Annual Progress Report on Malting Barley Research March, 2007

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Fractionation by differential centrifugation and aqueous two-phase separation of the microsome proteins located Rpg1 mainly in the cytosol, but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in vitro intramolecularly only serine and threonine amino acids with a preference for Mn 2+ cations and a Km of 0.15 and Vmax of 0.47 nmol·min -1 ·mg -1 protein. The inability of wild type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by site directed mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine residues in the ATP anchor of the two-kinase domains established that the first of the two tandem kinase domains is non-functional and that lysine 461 of the 2 nd domain is the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing requirement of both kinase domains for resistance. In planta expressed Rpg1 mutant protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of autophosphorylation. Molecular mapping and marker-assisted selection of genes for Septoria speckled leaf blotch resistance in barley. Septoria speckled leaf blotch (SSLB), caused by Septoria passerinii, has emerged as one of the most important foliar diseases of barley in the Upper Midwest region of the USA. To map and tag genes for SSLB resistance, we developed two populations derived from the resistant accessions CIho 4780 and CIho 10644 and the susceptible malting cultivar Foster. Segregation analysis of F2 plants and/or F2:3 families from the Foster/CIho 4780 and Foster/CIho 10644 populations revealed that a single dominant gene conferred resistance at the seedling stage. Bulked segregant analysis identified an AFLP marker, E-ACT/M-CAA- 170, that cosegregated with the SSLB resistance gene Rsp2 in the Foster/CIho 4780 F2 population. Southern hybridization analysis with DNA from the wheat/barley addition lines localized E-ACT/M-CAA-170 on the short arm of the barley chromosome 5(1H). RFLP analysis with DNA clones previously mapped to the short arm of chromosome 5(1H) placed Rsp2 at a position flanked by the markers Act8 and ksuD14. A Sequence Characterized Amplified Region (SCAR) marker (E-ACT/M-CAA-170a) was developed that not only cosegregated with Rsp2 in the Foster/CIho 4780 population, but also resistance gene Rsp3 in the Foster/CIho 10644 population. This result indicates that Rsp3 is closely linked to Rsp2 on the short arm of chromosome 5(1H). The utility of SCAR marker E-ACT/M-CAA-170a for selecting Rsp2 in two different breeding populations was validated. Molecular mapping of loci conferring resistance to different pathotypes of the spot blotch pathogen in barley. Spot blotch, caused by Cochliobolus sativus, is an important disease of barley in many production areas and is best controlled through the deployment of resistant cultivars. Information on the genetics of resistance in various sources can be useful in developing effective breeding strategies. Parents of the doubled haploid mapping population Calicuchima-sib/Bowman-BC (C/B) exhibit a differential reaction to pathotypes 1 and 2 of C. sativus. To elucidate the genetics of spot blotch resistance in this population, C/B progeny were evaluated to both 36
pathotypes at the seedling stage in the greenhouse and at the adult plant stage in the field. At the seedling stage, progeny segregated 84 resistant : 26 susceptible based on the qualitative analysis of infection response (IR) data to pathotype 1. This fit best to a 3:1 ratio, indicating that two genes were involved in conferring resistance. Quantitative analysis of the raw IR data to pathotype 1 revealed a single quantitative trait locus (QTL) on chromosome 4(4H) explaining 14% of the phenotypic variance. Adult plant resistance to pathotype 1 was conferred by QTL on chromosome 2(2H) and chromosome 3(3H), explaining 21% and 32% of the phenotypic variation, respectively. Bowman contributed the resistance alleles on chromosome 3(3H) and chromosome 4(4H), whereas Calicuchima-sib contributed the resistance allele on chromosome 2(2H). Resistance to pathotype 2 was conferred by a single gene (designated Rcs6) on chromosome 5(1H) based on qualitative analysis of data. Rcs6 was effective at both the seedling and adult plant stages and was contributed by Calicuchima-sib. This result was corroborated in the quantitative analysis of raw IR (seedling stage) and disease severity (adult plant stage) data as a single major effect (r 2 =0.93 and 0.88, respectively) QTL was identified on chromosome 5(1H). Progeny with resistance to both pathotypes were identified in the C/B population and may be useful in programs breeding for spot blotch resistance. Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley. Rpg1 is a stem rust resistance gene that has protected barley from severe losses for over 60 years in the United States and Canada. It confers resistance to many, but not all pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. A fast neutron induced mutant showing susceptibility to stem rust pathotype Pgt-MCC was identified in barley cv. Morex, which carries Rpg1. Genetic and Rpg1 expression level analysis showed that the mutation was a suppressor of Rpg1 and was designated Rpr1 (Required for Puccinia graminis resistance). Genome-wide expression profiling, using the Affymetrix Barley1 microarray containing approximately 22,840 probesets, was conducted between Morex and the rpr1 mutant. Southern analysis confirmed that three genes (Contig4901_s_at, HU03D17U_s_at, and Contig7061_s_at) are deleted in the rpr1 mutant. These three genes mapped to chromosome 4(4H) bin 5 and cosegregated with rpr1-mediated susceptible phenotype. The loss of resistance was presumed to be due to mutation in one or more of these genes. However, the possibility exists that there are other genes within the deletions, which are not represented on the Barley1 microarray. The Rpr1 gene was not required for Rpg5- and rpg4-mediated stem rust resistance, indicating that it is specific to the Rpg1-mediated resistance pathway. Analysis of ergosterol in single kernel and ground grain by gas chromatography-mass spectrometry. A method for analyzing ergosterol in a single kernel and ground barley and wheat was developed using gas chromatography-mass spectrometry (GC-MS). Samples were saponified in methanolic KOH. Ergosterol was extracted by “one step” hexane extraction and subsequently silylated by Ntrimethylsilylimidazole/trimethylchlorosilane (TMSI/TMCS) reagent at room temperature. The recoveries of ergosterol from ground barley were 96.6, 97.1, 97.1, 88.5, and 90.3% 37
Page 1 and 2: Annual Pro
Page 3 and 4: -i- INTRODUCTION The March 2007 <st
Page 5 and 6: USDA-ARS NORTHERN CROP SCIENCE LABO
Page 7 and 8: Spring Regional over two years at D
Page 9 and 10: Table 1. 2006 UCD Two-Rowed Malting
Page 11 and 12: environments was practiced, 2) elit
Page 13 and 14: Table 4. Selected six-rowed lines c
Page 15 and 16: Other Barley Research: Feed Program
Page 17 and 18: Executive Summary AMBA Prog
Page 19 and 20: Table 1. Agronomic performance of M
Page 21 and 22: Lines M122 and M124 were rated sati
Page 23 and 24: EARLY GENERATION LINES Forty F5 pop
Page 25 and 26: have evaluated a PCR based marker,
Page 27 and 28: Edward Schiefelbein, Research Scien
Page 29 and 30: small grains pathology input to thi
Page 31 and 32: Project Personnel Small Grains Path
Page 33 and 34: Investigations on Barley Diseases a
Page 35: Minnesota barley disease survey and
Page 39 and 40: germplasm with the highest level of
Page 41 and 42: Mirlohi, A., Brueggeman, R., Steffe
Page 43 and 44: Developing Improved Malt Barley Var
Page 45 and 46: Table 2. Montana’s Statewide Tria
Page 47 and 48: As a Barley CAP collaborator, our C
Page 49 and 50: 3.2. A barley nursery composed of d
Page 51 and 52: 5. Related Barley Projects Barley P
Page 53 and 54: Barley Breeding - Advanced Testing
Page 55 and 56: Table 3. Agronomic comparisons of N
Page 57 and 58: Preliminary Malting Barley Yield Tr
Page 59 and 60: dwarf genotypes with the convention
Page 61 and 62: STUDIES ON BARLEY DISEASES AND THEI
Page 63 and 64: inoculated plots. The inoculum used
Page 65 and 66: much of North Dakota in 2006, few f
Page 67 and 68: 5. Collect isolates of barley patho
Page 69 and 70: oot rot. In collaboration with Scot
Page 71 and 72: 2. United States Wheat and Barley S
Page 73 and 74: 6. Lee, S. and Neate, S.M. (2006) P
Page 75 and 76: Objectives Met in the One-Year Fund
Page 77 and 78: These automated methods have been t
Page 79 and 80: Beta-limit dextrin Alpha-Amylase Al
Page 81 and 82: M w kDa 800 600 400 200 0 0.4 0 2 4
Page 83 and 84: Figure 7. Time course of I2-binding
Page 85 and 86: 10 9 8 7 6 5 4 3 2 1 0 1993 1994 19
Page 87 and 88:
2. Other Research (A) Predicting Se
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forms (through enzymolysis during m
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PUBLICATIONS (2006-07) Schwarz, P.B
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CI9214, PI552963 (Heartland), and N
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showing good levels of resistance t
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Germplasm Enhancement for RWA Resis
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Gary Hein in Colorado and Nebraska.
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Kevin Hicks, Research Leader, USDA-
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Detailed Report on
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Other Barley Research and Future Di
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Table 1. 2006/2007 Oregon winter ba
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Table 4. Agronomic data for OSU win
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MALTING QUALITY ANALYSIS OF NEW BAR
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We recently secured an industrial c
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2006-2007 Progress
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enzyme it encodes. We found that se
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horseradish peroxidase-conjugated g
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found in plant cell walls. The degr
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Establishing that sequence variatio
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Charles Karpelenia, technician Joe
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Annual Progress Report on Malting Barley Research March, 2007

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