems have been optimized to produce
malts that have quality parameters that are similar to those from malts prepared with our
traditional malting system and comparable to commercial malts. The use of the JW
micromalters has increased our annual malting capacity by 1,500 samples. During this
year, we examined how changes in some steps of the malting process affected the malt
quality. In addition, we started using the JW machines to carry out the regular
micromalting of ‘experimental’ barleys from collaborating barley researchers.

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Methods.
Our malting schedule. We use the following protocol with the JW Micromalting
systems:
Steep: Air rest airflow rate of 30%, with 0% recirculation. All stages at 19ºC. 7
hours (h) wet, 8h air, 4h wet, 5h air, 2h wet, 2h air, and 2h wet (30h total).
Germination: Airflow rate of 30%, with 0% recirculation. 4 turns every 2h with a 30º
trim angle. 48h 18ºC, 24h 17ºC, and 24h 16ºC.
Kiln: All stages 40% airflow. 0% recirculation for stages 1-3, 50% for stage 4, and
75% for stage 5. 10h 49ºC, 4h 54ºC, 3h 60ºC, 2h 68ºC, and 3h 85ºC, with a 30 min. ramp
between stages.
Results.
High soluble protein levels. This schedule produced malt that was consistent with that
from our traditional system. However, the results of the 2001 AMBA Collaborative study
revealed that the malts produced in both our JW and traditional systems had elevated wort
soluble protein levels, compared to those from the other collaborators. We are, therefore,
still conducting research that is aimed at reducing the soluble protein levels in our
micromalts.
The Effect of air recirculation during germination. Researchers at the JW Maltings
had indicated that using less recirculated air during the steep air rests and germination
would decrease the grain modification rate. We have integrated this concept into our
malting schedule, but we needed to confirm this effect experimentally.
Multiple samples of each of four malting barley varieties (Harrington, Morex, Conlon, and
Robust) were steeped in one of the JW micromalting machines. Half of the samples were
then germinated in the same machine with 100% air recirculation, while the other half
were transferred to a second JW instrument and germinated with 0% recirculated air. All
of the samples were kilned with our normal 24-hour method. The quality parameters of
the finished malts are compared in Table 1.

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Table 1. A comparison of the malting quality of barley samples germinated with 0% or
100% air recirculation.
Fine
Wort
Variety or Percent Moisture Extract Wort Wort Protein S/T DP
AlphaBetaamylaseglucan Quality
Selection recirc'n adjusted (%) Color Clarity (%) (%) (ASBC) (20ºDU) (ppm) Score
Conlon 0 OS,d1,d2 81.6 1.8 1 5.37 49.1 88 71.9 93 39
Conlon 100 OS,d1,d2 81.7 1.6 1 5.29 47.5 97 76.1 86 43
Robust 0 OS,d1,d2 78.7 2 1 6.17 47.3 147 57.1 196 27
Robust 100 OS,d1,d2 78.6 1.9 1 6.17 47.3 155 56 195 32
Harrington 0 OS,d1,d2 81.6 1.4 1 5.22 47.1 87 66.6 94 32
Harrington 100 OS,d1,d2 81.4 1.3 1 5.16 45 97 68.1 88 41
Morex 0 OS,d1,d2 80.5 2.4 1 6.15 53.1 104 66.6 109 36
Morex 100 OS,d1,d2 80.2 1.9 1 6.23 54.2 131 73 83 40
Only the Diastatic Power values varied when the air flow was altered. The levels
increased markedly when the air flow was raised to 100%. Varying the air had no effect
on the moisture retention of the barley samples. Other than the DP values, the amount
of air recirculation during germination had little effect on the finished malt quality.
The effect of moisture levels during germination. The quality of malts prepared in
the JW micromalting units is affected by moisture fluctuations, and especially by
desiccation, during malting. It is thus important to monitor the moisture levels of the
barley during germination and to understand how moisture changes that occur
throughout the malting process affect the quality of the finished malt. An investigation
of how the out-of-steep (O-S) and germination moisture levels affected the malt quality
has been carried out. The moisture levels of Harrington barley samples were adjusted at
O-S, and every 24 hours afterwards, until germination was completed. The quality
values of the resulting malts are displayed in Table 2.
Table 2. The effect of germination moisture levels on Harrington malt quality
parameters.
Wort
Protein
Alphaamylase
Betaglucan
(ppm) FAN
Fine Coarse
S/T DP
Moisture Extract Extract F-C (%) (%) (ASBC) (20ºDU)
43% 81.0 79.0 2.0 5.37 48.7 89 63 296 218
44% 81.3 79.1 2.2 5.47 50.2 84 68 314 215
45% 81.2 79.4 1.8 5.47 49.0 85 64 256 212
46% 81.3 79.8 1.5 5.66 50.7 98 72 108 222
Statistical tests revealed that the Fine Grind Extract and Wort Clarity parameters were the
only ones that were not affected by the germination moisture level. All of the other grain

141
modification measures indicated that the barley was better modified when the higher
moisture levels were used.
The effect of altering the moisture levels at O-S or during germination was also studied.
The most important finding from this study was that, although water sprinkling during
germination improved malt quality, some of the quality parameters were irreversibly
affected by the O-S moisture level. The Fine and Coarse Grind Extracts, F-C, and
Diastatic Power levels of malts that were steeped and germinated at a consistent 43%
moisture did not differ from those of samples that were steeped at 43% moisture and then
hydrated to 47% at any time during germination. Conversely, the alpha-amylase values
were raised and β-Glucan levels lowered by increased hydration during germination. The
protein modification measures - wort protein, S/T, and FAN - increased when the barley
was sprinkled to increase its moisture level during germination.
The analysis of breeders’ samples. During 2001, 624 experimental barley samples were
received from collaborating plant breeders and these were processed, micromalted, and
analyzed for quality. Four hundred and forty samples have been received for testing in
the JW micromalters this year. The barley parameters of these samples have been
ascertained and their micromalting and quality analyses are underway.
Publications.
Hang, A., Burton, C.S., Hoffman, D.L. and Jones, B.L. Random amplified polymorphic
primer-generated embryo DNA polymorphisms among 16 North American malting
barley cultivars. J. Am. Soc Brew. Chem., 58: 147-151, 2000.
Hayes, P.M., Castro, A., Corey, A., Marquez-Cedillo, Jones, B.L., Mather, D., Matus, I.,
Rossi, C., and Sato, K. A Summary of Published barley QTL <strong>Report</strong>s.
http://www.css.orst.edu/barley/nabgmp/qtlsum.htm. 2000.
Budde, A.D. and Jones, B.L. Assessing differences in malting quality parameters when
malts are extracted under ‘standard ASBC’ and ‘high gravity’ conditions. ASBC
Newsletter 61: PB25. 2001 and Brewers Digest 76 (3); 40, 2001. (Abstact).
Budde, A.D., Jones, B.L., Goplin, E.D., Peterson, D.M. and Staff. Mississippi Valley
Uniform Regional Nursery - 2000 Preliminary Quality <strong>Report</strong>. 2000. (Technical report)
Budde, A.D., Jones, B.L., Goplin, E.D., Peterson, D.M. and Staff. Western Regional
Spring Barley Nursery - 2000 Preliminary Quality <strong>Report</strong>. 2000. (Technical report)
Budde, A.D., Jones, B.L., Goplin, E.D., Peterson, D.M. and Staff. Malting Quality of
Barley Varieties and Selections Grown at the Mississippi Valley Nursery and at Central
and Eastern Stations in 2000. 2000. (Technical report)
Budde, A.D., Jones, B.L., Goplin, E.D., Peterson, D.M. and Staff. Malting Quality of
Barley Varieties and Selections Grown in Rocky Mountain and Western Stations in 2000.
2000. (Technical report)

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Personnel
Dr. B.L. Jones, Supervisory Research Chemist Mr. A.D. Budde, Plant Physiologist
Mr. M.E. Marinac, Physical Science Tech. Ms. L.M. Short, Laboratory Tech.*
Mr. C.H. Martens, Biological Science Tech. Mr. E.D. Goplin, Laboratory Tech.**
Mr. L.E. Hornbacher, Physical Science Tech. Dr. D.M. Peterson, Research Leader
Ms. D.K. Schaefer, Secretary
*AMBA supported
** Partially AMBA supported

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CHARACTERIZATION OF THE PROTEIN HYDROLYZING SYSTEMS OF
MASHES AND MALTS
Berne L. Jones, Debora Fontanini and Laurie Marinac.
Cereal Crops Research Unit, USDA, ARService and
Department of Agronomy, University of Wisconsin, Madison, WI
Overall Objectives
To further define the biology and biochemistry of the protein hydrolysis processes that
occur during the malting and mashing operations of brewing. The hydrolysis of proteins
into metabolizable low-molecular-weight nitrogenous compounds is one of the most
important aspects of malting and brewing, yet there are still many aspects of this process
that are not known. This is an ongoing study to determine which proteolytic enzymes are
involved in these processes, what barley and malt endogenous inhibitors can repress the
activities of these proteinases, and which of the proteinases and inhibitors really affect the
release of ‘soluble protein’ during malting and mashing. Knowing these things will make
it possible to more scientifically and efficiently design malting barleys that are even
better suited for their particular end uses.
Purification and characterization of three cysteine proteases from green malt
Laurie Marinac and Berne L. Jones
Objective
The objective of this study is to purify and characterize some of the green malt cysteine
class proteases that have not been investigated previously. The enzymes being studied
have gelatinase activities that are stable throughout kilning and they maintain their
activities during mashing until they are inactivated during the ‘conversion’ step, when the
temperature is increased to 72°C. One of these enzymes hydrolyzes the substrate protein
gelatin in vitro at a very high rate during incubation at 40 o C. The others digest gelatin at
about the same rate as the two cysteine proteases that we have studied previously; the 30
and 31 kiloDalton proteases that were purified and characterized by Poulle (Plant
Physiol., 88: 1454_1460, 1988) and Zhang (Planta 199:565-572, 1996) respectively. All
three of the enzymes being studied, however, digest significant amounts of gelatin within
30 min of incubation at 40 o C after separation with a 2-D, IEF x PAGE analysis method,
and thus are some of the most active ones that occur in green malt extracts. The cysteine
proteases of barley are apparently the main enzymes that are responsible for breaking
down the insoluble storage proteins into low molecular weight compounds during
malting. The characteristics of the enzymes that are now being studied differ
significantly from those studied previously, and understanding the roles of the various
proteases is fundamental to understanding how the protein hydrolyzing system in barley
functions. This, in turn, is critical for gaining a more complete understanding of what
happens to the barley proteins during malting and mashing and how this affects brewing.

144
Selected Methods
Extracts of cysteine proteases prepared from Morex green malt were partially purified
using cation exchange and chromatofocusing chromatographies. The proteinase that
most actively hydrolyzed gelatin had a pI of about 4.5 and its molecular mass was
approximately 35 kDa. It has been purified to a single gelatinase activity, but the
preparation still contains some non-proteinase contaminants. It has not been possible to
remove all of these contaminants using size exclusion HPLC, so other purification
methods are now being tested.
The other two cysteine proteases that are being purified have higher isoelectric points
than that of the very active enzyme or those of the two previously purified proteinases, all
of which had pI values that fell between 3.5 and 4.5. These two, which migrate similarly
on IEF x PAGE gels, have pI values that are near 6. They thus have the highest
isoelectric points of any of the cysteine gelatinase activities that can be detected using the
2-D analysis system. Their molecular masses have not yet been determined, as we have
only recently begun to study them.
Discussion
The study of these proteases will add significantly to the information we have about the
roles of the different green malt cysteine proteases, since they are quite different from
those that have been studied previously. These enzymes may play especially significant
roles during mashing, since they retain their activities a little longer than the 30 and 31
kDa enzymes when the mash temperatures are being raised to carry out the ‘conversion’
step. This is especially true of the ‘most active’ proteinase form. When these three
proteinases are sufficiently purified, amino acid composition and sequence data will be
collected and the biochemistry behind their abilities to degrade storage proteins will be
defined.
Purification of a green malt serine endopeptidase
Debora Fontanini and Berne L. Jones
Objectives
In our continuing effort to define the barley seed proteolytic system and to ascertain the
roles that various proteolytic activities play in the germination of barley seeds and in
malting/mashing, we had previously isolated and studied a group of metallopeptidases
(MPs; Fontanini and Jones, 2001). These green malt enzymes digested some of the
barley storage proteins in vitro. In vivo, however, the MP activities were not present in
the barley starchy endosperm (SE) tissue, where most of the hordein storage proteins are
localized. Because the amount of soluble protein released in mashes that were carried out
in o-phenanthroline, a metallopeptidase inhibitor, decreased significantly (Jones and
Budde, 1999), metallopeptidases are apparently involved in degrading proteins during
mashing. Because the barley seed is thoroughly crushed before mashing, its tissue
compartmentalization is largely lost and the hordeins of the SE are exposed to hydrolysis
by enzymes, such as the metallopeptidases that we have isolated, that in the whole seed
were localized in the different tissues. Under these circumstances, the MPs are very
likely involved in the hydrolysis of storage proteins that occurs during mashing.

145
Purification of a serine endopeptidase from green malt
An endopeptidase (SEP-1) was purified from green malt using a series of
chromatographic separations. Some of the biochemical properties of SEP-1 have been
characterized, using either gelatin or a synthetic peptide substrate (N-succinyl Ala-Ala-
Pro-Leu p-nitroanilide [AAPLpNA]). The enzyme had a molecular weight of 70 kD, as
estimated both on size-exclusion chromatography and SDS-PAGE. The hydrolysis of the
synthetic peptide was optimal at pH 6.5 and 50 °C, with a Km of 2.6 mM. Inhibition
studies indicated that the enzyme belonged to the class of serine endopeptidases. Amino
acid sequencing of the SEP-1 polypeptide showed that its structure was similar to those
of a group of enzymes that have been recently identified in plants, the cucumisin-like
peptidases. The cucumisins are a subfamily of the subtilases, which are enzymes that are
found in bacteria, yeasts and mammals. The roles of these enzymes in plants have not yet
been elucidated.
By studying the localization of SEP-1 in the various tissues of resting barley seeds and in
seeds that were malted for several days, we discovered that the enzymes was present in
small amounts in the scutellum-embryo tissue fractions that were excised from dry seeds.
This scutellar activity increased considerably during the course of malting, and the
enzyme started showing up in other seed tissues. In particular, in green malt it was
present in the highest amounts in the rootlets and shoots, in low amounts in aleurone and
it was completely absent from the SE. Its absence from the SE, where the storage protein
are deposited, implies that the enzyme probably cannot digest storage proteins. Our own
in vitro experiments, in which purified SEP-1 was incubated with storage protein
extracts, also indicated that the enzyme can not hydrolyze hordein proteins. However,
even if SEP-1 is not directly involved in the solubilization of storage proteins, it could
still indirectly affect this process by carrying out the limited hydrolysis of specific seed
proteins. Such hydrolyses could lead to: 1) the activation of other proteolytic enzymes
that, in turn, can directly hydrolyze hordeins, or 2) a limited processing of the storage
proteins that could render them more susceptible to proteolysis by the other malt
proteinases whose job it is to carry out the majority of the hydrolysis of the storage
proteins.
Publications
Jones, B. L. and Marinac, L. A. Purification and Partial Characterization of a Second
Cysteine Proteinase Inhibitor from Ungerminated Barley. J. Agric. Food Chem., 48 (2),
257 -264, 2000.
Fontanini, D., and Jones, B.L. A study of metallopeptidase isozymes from malted barley
(Hordeum vulgare cv. Morex). J. Agric. Food Chem., 49, 4903 - 4911, 2001
Jones, B.L., and Marinac, L. The effect of mashing on malt endoproteolytic activities.
J. Agric. Food Chem., 50, 858-864, 2002.
Jones, B. L. Interactions of malt and barley (Hordeum vulgare L.) Endoproteinases with
their endogenous inhibitors. J. Agric. Food Chem., 49, 5975-5981, 2001.

146
Mikola, M., Brinck, O., and Jones, B.L., Characterization of oat endoproteinases that
hydrolyze oat avenins. Cereal Chemistry, 78: 55-58, 2001.
Personnel
Dr. B.L. Jones, Supervisory Research Chemist
Ms. L.A. Marinac, Plant Biologist
Ms. D. Fontanini, Graduate Student

147
STUDIES ON THE PROTEINASES THAT ARE PRODUCED WHEN FUSARIUM
GROWS ON CEREAL PROTEINS AND BARLEY PROTEINS THAT INHIBIT THEM
Anja I. Pekkarinen and Berne L. Jones
Cereal Crops Research Unit, USDA, ARS, Department of Agronomy,
University of Wisconsin, Madison, and VTT Biotechnology, Espoo, Finland
Objectives
The protein matrix of the wheat grain endosperm is degraded when the kernels are
attacked by the disease Fusarium head blight (FHB) or ‘scab’. It seems likely that the
same phenomenon also occurs in heavily infected barley kernels and that it may be
related to the method whereby Fusarium attacks the barley plant. We have shown that
two alkaline serine proteinases are synthesized by F. culmorum when it is cultured on
cereal storage proteins, and it seems likely that these enzymes are the ones that degrade
the proteins of infected barley grains. The proteinases are not produced when the
Fusarium is grown in media that lack protein. If, as seems probable, one function of these
proteinases is to help the fungus attack the barley head, then it seemed possible that the
grain might have evolved to produce compounds that could inactivate these enzymes and
thus retard its infection by the Fusarium. This study was carried out to purify and
characterize the Fusarium proteinases, to use the purified enzymes to test for the presence
of inhibitors in the barley and to determine whether it might be possible to manipulate the
inhibitors to increase the resistance of barleys to Fusarium attack.
<strong>Progress</strong> in studies on Fusarium culmorum endoproteinases
In the 2001 AMBA <strong>Annual</strong> Research <strong>Progress</strong> <strong>Report</strong>, we described how two alkaline F.
culmorum serine proteinases were purified from a culture medium and characterized.
These enzymes were denominated proteinase C (chymotrypsin-like) and proteinase T
(trypsin-like). At that time, we already had analyzed the N-terminal amino acid sequence
of the proteinase T, but our attempts to sequence the N-terminal amino acids of the other
protein had failed because the N-terminus of the enzyme was blocked and couldn’t be
sequenced by the Edman degradation. To get around this problem, the purified enzyme
has been digested into small peptides with a modified trypsin. These peptides were
separated and their amino acids were sequenced at the Protein Chemistry Laboratory in
the University of Texas Medical Branch (UTMB), Galveston, TX. The amino acids of the
peptides were highly homologous with those of several fungal subtilisins, but no
completely identical matches were found among the proteins that are listed in the various
amino acid sequence databases, indicating that it was a novel enzyme. Hence, it was
renamed proteinase S (subtilisin-like). In addition, we demonstrated that both proteinase
S and T hydrolyzed some of the barley storage proteins, the C- and D hordeins, in vitro.
The description of the purification and characterization of the proteinase S has recently
been published in the European Journal of Biochemistry (2002, Vol. 269, p. 798-807). A
manuscript covering the characterization of the second enzyme, proteinase T, has been
submitted to the Journal of Agricultural and Food Chemistry.
Recently, we discovered that the F. culmorum culture medium contained a third
proteinase enzyme. Preliminary studies have indicated that it is very different from the

148
two serine proteinases that we had characterized earlier. Unlike the proteinases S and T,
this enzyme was not inhibited by a barley extract when tested using the experimental
conditions that readily detected the S and T enzyme inhibitions. This was interesting,
because it indicates that if this enzyme is produced during FHB-infection, the barley may
not be able to stop it from functioning. However, we have not yet determined whether
this new enzyme is actually produced by the fungus in infected barley grain. The
properties of the enzyme will be studied in the near future, if time allows.
Fusarium proteinases are produced in FHB-infested barley. Polyclonal antibodies against
the purified S and T serine proteinases were produced in rabbits at the University of
Wisconsin Medical School Animal Care Unit. These antibodies have been used to show
that the proteinases do occur inside Fusarium infected grain (as opposed to only being
present in the Fusarium culture medium, from which they were purified). Both antibodies
specifically recognized their respective S and T antigens in extracts that were prepared
from artificially inoculated, field grown, barley grain. The tested grain samples were
obtained from the VTT Microbiology group in Finland. The enzymes were detected only
in material that was heavily Fusarium infested, implying that the fungus produced them
only after it was well established within the kernel. On the other hand, this may simply
indicate that the sensitivity of the method that we used was not adequate for recognizing
very small amounts of the proteinases. To determine exactly where the proteinases are
produced within the infected grains, the antibodies are now being used to carry out
immunomicroscopic studies. This study is being carried out in collaboration with Dr
Salla Marttila, a researcher at the Swedish University of Agricultural Sciences in Alnarp,
Sweden. Preliminary immunolocalization experiments have shown that the proteinase T
was produced during all of the infection stages when Fusarium was applied to barley
plants that were grown in a glass house. Similar studies using the proteinase S antibodies
have not yet been carried out. A manuscript reporting the results of studies that we have
carried out with infected grain from the field experiment is now being prepared.
Inhibitors of the Fusarium proteinases
Previously, we reported how we had purified five barley proteins that inhibited the
activity of proteinase S. These were purified by size-exclusion and ion exchange
chromatographies and reverse phase-HPLC. By improving these purification methods we
have been able to isolate several other proteins that also inhibited this enzyme. We have
also purified a single protein that inhibited the proteinase T. The N-terminal amino acid
sequences of the various purified inhibitor samples were analyzed at the UTMB. The
proteinase S inhibitors were the barley α-amylase/subtilisin inhibitor (BASI) and the
chymotrypsin/subtilisin inhibitors (CI) 1A, 1B and 2A. Some of the inhibitors were CIs
that contained all of their amino acids and some were the same molecules from which
various numbers of amino acid residues had been removed. The proteinase T inhibitor
was a protein called the Bowman-Birk inhibitor (BBI). All of these inhibitors have been
described previously in the literature, but it was not known that they affected any of the
Fusarium proteinases. Previous studies of the kinetic properties of the CI inhibitors by
other reseachers had shown that they were slow-binding inhibitors that form a tight
complex with bovine chymotrypsin or bacterial subtilisin. We have now shown that they
use a similar mechanism to inhibit the Fusarium proteinase S. The interaction of the BBI

149
with the proteinase T will be studied shortly. We are producing antibodies against the
BBI and CI-2A proteins in rabbits so that we can use immunomicroscopic methods to
study their interactions with the Fusarium proteinases during the FHB-infection process.
Summary
We have shown that two alkaline serine proteinases, called S and T, are produced in
Fusarium-infested barley grain and that both are able to hydrolyze the storage proteins of
barley grains in vitro. Hence, these enzymes apparently participate in the degradation of
the barley endosperm protein matrix after the Fusarium attacks the barley grain. We have
also demonstrated that barley contains several small proteins that can bind tightly to these
enzymes and, by doing so, prevent them from functioning under experimental conditions.
Whether or not these inhibitors and the proteinases really do interact in the grain during
fungal invasion will have to be determined by immunomicroscopy studies, which we are
preparing to carry out with a collaborator. In addition, we have discovered that there is a
third proteinase present in F. culmorum culture medium that contains cereal proteins and
that this enzyme may not have a counteracting inhibitor in barley grain. We are very
interested in examining whether this third proteinase is produced by Fusarium when it
grows in the infected grain, and will examine this question if we have time.
Publications
Pekkarinen, A.I., Niku-Paavola, M-L. and Jones, B.L. Purification and Properties of an
Alkaline Proteinase of Fusarium culmorum. Eur. J. Biochem. 269, 798-807, 2001.
Pekkarinen, A.I. and Jones, B.L. Some Characteristics of a Trypsin-like Proteinase of
Fusarium culmorum. Submitted to J. Ag Food Chem.
Pekkarinen, A.I., Jones, B.L. and Niku-Paavola, M-L. Two Fusarium proteinases that
probably hydrolyze the storage proteins of scab-infested barleys. ASBC Newsletter 61:
PB10. 2001 and Brewers Digest 76 (3); 32 -33, 2001. (Abstract).
Pekkarinen, A.I. and Jones, B.L. Alkaline proteinases of fusarium infected barley. Poster
presented at the 2nd General Meeting of the International Proteolysis Society (IPS),
associated with the International Conference on Protease Inhibitors (ICPI). 2001,
Munich-Friesing. IPS abstracts p110, 2001. (Abstract).
Personnel
Dr. B.L. Jones, Supervisory Research Chemist
Ms. A. I. Pekkarinen, Research Intern*
* AMBA supported

150
STUDIES OF STARCH DEGRADATION AND FERMENTABLE SUGAR
PRODUCTION IN MALTING BARLEY
Cynthia A. Henson
USDA-Agricultural Research Service - Cereal Crops Research Unit
and Department of Agronomy, University of Wisconsin, Madison, 53706
Four carbohydrases (α-amylase, β-amylase, α-glucosidase and limit dextrinase) are
involved in degrading starch and producing fermentable sugars during the processes of
malting and mashing. α-Amylase is the only one of these enzymes that is sufficiently
thermostable to remain active throughout the latter stages of mashing when most of the
starch is degraded. Hence, it is often said that the yield of fermentable sugars from
adjunct and malt starches is probably limited by the thermolability of some or all of the
other three enzymes.
Numerous labs around the world are working to enhance the thermostability of two of the
three labile enzymes in order to maximize starch degradation and fermentable sugar
production during mashing. Most of these labs are either creating better proteins via
mutagenesis or trying to find alleles that encode more stable or more active enzymes. The
first to succeed was Yoshigi et al. (1995) who engineered the βamy1 gene isolated from
barley to produce β-amylases with thermostabilities õ 10°C higher than the wild type
enzyme. Alleles encoding β-amylases with enhanced thermostability, albeit less than
Yoshigi and coworkers achieved via mutagenesis, have been identified by Ma et al.
(2001) in Australian germplasm and by Clark, Hayes and Henson (manuscript in
preparation) in North American germplasm. The thermostability of barley ∀-glucosidase
has been increased by 10°C relative to the wild type enzyme via site directed mutagenesis
of Agl1 (Muslin et al., 2002). To date, no alleles encoding ∀-glucosidases or limit
dextrinases with enhanced thermostability or enzymatic activities have been reported. A
project, funded by the NABGP program, to screen for variation in ∀-glucosidase alleles
has been initiated but will not be reported on here.
AMBA supported research in my lab established the basis of the thermolability of αglucosidase
which led to the successful engineering of a more thermostable enzyme
(Muslin et al., 2002). We have presented preliminary data on the thermostability of this
mutant α-glucosidase (2001 AMBA <strong>Progress</strong> <strong>Report</strong>, 2001 Barley Improvement
Conference <strong>Report</strong>). In this report I present data showing the impact of the thermostable
∀-glucosidase upon the production of fermentable (DP 1 - 3) and nonfermentable (DP 4 -
7) maltodextrins during mashing and upon the calculated RDF values. The
thermostabilities of the recombinant wild type ∀-glucosidase and the recombinant
mutated ∀-glucosidase are compared in Figure 1. The temperature at which the wild type
enzyme has only 50% of its maximal activity (T50) was 48°C whereas the T50 of the
mutant α-glucosidase was 58°C. It is noteworthy that this increase in stability was
achieved by mutating only a single base to result in a change in one amino acid. Not only
is this result atypical (Yoshigi et al.’s work required changes in 7 amino acids to achieve
a 10°C increase in stability), but it is encouraging. Since we showed that α-glucosidases
exist in three plant genera that have this base change (Muslin et al., 2002) and since a

151
10°C increase in thermostability was achieved with just this mutation, it is possible that
an allele with this single nucleotide mutation exists in the genus Hordeum. Finding this
allele, should it exist, could be a significant step in the enhancement of malting barley
germplasm.
The efficacy of this thermostable α-glucosidase during mashing was tested by adding the
mutant enzyme or the recombinant wild type α-glucosidase (control) to mashes at the
beginning of protein rest and monitoring the production of appropriate sugars during
mashing. The mashing schedule used is shown in Figure 2. The adjunct grain was rice
and the malt was Morex. Mashes spiked with the control enzyme or the mutant enzyme
were conducted in triplicate in the automated MA-002 mashing apparatus (LG-Automatic
APS, Denmark). The sugars produced, shown in Figure 3, were detected and quantified
by pulsed amperometry after separation on an anion exchange column by HPLC (Im and
Henson, 1995). The specific gravity of the filtered worts was determined with an Anton
Paar digital density meter at 20°C.
The addition of the recombinant thermostable α-glucosidase to mashes resulted in a
significant increase in glucose production that was evident as early as the end of protein
rest (Fig. 3A). The only starch present during this stage of mashing is nongelatinized
starch in malt. The most important enzyme in hydrolysis of nongelatinized starch is αamylase
and the second most important enzyme is α-glucosidase (Sun and Henson,
1991). Furthermore, in vitro studies conducted at lower temperatures than used here for
protein rest showed that α-amylase and α-glucosidase function synergistically to
hydrolyze nongelatinized starch (Sun and Henson, 1990; Sissons and MacGregor, 1994).
The enhanced production of glucose from nongelatinized malt starch likely resulted from
both the individual action of α-glucosidase and its synergistic action with α-amylase.
The amount of glucose produced at the end of starch conversion was also significantly
increased by the action of the thermostable α-glucosidase compared to the amount
produced by the recombinant wild type enzyme (Fig. 3B). Since glucose is a sugar
preferentially taken up and metabolized by brewer’s yeast, increasing its yield from raw
materials is potentially important.
The production of sugars other than glucose during mashing was also affected by the
thermostable α-glucosidase (Fig. 3). At the completion of starch conversion the
production of glucose, maltose, and maltotriose were all significantly increased by
addition of the thermostable α-glucosidase relative to the amount of these same sugars
produced by an equivalent amount of wild type α-glucosidase. Although the addition of
thermostable α-glucosidase did result in differences in the amounts of maltodextrins with
DP õ 4 (Fig. 3C,D), these differences were not significant.
The additions of thermostable α-glucosidase to mashing also influenced the calculated
RDF values of worts (Table 1). When thermostable α-glucosidase was add to the mashes
the RDF values were 10.7% greater than the RDF values obtained by adding the wild
type α-glucosidase (control) to the mashes and these differences were significant at the
P=0.03 level. Because the amount of wild type α-glucosidase activity added to the
control mashes was actually slightly greater than the activity of the thermostable αglucosidase,
we also calculated the RDF values based on the unit of activity added. The

152
adjusted RDF value obtained for mashes with added thermostable α-glucosidase were
69% greater than those obtained for mashes with wild type α-glucosidase added.
This study shows that the yield of fermentable sugars from the degradation of adjunct and
malt starches can be increased by the presence of thermostable α-glucosidase.
Additionally, this work shows that the thermostable α-glucosidase produced significantly
more glucose when nongelatinized starch from the malt was present and when the
starches from the malt and adjunct were gelatinized. In other words, the thermostable αglucosidase
was a significant contributor to the production of fermentable sugars and
maltodextrins with DP 4-7 throughout mashing as long as starch and/or large
polysaccharides were present.
Additional research funded by AMBA supports the production of transgenic barley
containing the mutated α-glucosidase gene imparting thermostability. To meet this goal
we are subcloning the mutated barley α-glucosidase DNA into the pAHC25 plasmid
(Ubi:Gus:Nos/Ubi:Bar:Nos) which will later be used to transform barley calli using
particle bombardment. Site directed mutagenesis was used to add a start codon and a
Sma1 restriction site to the 5' end of the T340P/PTZ18U construct. During this stage of
the research the vendor that supplied our preferred mutagenesis kit and reagents
discontinued their production. Replacement sources of helper phage and the enzymes
required for mutagenesis have now been found and the research is again progressing.
The T340P was cut out of PTZ18U at both the 5' and 3' Sma I sites and purified using
GENECLEAN II. pAHC25 was digested with Sma I and Sac I, thus removing the GUS
gene, and purified using GENECLEAN II. The sticky end of the pAHC25 plasmid
caused by cutting with the SacI will be made blunt-ended with T4 DNA polymerase. The
T340P DNA will then be ligated into the pAHC25 vector using T4 DNA ligase. Upon
complete generation of this construct it will be sent to Dr. Lynn Dahleen who has agreed
to collaboratively produce barley (cv. Conlon) containing this gene for thermostable αglucosidase.
Publications
Clark SE, Hayes PM, Henson CA (2002) A comparison of β-amylase alleles from two
North American barley cultivars. Manuscript in preparation.
Im H and Henson CA (1995) Characterization of high pI α-glucosidase from germinated
barley seeds. Carbohydrate Research 277:145-159
Muslin EH, Clark SE, Henson CA (2002) The effect of proline insertion on the
thermostability of a barley α-glucosidase. Protein Engineering 15:29-33
Sun Z and Henson CA (1990) Degradation of native starch granules by barley αglucosidases.
Plant Physiology 94:320-327
Sun A and Henson CA (1991) A quantitative assessment of the importance of α-amylase,
β-amylase, debranching enzyme and α-glucosidase in starch degradation. Archives of
Biochemistry and Biophysics 28:298-305

153
Sissons MJ and MacGregor AW (1994) Hydrolysis of barely starch granules by αglucosidases
from malt. Journal of Cereal Science 19:161-169
Yoshigi N, Okada Y, Maeba H, Sahara H, Tamaki T (1995) Construction of a plasmid
used for expression of a sevenfold mutant barley β-amylase with increased
thermostability in E. coli and properties of the sevenfold mutant β-amylase. Journal of
Biochemistry 118:562-569
Table 1. The effects of α-glucosidase additions to mashing on the calculated RDF values
of worts.
RDF RDF/unit α-glucosidase
added
wild type α-glucosidase 63.4 13.5
thermostable α-glucosdiase 70.2* 27.9**
* P = 0.03, ** P = 0.0002

Residual α-Glucosidase Activity
175
150
125
100
75
50
25
Mutant
Wild Type
154
0
0 10 20 30 40 50 60 70
Temperature (°C)
Figure 1. Effect of temperature on the activities of recombinant wild type (squares) and
mutant (triangles) α-glucosidases at pH 6.0. Enzymes were incubated for 10 minutes at
temperatures ranging from 0 to 60°C at pH 6. The residual rates of maltose hydrolysis
were assayed for 18 hours at 30°C at pH 4.5. Residual activities are based on % of nonheated
controls.
Temperature (°C)
110
100
90
80
70
60
50
40
0 20 40 60 80 100 120 140 160 180 200
*
Time (min)
**
Adjunct Mash
Main Mash
Figure 2. Changes in mash temperature as a function of time. The adjunct mash (open
circles) and main mash (closed circles) are shown. Samples for carbohydrate analysis
were taken after protein rest (*) and after completion of starch conversion as determined
by iodine testing (**).

µM Carbohydrate / Unit of α-Glucosidase Added
120
100
80
60
40
20
0
120
100
80
60
40
20
0
A
B
*
Glucose
*
Glucose
Maltose
*
Maltose
WT
T340P
Maltotriose
WT
T340P
*
Maltotriose
155
µM Carbohydrate / Unit of α-Glucosidase Added
2.0
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
C
D
Maltotetraose
Maltotetraose
Maltopentaose
Maltopentaose
Maltohexaose
Maltohexaose
Maltoheptaose
Maltoheptaose
Figure 3. Effect of the addition of recombinant wild type (WT) or mutant (T340P)
α-glucosidases on the production of fermentable (A,B) and non-fermentable (C,D) sugars
during mashing. Carbohydrate concentrations were determined after protein rest (30
minutes at 46°C) (A,C) and after starch conversion (B,D). The * indicates significant
differences between the means at the 0.05 level.

156
DIRECTED EXPRESSION OF ANTIFUNGAL GENES IN BARLEY AND INFLUENCES
OF NATIVE ANTIFUNGAL SEED PROTEINS ON MALTING QUALITY
Ronald W. Skadsen
USDA, Agricultural Research Service, Cereal Crops Research Unit; 501 N. Walnut St.,
Madison, WI 53705
INTRODUCTION AND OBJECTIVES
The objective of this research is to produce Fusarium-resistant barley through genetic
transformation with specifically targeted native antifungal protein genes. Tissue-specific
promoters will ultimately be needed to target antifungal protein gene expression to spike
tissues. Our studies with a strain of F. graminearum expressing the green fluorescent
protein gene (gfp) point to the lemma/palea and pericarp (particularly the ovary epithelial
hairs) as important targets for antifungal gene expression. The targeted expression of
antifungal proteins will reduce metabolic burdens on the plant and minimize pressures
that select for resistant pathogen strains.
We have focused on the cloning and expression of two antifungal proteins found in
barley endosperm, permatin and hordothionin (HTH). HTH is a highly basic, sulfur-rich
low molecular weight protein. Barley leaves and roots produce thionins related to seed
HTH. These can be induced by fungi and have antifungal properties in vitro. Despite
these properties, the non-seed thionins apparently do not protect existing barley cultivars
from F. graminearum. Although we have demonstrated the effectiveness of HTH against
Fusarium, HTH is found only in the starchy endosperm and, as normally expressed, is
not available to prevent colonization by Fusarium on outer floret tissues. The same is
true of permatin. Permatins are thaumatin-like proteins (TLPs) found in many cereals
and are homologous with PR-5 proteins. Permatin has been purified from maize and
found to have antifungal properties. However, purified permatin has not been produced
and tested against Fusarium. Because thionin is the only protein shown to be effective
against Fusarium, barley transformation efforts having been concentrated on expressing
this particular gene. However, even though Hth can be expressed at the mRNA in
transformants, there are considerable barriers in leafy tissues to its expression at the
protein level (see Results). In addition, besides their antifungal properties, the effects of
permatin and HTH on malting quality have never been analyzed.
METHODOLOGY
Tissue-specific promoters - In order to develop gene promoters to target antifungal
proteins to specific tissues, genes expressed only in the lemma/palea, and not in leaves,
were cloned. This effort was also extended to the pericarp. The differential display
screening procedure used to select these genes was described in the 1999 AMBA <strong>Annual</strong>
<strong>Progress</strong> <strong>Report</strong>. We have now adopted the PCR-based suppressive subtractive
hybridization (SSH) method to detect tissue-specific genes. The corresponding promoter
sequences were derived from BAC clones and analyzed for tissue-specificity.
Ultimately, vectors will be constructed to express an HTH gene under the control of
tissue-specific promoters, and transformants will be tested for Fusarium resistance.

157
Tissue-specific gene products from the above differential display PCRs were used by Dr.
Andris Kleinhofs (Washington State U.) as probes to find the corresponding Morex
genomic clones in a BAC library. We have also adopted additional options, the inverse
PCR procedure and PCR-based gene walking. Before the promoter region can be
subcloned from a BAC genomic clone, it is necessary to determine the 5’ end of the
mRNA. Determination of the 5' (and, where needed, 3') end sequences of mRNAs are
performed by Rapid Amplification of cDNA ends (RACE) using the GeneRacer TM kit
(InVitrogen), as described by the supplier. These techniques were described in the 2001
AMBA progress report.
Molecular cloning and vector construction - Vectors for stable expression of antifungal
genes were prepared from the Ubi/GUS+Ubi/BAR vector pAHC25 by replacing the GUS
gene. For stable tissue-specific expression of Hth, a barley promoter/Hth fusion will
replace the Ubi/GUS fusion in pAHC25. Transient expression studies of promoter
activity are conducted with the pAHC17 vector, which contains the Ubi promoter but has
no selectable marker. We have modified this vector so that the Gfp gene occurs
downstream from the Ubi promoter, allowing constitutive expression of Gfp
(pAHCSGFP vector). To test our promoters, the Ubi promoter is removed and replaced
with lemma/palea or pericarp promoter candidates. The modified vectors are used to
transform barley through the biolistic (gene gun) approach. The particle delivery and
tissue culture is performed essentially as described by Wan and Lemaux (1994).
Screening of putative transformants was conducted using PCR on leaf DNA extracted by
the CTAB procedure.
Transient expression analysis of promoters and antifungal testing - The putative
promoter regions typically include up to 1500 bp of upstream sequence (with respect to
the mRNA 5' end). In addition to the upstream region, regulatory sequences are
sometimes found in the mRNA 5' UTR and in the first intron. Initially, the upstream
region are subcloned from BAC clones or from Gene Walker PCR products and ligated in
front of the Gfp gene in our pAHCSGFP vector (above). Particle bombardments of
spikelets from emerged spikes, pericarps of intact seeds (after removing lemma and
palea), and leaves are conducted and monitored for up to 72 h with short-wave blue light.
Sequencing of cDNAs and transcribed regions of BAC clones is used to determine the
location of introns. We have now encountered two genes, Lem2 and EpiLTP, in which
introns near the 5’ end may influence expression. In these cases, additional tests are
conducted by joining 5' UTR and/or first intron sequences with the upstream sequence, in
which the first intron is ligated between the promoter region originally tested and the Gfp
coding sequence.
In order to detect Hth gene expression in transformants, northern blots and RT-PCR were
conducted. Total RNA was isolated from leaves by the GT procedure. First strand
cDNA was made by priming a reverse transcriptase reaction with oligo d(T). For RT-
PCRs, reactions were then conducted with an upstream Hth primer and a downstream
NOS primer. As an internal control, actin primers were used simultaneously. Since
much more mRNA was produced in Hth2 transformants, northern blots were used to
analyze expression.

158
RESULTS
Tissue-specific promoters - Ultimately, tissue-specific promoters will be needed to
restrict expression to discrete spike tissues. The pericarp epithelium was previously
identified as a possible route of Fusarium infection. In the ’01 grant period, a gene
preferentially expressed in the pericarp epithelium was cloned and sequenced by Maria
Laura Federico. This gene (EpiLTP) was found to be homologous to non-specific lipid
transfer protein genes. Northern blot analysis also showed that EpiLTP is also expressed
in the coleoptile and in the root and shoot axes of the developing embryo (Fig. 1). The
promoter was cloned, sequenced and ligated to the gfp reporter gene in order to test it
through transient expression (Fig. 2). Following particle bombardment of pericarps and
other tissues, GFP fluorescence occurred strongly in the pericarp. To identify the core
promoter region, deletions were made in the promoter from the 5’ and 3’ directions. A
relatively short sequence in the 5’ proximal region was found to be necessary and
sufficient to drive gfp expression in the pericarp. The promoter is now being used for
stable transformation of barley to test its functions in intact plants. If this proves to be
functional and specific, it will be used to drive expression of a hordothionin gene.
Transformation with hordothionin - We previously found that the Hth1 cDNA clone
does not produce protein in a pET/E. coli system. This could explain why our previous
transformation of barley with Hth1 did not produce transformants that would synthesize
HTH1 protein constitutively. Hth1 has two 5’ methionine codons separated by 18
nucleotides. When the sequence leading to the second methionine codon was deleted to
produce an Hth2 clone, high levels of Trx-HTH fusion protein were produced (Fig. 3A).
Surprisingly, the fusion transcript was produced constitutively, and higher levels occurred
after induction with IPTG (Fig. 3B). When the Hth2 clone was then used to transform
barley, leaves produced high levels of Hth2 mRNA but again did not produce HTH
protein, even though the mRNA occurred on membrane-bound polyribosomes and was
found in the expected size range of polyribosomes (Fig. 4; manuscript submitted to Plant
Mol Biol). Expression of this seed-specific gene in leaves appears to be inhibited by a
two-fold mechanism, one involving a sequence element that limits mRNA production or
stability and one that limits translation product stability. We are presently constructing
new Hth genes that may overcome these limitations.
HTH antibodies to a 28 amino acid peptide representing the C-terminal half of the mature
HTH protein have been produced. The synthetic peptide and antibodies were produced
by AnaSpec Corp. (San Jose).
Other related research - 1) Tilahun Abebe has used a suppression subtraction
hybridization method to clone over 300 genes expressed in the lemma/palea and not in
leaves. Fifty of the clones have been sequenced, providing insights into the activities of
the lemma and palea organs (manuscript submitted to Plant Physiol). These appear to be
highly photosynthetic organs that need to protect themselves from associated free radical
damage. However, by limiting free radicals, these organs may forgo the use of antipathogen
defensive mechanisms involving oxy radicals. Two genes (1-5 and 2-6) that are
specifically and strongly expressed in the lemma/palea were chosen for further study.

159
The promoters were subcloned from BAC clones supplied by Andy Kleinhofs. The 1-5
promoter is currently being ligated to the gfp reporter gene and will be tested using
transient expression assays. Clone 1-5 is particularly interesting because it may encode a
defensive protein that binds the carbohydrate moiety of fungal cell walls. This could be
part of an alternative defensive mechanism suggested by the above study. 2) We have
conducted developmental western blot analysis of permatin protein in germinating seeds.
Permatin appears to be extremely stable, with no signs of degradation for at least six
days. We are now planning to analyze its presence in malt, during brewing, and in hazy
beer.
PERSONNEL
John Herbst, USDA Technician Jianming Fu, Post Doctoral
Associate
Laura Oesterle, USDA Technician Maria Laura Federico, Ph.D. Student
Tillahun Abebe, Post Doctoral Associate
PUBLICATIONS
Skadsen RW, Sathish P, Federico ML, Fu J, Kaeppler HF. Cloning of the promoter for a
novel barley gene, lem1, and its organ-specific promotion of Gfp expression in lemma
and palea. Plant Molecular Biology (in press).
Federico ML, Fu J, Kaeppler HF, Skadsen RW. Cloning and analysis of the promoter of
a barley gene preferentially expressed in the pericarp epidermis. Abs. 729, p. 151. Am
Soc of Plant Biol, Providence, July, 2001.
Fu J, Sathish P, Federico ML, Kaeppler HF, Skadsen RW. Constitutive expression of an
antifungal protein alpha-hordothionin in transgenic barley. Abs. P-1012. Congress of the
Society for In Vitro Biology, June, 2001. In InVitro 37: 25-A.

Stem
Roots
Leaves
Flag leaf
Developing Spike
Coleoptile (3d)
First Leaf (3 d)
160
Anthers
Ovary
Epicarp
Pericarp/Testa
Endosperm
Embryo
Rachis
Lemma/Palea
Fig. 1. EpiLTP gene expression. Left: Barley EpiLTP gene expression in
various vegetative tissues EpiLTP is highly expressed in the developing spike
but not in leaf, stem or root tissues. Center: Barley EpiLTP gene expression
in 3-day old seedlings. EpiLTP is highly expressed in the coleoptile tissue.
Right: Barley EpiLTP gene expression in various spike tissues. EpiLTP is
highly expressed in the epicarp and embryo, but not in endosperm, anther,
rachis or awn tissues. Low expression was found in the ovary.
Fig. 2. Barley EpiLTP gene structure. Homology
search of putative cis-acting DNA elements was
performed using PLACE database (Higo et al.,
1999). The positions of different putative cis-acting
DNA elements are represented by triangles. Name,
code and potential response or tissue-specificity of
each of these DNA elements is summarized in table
(right).
Awns

161
Fig. 3. Expression of Trx-Hth fusion protein genes in E. coli. A: Separation of bacterial proteins by SDS-
PAGE. Soluble protein from induced cultures Lane 1, standards. Proteins from bacteria harboring Trx
(wedge) only (lane 2), Trx-Hth1 (lane 3), and Trx-Hth2 (lane 4; wedge marks Trx-HTH fusion protein). B.
Top: Northern blot using RNA extracted from transformed E. coli and probed with 32 P-labeled Hth1 cDNA.
RNA from the uninduced (lane 1) and IPTG-induced (Lane 2) bacteria harboring Trx-Hth1 and uninduced
(lane 3) and induced (lane4) bacteria harboring Trx-Hth2. B: Bottom: The stripped northern blot was
rehybridized with 32 P-labeled cDNA probes synthesized from bacterial total RNA.
A
B
B
16S
(1.5kb
)
80S
Trx-HTH1 Trx-HTH2
IPTG - + - +
1 2 3 4
8-mer
14-mer
16S
Fig. 4. Analysis of Hth2 mRNA on polyribosomes. A: Leaf polyribosomes from transformant B03 were
separated on a sucrose density gradient. 8-mer refers to mRNA with 8 ribosomes attached. B: Equal
volume fractions were collected, and RNA was applied to a Nytran filter through a slot blot apparatus. The
blot was probed with 32 P-dCTP labeled Hth1 cDNA. C: RNAs from free and membrane-bound (mem)
polyribosomes from leaves of lines B01 and B03 were northern blotted and probed as above. Control lane
(con) is total leaf RNA from line B03. D: Blot for panel C was stripped and reprobed with labeled total
RNA cDNA, as above. Dark band is 18S rRNA; light upper band is 28S rRNA.
C
D
A
kDa 1 2 3 4
9
4 6
7 4
3
3
Trx 0
2
0
B03 B01
free mem free mem con