Annual Progress Report on Malting Barley Research March, 2007

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Other Barley Research and Future Direction of Program The major long term objective of the PIs research is to provide sufficient information to barley breeders that they can produce genotypes whose seeds contain the best (e.g.- most active, most thermostable, most desirable substrate specificity) combination of alleles of the enzymes that produce fermentable sugars from starch, their promoters, and their regulatory DNA-binding proteins. Henson’s prior research demonstrated that the endosperm specific β-amylase from Steptoe was both higher in activity and thermostability than the endosperm specific β-amylase from Morex and determined that the favorable characteristics of the Steptoe enzyme were due to having an arginine in amino acid position 115, an aspartate in position 165 and a valine in position 430 (Clark et al., 2003). The enzyme from Morex had CEA in these three positions. The PIs are conducting collaborative research, with others funded by NABGP or Barley CAP, to determine the distribution of the RDV and CEA haplotypes in a collection of important barley germplasm known as MaltGenes and to correlate these amino acid footprints with levels of diastatic power plus β-amylase activities and thermostabilities in malts of these genotypes. This research is testing the hypothesis that the RDV haplotype is higher in malt β-amylase activity/thermostability and in DP values. The PIs are collaborating, with Prof. Patrick Hayes, to test this same hypothesis in breeding lines of known ancestry containing three 115, 165 and 430 alleles and two intron III alleles. Based on data from our study of Steptoe and Morex (Clark et al., 2003) and on data from the first year’s trials of the breeding lines, it appears more likely than not that the null hypothesis will be accepted which forces us to conclude that other regions of the genome either individually determine β-amylase activity and DP or interact with bmy1, the gene encoding endosperm specific β-amylase, to determine these two traits. The PIs are collaborating to determine if the allelic variation in intron III of bmy1 regulates expression levels or thermostabilities of the enzyme. Numerous publications correlate two intron variants with these enzyme properties, yet no mechanistic data prove that structural features of intron III have any affect on gene expression. 122
Establishing that sequence variations in this intron do regulate gene expression, via recruiting transcription factors or other DNA-binding proteins, will provide additional gene sequences to fix in malting barley germplasm as we proceed to assemble the best package of alleles for the conversion of starch to fermentable sugars. Henson’s lab has established that there are two amino acid positions in α- glucosidase1, the product of Agl1, that, when the optimal amino acids are in those positions, impart increased thermostability (Muslin et al., 2002; Clark et al., 2004). We further demonstrated that α-glucosidase with one of these substitutions increases the yield of fermentable sugars from malt starch during mashing (Muslin et al., 2003). We created these mutant enzymes based upon sequences of α-glucosidases from other plant species whose activities we demonstrated were more thermostable than the α- glucosidase from barley. Because these thermostable α-glucosidases with these sequences exist in plants, we’ve embarked upon an effort to sequence Agl1 from the MaltGenes collection. To date we have sequenced 21 cultivars, analyzed 20 of the sequences and found no sequence variation. Interestingly enough, we’ve shown that the extractable α-glucosidase activities from 7 of these cultivars show considerable variation in enzyme thermostability. As is the case for β-amylase, the emerging picture is that sequence variation in the structural gene alone is insufficient to account for variations in the phenotypes of α-glucosidases, the enzymes responsible for the production of at least 30% of the glucose produced during mashing. The PIs are working together to establish metabolic profiles that distinguish between the best of elite malting barley cultivars and other commercially available malting barley cultivars that produce less RDF under commercial brewing conditions. The goal of this research is to identify 1 – 10 candidate metabolites for which we can develop simple quantitative assays as novel measures to predict malt quality. The PIs are working together to assess novel measures of malt quality. We are testing the hypothesis that malt and seed osmolyte concentrations (OC) will be good indicators of malt quality and pre-harvest germination. These hypotheses are based on the knowledge that the degradation of storage compounds (starch and protein) that 123
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Annual Pro
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-i- INTRODUCTION The March 2007 <st
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USDA-ARS NORTHERN CROP SCIENCE LABO
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Spring Regional over two years at D
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Table 1. 2006 UCD Two-Rowed Malting
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environments was practiced, 2) elit
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Table 4. Selected six-rowed lines c
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Other Barley Research: Feed Program
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Executive Summary AMBA Prog
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Table 1. Agronomic performance of M
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Lines M122 and M124 were rated sati
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EARLY GENERATION LINES Forty F5 pop
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have evaluated a PCR based marker,
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Edward Schiefelbein, Research Scien
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small grains pathology input to thi
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Project Personnel Small Grains Path
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Investigations on Barley Diseases a
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Minnesota barley disease survey and
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pathotypes at the seedling stage in
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germplasm with the highest level of
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Mirlohi, A., Brueggeman, R., Steffe
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Developing Improved Malt Barley Var
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Table 2. Montana’s Statewide Tria
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As a Barley CAP collaborator, our C
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3.2. A barley nursery composed of d
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5. Related Barley Projects Barley P
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Barley Breeding - Advanced Testing
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Table 3. Agronomic comparisons of N
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Preliminary Malting Barley Yield Tr
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dwarf genotypes with the convention
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STUDIES ON BARLEY DISEASES AND THEI
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inoculated plots. The inoculum used
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much of North Dakota in 2006, few f
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5. Collect isolates of barley patho
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oot rot. In collaboration with Scot
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Page 75 and 76: Objectives Met in the One-Year Fund
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Page 79 and 80: Beta-limit dextrin Alpha-Amylase Al
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Page 83 and 84: Figure 7. Time course of I2-binding
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Page 87 and 88: 2. Other Research (A) Predicting Se
Page 89 and 90: forms (through enzymolysis during m
Page 91 and 92: PUBLICATIONS (2006-07) Schwarz, P.B
Page 93 and 94: CI9214, PI552963 (Heartland), and N
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Page 97 and 98: Germplasm Enhancement for RWA Resis
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Page 101 and 102: Kevin Hicks, Research Leader, USDA-
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Page 107 and 108: Table 1. 2006/2007 Oregon winter ba
Page 109 and 110: Table 4. Agronomic data for OSU win
Page 111 and 112: MALTING QUALITY ANALYSIS OF NEW BAR
Page 113 and 114: We recently secured an industrial c
Page 115 and 116: 2006-2007 Progress
Page 117 and 118: enzyme it encodes. We found that se
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Page 121: found in plant cell walls. The degr
Page 125: Charles Karpelenia, technician Joe
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Annual Progress Report on Malting Barley Research March, 2007

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