2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Barley Improvement Kevin Smith, University of Minnesota The overall aim of this research is to develop new barley varieties with acceptable malt quality, improved disease resistance, and high yield potential. At present we are developing six-row spring and two-row and six-row winter malting barley for the Midwest region. Our long term goal is to release winter barley for the Midwest that will fit into cropping systems that are desirable to producers in the region such as a winter barley-soybean double cropping system. This research will directly assist in AMBA’s mission to provide an adequate supply of high quality malting barley. Barley improvement at the University of Minnesota is a cooperative effort of the Departments of Agronomy and Plant Genetics, Plant Pathology, and Research and Outreach Centers of the University of Minnesota. Specific breeding goals include high yield, enhanced lodging resistance, resistance to Fusarium head blight (FHB), net blotch, spot blotch, stem rust, and Septoria speckled leaf blotch (SSLB), and favorable malting and brewing characteristics. In addition, in our winter barley program we seek to enhance winter hardiness and combine it with high yield and good malting quality. To meet these objectives, we are conducting a comprehensive breeding and genetics research effort funded by industry, state and federal grants. This AMBA proposal is designed to address research objectives not funded through other sources, to maximize the effectiveness of resources, and to insure that germplasm, information, and technological discoveries are translated rapidly toward the development of new malting barley varieties. In our winter barley programs, we have just made crosses in our two-row program and just planted our first preliminary yield trials in our six-row program. In our six-row program, we are utilizing genomic selection (GS) which should dramatically shorten the breeding cycle, which is the time from which we make a cross to when a breeding line from that cross is selected to be used as a parent. We have just completed our third cycle of selection in this program. Lines enter preliminary yield trials 2 years after selection and could be entered into AMBA testing two years after that. We will harvest our cycle 1breeding lines in preliminary yield trials next summer and have them evaluated for quality. Selected lines from this set would go to 2nd year yield trials in the 2013-14 field season and then enter AMBA Pilot testing with the 2015 crop. Our two row winter program is about three years behind the time line for the six-row program. Preliminary evaluation of the GS predication accuracy for winter survival or low temperature tolerance (LTT) is encouraging. We have worked out some of the bugs in the very tight timeline required to plant, genotype, make genomic predictions, select parents, and make crosses in about a seven week period of time. Based on this success, we will do two cycles of selection in our winter six-row program this year which compares to one cycle every four years in our traditional breeding program. We are also implementing GS in our spring six-row program. We just made selections for cycle 3. Our cycle 1 selections from that program were evaluated in 2nd year yield trials this year and will be considered for entry into AMBA Pilot testing with the 2013 crop. Selections from cycles 2 and 3 would enter AMBA testing in 2014 and 2015, respectively. In the next funding cycle, we hope to continue to scale up our winter breeding programs and collect more data on our genomic selection procedures to verify prediction accuracy and measure effectiveness. We will also be introducing genetic diversity for LTT based on a large scale study that will identify the most winter hardy barley lines from a world-wide collection of over 1,000 lines. Determining the optimum window for planting winter barley in the Northern Plains Jochum Wiersma, University of Minnesota Winter malting barley has potential benefits for producers, end-users, and the environment. Winter barley has the potential to yield more than spring barley, avoid some diseases, reduce soil erosion during winter and 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 2 early spring, and help diversify cropping systems. Thus, production of winter malting barley in the Northern Plains could complement spring barley production and help support an adequate supply of high quality malting barley for the malting and brewing industry. The success of winter barley in the Northern Great Plains will largely depend on its ability to survive freezing temperatures. Cold hardiness in winter wheat and other winter annuals is not a static condition; it’s controlled genetically and influenced by management practices. Agronomic practices, including planting date and availability of standing crop residues that have the ability to trap snow influence winter survival. A first step towards successful introduction of winter barley in the Northern Plains is to determine a planting date window for winter barley that maximizes winter survival. Our objective is to determine the effect of planting date on the winter survival and yield of winter barley varieties with know differences in winter hardiness. Because of uniqueness of soils and climates throughout Minnesota, trials will be conducted in unique environments and repeated over several years to sample of range of fall weather conditions. The optimum planting window for winter wheat into standing stubble in Minnesota is between September 10th and September 30th south of Interstate 94 and between September 1st and September 15th north of Interstate 94. Planting delays have shown to increase winterkill and delay maturity the following growing season. The proposed research will answer whether the same is true for winter barley. First year results point to an optimum window much the same as winter wheat. The earliest planting date not only produced a very lush fall stand but was also infected BYDV, both factors known to reduce winter hardiness. The latest planting dates showed more winter injury for the less winter hardy cultivar Charles compared to McGregor. Although this is not one of the objective of the current research, the opportunity to harvest a crop in early July - as was the case in St. Paul in 2011 - may allow winter barley to be double cropped with, for example, short-season soybeans or peas. Double cropping soybeans following winter wheat is common in the Eastern United States but has not feasible in Minnesota to date; the earlier maturity and harvest of winter barley may change that. The very dry fall in 2011 caused uneven and delayed emergence in St. Paul. The no-till trial in Crookston showed a very even emergence despite the dry conditions. Incidence and severity of BYDV at both locations was lower in the earlier planting dates compared to the year prior. The advantages of no-till seeding to protect the seedlings from lethal temperatures was showcased when mild temperatures in early January caused most of the snow cover to disappear in the tilled field surrounding the trial in Crookston (Photo1). Winter survival was excellent at all locations. The trials in St. Paul and Crookston were harvested and samples are being processed. At this point in time, the emergence in Crookston is uneven and delayed as this summer's drought extended well into September. Although we have received substantial precipitation since October 4th it may be 'too little too late' for a good emergence as temperatures in October have been well below average. Only some 224 Growing Degree Days (GDD) have been accumulated since the initial precipitation fell in the form of heavy wet snow on October 4th. Barley Trials in Wisconsin Dean Volenberg & Matt Stasiak, University of Wisconsin The lakeshore counties of Northeastern Wisconsin have historically produced malting barley. Over time, other agricultural enterprises became more profitable and malting barley moved west in the United States and north into Canada. Currently, there is renewed interest in malting barley in Wisconsin. This interest is 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 3 favored by the Craft breweries that are looking to produce a unique local Wisconsin product. Presently, there is little data on either winter or spring malting barley varieties for Wisconsin. The objective of this research is to continue evaluation of winter malting barley selections from Dr. Kevin Smith, University of Minnesota and also evaluate 2-row spring malting barley varieties -- Pinnacle, Newdale, and Scarlet in replicated field trials at four different locations. Pinnacle, in a 2011 preliminary trial, showed promise, providing a suitable malt quality profile. The replicated winter malting variety trial seeded on October 4, 2011 will screen selections and named varieties for winter hardiness, yield, growth characteristics and malt quality will be evaluated in cooperation with the USDA Cereal Crops Research Unit in Madison, Wisconsin. Similarly, the spring malting barley trials will screen varieties for stand establishment, yield, growth characteristics, disease susceptibility, and malt quality. This project will produce agronomic data and malt quality profiles on winter and spring malting barley selections and named varieties for Wisconsin. The agronomic data from this project will be completed by November, 2012 and malt quality data will be completed prior to 2013. This project fulfills the primary objective of AMBA and also fills the strategic goal of quality evaluation for a breeding program. This is the second year of successfully establishing and producing winter malting barley in Northeast Wisconsin. Yields of some entries produced over 100 bushels per acre in the presence of heavy disease pressure from powdery mildew. The trial was conducted without foliar fungicide applications, making the yields very impressive. Malt quality analysis has been completed by the Cereals Crops Research Unit in Madison, Wisconsin. Successful winter malting barley production has been demonstrated at the plot-scale level and future research needs to focus on field scale production. This will not only provide valuable research data, but provide an opportunity to create awareness and build grower support for malting barley production in Northeast Wisconsin. Five varieties of spring malting barley were established in April at four different geographic locations that vary climatically; Sturgeon Bay, Chippewa Falls, Alma, and Bayfield. In three of the four locations, field- scale trials were established in cooperation with growers. The fourth trial was a large scale plot trial conducted in Sturgeon Bay. Varieties included were Pinnacle, Stellar, Robust, Lacey, and Newdale. Pinnacle in terms of yield, outperformed the other varieties. Malt quality analysis has been completed only for the Sturgeon Bay site as of this report. Although varieties were established under excellent growing conditions in April, drought and powdery mildew were likely the main factors depressing yields. Preliminary spring barley trial results in Wisconsin during 2010-2011, and these current trial results, suggest that Pinnacle is a good choice for Wisconsin. Moving forward, research objectives will be to evaluate fertility programs, especially nitrogen, to maximize yield while maintaining malt quality. Management and Epidemiology of Barley Diseases Ruth Dill-Macky, University of Minnesota This is an applied research program directed at the management of the diseases of economic importance to the barley industry in Minnesota and the Upper Midwest. The project is instrumental in screening barley germplasm for resistance to Fusarium head blight, net blotch, bacterial leaf streak and other diseases of economic importance. This project; surveys commercial barley crops for disease; develops and maintains a collection of barley pathogens; examines the structure and diversity of pathogen populations; examines the epidemiology of barley diseases; conducts field and greenhouse tests to identify and introgress sources of genetic resistance; and conducts experiments to develop and evaluate chemical, biological and cultural control options for the management of barley diseases. The screening of germplasm, conducted in collaboration with the Minnesota barley breeding program, serves both to identify progeny suitable for advancement and/or release as commercial malting barley cultivars and 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 4 provides information for the genetic characterization of resistance and the development of markers that may be utilized in marker assisted selection (MAS). In addition to providing services to the breeding program this program conducts independent research aimed at the development of effective and efficient techniques for the identification of resistance to a number of barley diseases. Monitoring barley crops in the Upper Midwest annually for the prevalence of diseases serves to identifying potential new and emerging threats to the industry and insure against the unexpected upsurge of diseases that may impact barley yield and/or quality. Studying the epidemiology of diseases and testing a variety of management options facilitates the implementation of effective disease control practices for the diseases of economic importance to barley. The development of new barley cultivars, like other studies undertaken in this project, are ongoing activities, the scope of which extend beyond a one-year project. Annual outcomes from this project may however be measured through the release of new malting quality barley varieties, such as Quest released in 2010, through the numbers of lines screened for their reaction to various diseases, and through research outcomes published in peer-reviewed scientific journals and extension publications including the variety trails bulletin. In 2012 we had a successful year working on a number of diseases including Fusarium head blight (FHB), net blotch and bacterial leaf streak (BLS). Our screening nurseries conducted in collaboration with the plant breeding programs aided in the selection of germplasm with improved resistance that advanced the breeding programs efforts to develop barley cultivars with resistance to multiple diseases prevalent in the Upper Midwest. A study, funded by the USWBSI, examined integrated management of FHB in barley integrating cultivar selection and fungicide treatments and demonstrated the efficacy of combined management practices for disease control. We also examined the impact of BLS on yield in a field experiment in 2012. Preliminary analysis of the data indicates that barley yields were not significantly impacted by BLS but that kernel weights may be reduced by up to 5% by the disease. Severe Barley Yellow Dwarf Virus (BYDV) and/or Aster Yellows (AY) developed in Minnesota's barley crops in 2012. The high levels of BYDV and AY likely resulted from an early warm spring that facilitated the arrival of vectors (aphids and leafhoppers) from southern states. Although this was likely an atypical event, my program is working with our new extension plant pathologist, Dr. Madeleine Smith, to examine ways to more rapidly determine the causal agent(s) of these virus- and phytoplasma-incited diseases. Identification of the diseases facilitates better monitoring of the diseases and will improve out understanding of the prevalence and relative importance of these diseases. Ultimately this work could lead to improved control measures in future years. I anticipate that the focus of our research effort in 2013 will be largely similar to 2012 with the exception of the addition of a modest research effort to address BYDV and AY. Breeding and Genetics of Six- and Two-rowed Malting Barley Richard Horsley, North Dakota State University The basic objective of the malting barley breeding program at North Dakota State University (NDSU) is to develop and release improved varieties acceptable to barley producers in the northern Great Plains and to those who use or process this barley. Because growers in the region have choices on which crops to grow on their farms, it is our responsibility to develop improved malting barley varieties that are competitive with other crops so barley is an economically viable choice for them to grow. Plant characters that receive the most attention are those that affect quality of the grain, resulting in losses to producers and end users. These characters include yield, grain protein, kernel plumpness, resistance to Fusarium head blight (FHB) and foliar diseases, and resistance to preharvest sprouting (PHS). Our barley breeding project also conducts research that leads to improved farming systems that promote production of barley with acceptable malting quality. 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 5 Since the inception of our work on breeding for FHB resistance, a main objective has been to transfer FHB- resistance from unadapted resistant accessions to our elite Midwest six-rowed malting barley germplasm. In the 2011 fall greenhouse, 100% of the six- and two-rowed crosses made had a least one parent with improved FHB resistance and lower DON accumulation. In 2011, four of the five six-rowed lines and two of the three NDSU two-rowed lines submitted for AMBA Pilot Scale evaluation came from our FHB-resistance breeding program. Over the next few years, our goal is to increase the frequency of lines with improved resistance to 100% from both programs. The number of growers producing barley in North Dakota and the number of acres they produce has been declining over the last 13 years. One of the reasons growers expressed for not growing barley was rejection of their grain for malting due to quality issues. Development of varieties with improved resistance to FHB will help in reducing the impacts due to disease, but growers also need to know the proper management decisions they can make to increase the likelihood that their crop will be accepted. Two of the easier management decisions a grower can make are what crop should precede barley and how should corn residue be managed if corn precedes barley. Two separate studies were initiated in 2009 at four sites in North Dakota to address these questions. Each of the studies requires two years to complete (i.e. one year for the corn or previous crop treatments and the second year for the barley treatments) and our goal is to repeat each experiment three times at each site over a four-year period. In total, about 1,260 new entries (735 six-rowed and 525 two-rowed) were tested in replicated yield trials (Preliminary Yield Trial; PYT) for their first time. Intermediate (IYT), Advanced (AYT), and Varietal Yield Trials (VYT) were grown at Fargo, Carrington, McVille, Minot, Osnabrock, Tioga (Nesson Valley), and Williston, ND. The six- and two-rowed VYT and AYT also were grown at Sidney, MT. There were 67, 20, and 10 six-rowed experimental lines in the Intermediate, Advanced, and Varietal Yield Trials, respectively. There were 75, 20, and 8 two rowed experimental lines in the Intermediate, Advanced, and Varietal Yield Trials, respectively. Plant growth was uniform throughout the growing season at all locations, but below average precipitation following heading limited yields. Foliar diseases were minimal at all locations due to the dry growing conditions. Fusarium head blight (FHB) was observed at our Osnabrock research site, likely promoted by very heavy dues during grain fill and crop dry down. Six-rowed Malting Barley Program: In January 2012 the NDSU Variety Release Committee recommended to released ND22421, with the condition being acceptable brewing data from the 2010 crop. Since we have limited commercial scale brewing data, I recommended in June 2012 that we wait on releasing the line until we have brewing data from the 2012 crop. We produced a small Foundation seed increase in 2012 and will have sufficient seed for release if we get acceptable malting and brewing data from the 2012 production. Averaged across our 2012 yield trials across North Dakota, ND22421yielded similar to Tradition (Table 1). The advantage ND22421 has over currently grown cultivars is its shorter and very strong straw (Table 2). This feature should make it especially suitable for growers producing barley under irrigation or wanting a cultivar that they can straight combine. Five lines will be submitted for AMBA Pilot Scale evaluation in 2012. The lines ND26891 and ND27177 are in their second year of evaluation. Each was rated satisfactory in 2011. ND28554, ND28555, and ND28993 are each in their first year of Pilot Scale evaluation. All entries accumulate less DON than Tradition and Stellar-ND and ND28993 is a semi-dwarf line with very strong straw strength during the growing season and at harvest (see attached tables). Two-rowed Malting Barley Program: 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 6 After several years of Pinnacle being the top yielding two-rowed barley cultivar across the state, Rawson and Haxby surpassed it in yield. This is the first time Pinnacle has been subjected to late season drought and high temperatures across the state. We will have to see if this response continues. Cargill contracted the two-rowed line 2ND25276 for production on about 300 acres in North Dakota. Grain from this production will be used for the first year of AMBA Plant Scale evaluation. Across all locations in 2012, 2ND25276 had a 5% yield advantage over Pinnacle. Since 2006, average yield of 2ND25276 and Pinnacle have been similar. Advantages of 2ND25276 over Pinnacle include lower concentrations of malt beta-glucan and DON accumulation (Table 3), and improved resistance to the spot form of net blotch. 2ND25276 could be a candidate for release in January 2014 if we receive favorable commercial scale malting and brewing results from the 2012 crop. We produced Breeder seed of 2ND25276 in Casselton last summer and we will plan on having a small Foundation seed increase during the 2013 growing season. Four NDSU developed two-rowed lines will be submitted for their first year of AMBA Pilot Scale evaluation in 2012. The lines are 2ND27705, 2ND28065, 2ND28071, and 2ND28131. Compared to Pinnacle, each of these lines yield more, have lower concentrations of malt betaglucans, and lower DON accumulation. Especially promising lines are 2ND28065 because of the 13% yield advantage it has over Pinnacle and 2ND28131 because it is a semi-dwarf line with lower DON accumulation than Conlon. The semi-dwarf character of the line will make it suitable for production under irrigation and/or straight combining. Barley Disease and Their Control Robert Brueggeman, North Dakota State University Diseases are the most important factor limiting yield and quality of malting barley production in the upper Midwestern United States. The primary goal of the Barley Pathology Program at North Dakota State University is to conduct research focused on the development of effective and durable genetic resistance to barley diseases important to the economics of production in the state and surrounding regions. To ensure that diseases effecting yield and quality of malting barley do not influence the choice producers make to grow barley we are developing economically feasible methods and practical tools for disease control. We are achieving this goal by working with the NDSU barley-breeding program (six- and two-rowed) to screen experimental germplasm against a range of economically important diseases in the Upper Midwest. The Barley Pathology Program screens material for both seedling and adult plant resistance in the greenhouse and field. Our efforts also involve screening germplasm collections for new sources of resistance as well as developing mapping populations to characterize resistance and develop molecular markers for the rapid deployment of disease resistance. Collections of pathogen populations are being made to monitor virulence spectra to ensure that any race changes in the population that threaten the effectiveness of deployed resistance are detected. In addition we are developing a website that forecasts the probability of FHB epidemics to assist producers in predicting when to apply fungicides. We also engage in applied research to develop integrated pest management practices that include cultural practices, fungicide application and genetic resistance. These activities are conducted in close collaboration with other programs at NDSU and with pathology, molecular genetics and breeding programs from other institutions. In the one year funding period, we will evaluate current elite breeding material and advanced lines in the NDSU breeding program for resistance to four of the most important barley diseases; spot blotch, net form net blotch, spot form net blotch and Fusarium Head Blight. These screening efforts help ensure that only the elite lines with adequate disease resistance will be advanced. Crosses will be made between lines containing new sources of net form net blotch and spot form net blotch resistance and stacked with two sources of Ug99 resistance to develop lines with resistance to all three diseases. Bi-parental populations will be produced and advanced for mapping and molecular marker development. The susceptible parents in all crosses will be elite 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 7 malting barley varieties to expedite the process of deploying these resistance sources into adapted Midwestern malting varieties. The virulence and genetic variability of major pathogens will be monitored to look for shifts that indicate the need for new resistance genes in barley cultivars. The recent epidemic of a new virulent form of spot form net blotch on the six-rowed malting barley variety “Tradition” was reported to cause unprecedented yield losses of 50-75% in eastern Montana during the 2011 growing season. This is an excellent example of why pathogen population monitoring is important. Genotype by sequencing will be utilized on large population of Pyrenophora teres f. teres and Pyrenophora teres f. maculata to determine the genetic diversity and virulence factors present in the pathogen populations in North Dakota and Montana. This information will help determine what resistance genes to deploy and susceptibility factors to eliminate to ensure durable resistance against the net and spot forms of net blotch. Our efforts to screen barley lines for the breeders to improve and/or maintain current levels of resistance while making gains in yield and quality are essential for the malting barley industry. We will help ensure that cultivars are released with effective and durable resistance. The barley pathology program has sustained its previous level of disease screening efforts by evaluating and/or setting up disease nurseries containing the NDSU breeding program’s two- and six-rowed materials. Field nurseries were set up to evaluate for reaction to spot blotch and Fusarium head blight. We also continued our greenhouse screening for spot blotch and net form net blotch. To meet the challenges of newly emerging diseases last year we expanded our screening efforts to include greenhouse screening for spot form net blotch resistance and this year plan to continue this screening and include screening for barley leaf rust. In collaboration with the NDSU breeding program the phenomics data of multiple diseases will be utilized by Dr. Rich Horsley’s Ph.D. student for association mapping. Research funding provided by the Triticeae Coordinated Agriculture Project has allowed us to identify eight new sources of SFNB resistance from the world barley collection effective against a diversity of pathogen isolates from around the world. These sources of resistance have been crossed with multiple adapted malting barley varieties (Tradition, Lacey, Celebration, AC Metcalfe, Conlon and Pinnacle) to develop bi-parental populations that will be utilized to validate resistance QTL identified by our association mapping analysis and possibly identify new QTL. The association mapping data will be utilized to develop molecular markers at six significant resistance loci that will be used in marker assisted selection to develop elite pre-breeding lines. The progeny from the SFNB resistant lines and elite lines will be screened by marker-assisted selection to develop adequate SFNB resistance through a rapid back crossing strategy. We have also introgressed stem rust race TTKSK (aka Ug99) resistance from two sources (rpg4/Rpg5 and RpgSw645) into Tradition, Conlon and Pinnacle and are developing markers that will enable us to combine resistance to both pathogens in these elite backgrounds. We have collected >150 NFNB and SFNB isolates from several geographically distinct locations in North Dakota and in collaboration with Dr. Timothy Friesen have collected isolates from Montana. These collections have been put through two rounds of single spore isolation and we are currently in the process of phenotyping them on a barley differential set. DNA has been isolated from the isolates and we have begun the process of genotyping using a new genotyping by sequencing (GBS) technology that we have developed using the Ion Torrent Personal Genomics Machine (PGM) next generation sequencing technology. We have invested ~$100,000 into buying this piece of equipment in collaboration with three barley researchers (Rich Horsley, Tim Friesen and Robert Brueggeman) and it will give us great capability in genotyping both barley lines and the pathogens that threaten production. CHALLENGES 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 8 In order to identify markers associated with disease resistance loci and QTL it is important that we develop bi-parental mapping populations and have the resources to genotype them. Robust genotyping has been the bottleneck because the cheapest technology available (Illumina Bead Express) still exceeds $60 per progeny line. To overcome this research bottleneck access to the newest genotyping technology is needed at NDSU. We removed this obstacle by organizing a group of barley researchers from The Departments of Plant Pathology, Plant Sciences and the Northern Crops Research Unit-USDA to contribute towards the purchase of an Ion Torrent PGM sequencer. A barley genomics/molecular pathology lab has been set up at NDSU with the capabilities to run the GBS analysis bringing the cost down to ~ $17/ barley line or $15/pathogen isolate. However, the large data sets we are now capable of generating has created a new bioinformatics challenge. The funding to hire a technician to run the PGM and also to perform the down stream data analysis is the major challenge facing NDSU barley pathology program at NDSU in 2013. Incorporation and Characterization of Barley Loci Associated with Stem Rust Resistance Enhancement Maricelis Acevedo, North Dakota State University Wheat stem rust is an economically important disease of barley everywhere barley is grown. To date only two highly effective P.graminis f. sp tritici (Rpg) resistance genes have been identified. Of those, only one, the rpg4/Rpg5 locus provides resistance to the highly virulent stem rust races of the TTKS (Ug99) lineage (Steffenson et al 2009). Despite those resistance being effective against current stem rust pathogen races, it is alarming to think that the entire barley crop depends on two major resistances against a pathogen that is highly variable and constantly evolving. Newer races ofthe pathogen such as QCCJ in the 90's in the US and the emerging Ug99 lineage in Easter Mrica serve as a reminder of the vulnerability of the crop. Recently, evaluation of a double haploid population of a cross between the barley lines Q21861 (stem rust resistant) and SM89010 (stem rust seedling susceptible) identified a "hot-spot" for genes associated with an enhancement of stem rust resistance in the barley chromosome 2H (Moscou et al., 2011). An earlier study, utilizing a different mapping population had also identified a region associated with stem rust resistance in a second location of this chromosome (Druka et al., 2008). Surprisingly this genetic region seems to not been actively utilized in breeding programs in the past. The objective of the proposed project is to incorporate these two genetic regions to expand the genetic basis of the stem rust resistance response in adapted barley cultivars with superior agronomic, malting, and brewing characteristics which already combine the Rpgl and the 7pg-I!Rpg5 resistances. By incorporation of this "resistance enhancer" we will be able to increase the resistance to Ug99 as well as local races. To be able to efficiently select and pyramid these resistances in breeding programs, highly reproducible genetic markers tightly associated with the new resistance enhancer regions need to be identified. This step is extremely important for an efficient incorporation of these genetic regions since their effect seems to only be noticeable in the presence of other genes with a larger effect on resistance. This project fits with AMBA' s mission and primary goals of developing improved varieties with enhanced, longer lasting and broader disease resistance, specifically Ug99 resistance. If one year of funding is allocated, we will start the initial crosses of SM to adapted lines of the cultivars Conlon, Pinnacle and ND25160 that Dr. Robert Brueggeman has already crossed (BC3) to a source of the 7pg4;Rpg5 resistance. After this initial cross, three additional backcrosses will be done to the adapted cultivars carrying the additional7pg4/Rpg5 under greenhouse conditions. Initial rust resistance evaluation to Ug99 will be done in Kenya during the spring season of2013 after the second backcrosses to the cultivars have been performed. After the third backcross has been performed, preliminary evaluation for agronomic traits will be done in summer on 2013 in field plots in Fargo. 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 9 The major concern for the success of this project is being able to obtain good disease reaction data that can be utilized for the mapping and development of genetic markers associated with the rust resistance enhancement trait. However, we have utilized the nursery for other projects without major problems in the past so it is likely that we can accomplish our objective. Additionally, we will need to be able to develop a good way of quantifying and measuring small differences in disease reaction. Do to the previous experiences on similar types of resistance in oats and wheat rust systems we envision a successful outcome. Objective 1) Incorporation and pyramiding of genetic regions associated with enhancement of stem rust resistance into adapted varieties. All parental lines being utilized to incorporate additional stem resistance QTL into adapted malting barley lines (as described in the executive summary) have been planted and will be crossed during the month of November. An earlier attempt to make crosses during the field season was unsuccessful do to plant stress caused by drought and high temperatures. After the initial crosses are accomplished, two additional backcrosses to the adapted lines will be necessary before disease evaluation. Objective 2) Identification/development of genetic markers highly associated with enhancement of stem rust resistance to Ug99 and other Puccinia graminis f. sp. tritici (stem rust pathogen). To identify candidate genes associated with the stem rust resistance enhancement, a total of 18 sets of PCR primers were developed based on barley unigenes localized to the 2H region. A NCBI BLAST search of the barley genome assembly produced alignments enabling the identification of intron-exon junctions. Eighteen sets of gene specific exon spanning primers were developed to screen the 2H loci in Q21861, Conlon, Pinnacle and ND25160. We are currently screening all parental lines to detect polymorphism that may be associated with the stem rust resistance enhancement. Next steps and challenges: Marker development If the current approach of using unigene sequences to develop markers associated with the stem rust resistance doesn’t provide the expected results, additional marker development will be performed using single nucleotide polymorphism (SNP) evaluation between Q21861, Conlon, Pinnacle ND25160 and SM89010. Since the region we are trying to saturate with markers has been previously delimited to barley bins 16-18 (Moscou et al., 2011) a relatively low-cost SYBR-green assay couple with a high resolution melt curve analysis approach will be utilized to detect SNPs. The thermo-cycler and software necessary to develop this assay are available in Dr. Acevedo’s laboratory. Disease screening: To confirm the usefulness of the markers developed, the new barley populations and lines will be phenotyped at the adult stage in the international stem rust nursery in Kenya. As an initial step, seedling test can be performed at the U of Minnesota facilities during the winter of 2013 but there are space, time and disease phenotype limitations that can only be overcome by field testing. Additional funding as stated in the initial grant proposal will be necessary to accomplish the field screening and finalize genetic marker development currently under way. Malting Barley Germplasm Development Lynn Gallagher, University of California-Davis The barley breeding program at UC Davis will develop both six-rowed and two-rowed germplasm which combines multiple disease resistances and malting quality with good agronomic performance in the Western Region. The developed germplasm will be distributed to both private and public institutions which express 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 10 an interest in evaluating germplasm for breeding purposes. Disease resistances for which selection will be practiced include the following: primary foci are 1) cereal yellow dwarf virus (CYDV); 2) barley yellow dwarf virus (BYDV), adding genes Ryd2 and Ryd3 together; 3) stem rust (Ug99); 4) barley stripe rust, and secondarily, net blotch, scald, leaf rust, and powdery mildew. The variety Madre Selva (MSsel) will provide some resistance for the first three of these diseases. A second source of resistance CI 11797 will be used as a donor of Ryd3. The genes Ryd2 and Ryd3 act in an additive manner, conferring a higher level of resistance to BYDV than either gene acting alone. It is doubtful that the primary objective of developing malting barley varieties broadly adapted for production areas in North America can be achieved without incorporating these resistances. Within one year I will create new advanced lines having various combinations of disease resistances that will be ready for evaluation in the 2012-13 crop season (November –June). The UC Davis barley breeding program also strives to increase secondary uses of barley through the improvement of both feed and food (hulless) barley varieties. The breeding pipeline is now full of advanced two-rowed and six-rowed lines which will be evaluated in the field for agronomic appearance and freedom from diseases. The UC Davis malting barley improvement will focus mainly upon two-rowed germplasm development which combines disease resistance and malting quality in a good agronomic background. The main foci are the following resistances: 1) cereal yellow dwarf virus (CYDV), 2) barley yellow dwarf virus (BYDV), 3) stem rust (Ug99), and 4) scald. Other diseases include stripe rust, net blotch, leaf rust and powdery mildew. The resistance gene Ryd3 is being incorporated into malting germplasm as rapidly as possible to increase resistance to BYDV. Advanced lines from the mapping population Butta 12 X Madre Selva appear to have combined CYDV resistance with malting quality in a good agronomic background based on USDA Malt Lab results of October 2012. The CYDV disease resistance of Madre Selva has been combined with the malting quality of Butta 12. The resistant lines will be used in April 2012 crossings. The mapping population is being exposed to the R. padi aphid viruliferous for CYDV to confirm initial observations. Additional money is needed to inventory UCD gemrplasm at the molecular level for the presence of genes conferring resistance to these two viruses. Secondary uses of barley include the development of six-rowed feed, food (hulless or naked grained), and forage types. Five forage types will be grown in half-acre plots by a private company which is cooperating in an agronomic evaluation in the 2012-13 growing seaon. UC Davis germplasm has been distributed to BARI. CO; Mt Vernon, WA; APB, Arizona; KARC, Ethiopia, and Egypt plus ICARDA-Morocco. Malting Barleys for the Intermountain west: two- and six-rowed spring and winter cultivar and germplasm development Gongshe Hu, USDA-ARS, Aberdeen, ID Our project mission is to 1) develop superior widely adapted spring and winter two-rowed and six-rowed malt barley cultivars, and 2) develop improved spring and winter barley germplasm for use by public and private barley breeding programs. In addition to cultivar and germplasm development, our work also provides valuable information to industry, private and public breeders, and barley producers via multi- location testing of lines from other breeding programs. Therefore we assist in meeting the mission of AMBA by the development of improved cultivars and germplasm, and assisting other breeding programs in their effort to release improved cultivars. 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 11 We are specifically addressing the development of improved winter and spring malting cultivars for the Intermountain West (IMW) areas of Idaho, Montana, Oregon, and Washington with the goal of releasing lines which not only meet industry standards for malt quality, but also exceed existing cultivars in agronomic performance, benefiting both the malting industry and the barley producer. Included in these agronomic characteristics are improved yield, lodging resistance, and disease resistance for both spring and winter cultivars. In addition, improved winter hardiness is a major objective, especially for the two-rowed winter program. AMBA funding allows us to evaluate lines at multiple locations leading to the development of cultivars with wide environmental stability for use across large growing areas. Specific goals in 2012-13 for the winter malt effort were to propagate enough seeds for the field planting in 2012 for the plant-scale evaluation of 02Ab17271 for targeted plant-scale evaluation production in the fall of 2012. Seed supplies were limited and so another increase is underway for actual planting for plant-scale evaluation in the fall of 2012. The general goal in one-year funding period is to advance breeding materials in all developing stages for the 2-row spring and winter types. In addition to the 02Ab17271, other highlights in the Aberdeen program include: 1) another 2-row spring malting line of 2Ab04-X010084-27. This 2-rowed spring malting barley was approved for the 3rd year of AMBA pilot-scale evaluation. The current data indicated that this line is similar to Harrington in malting quality except that the beta-glucan is significantly lower and yield potential is higher; 2) Two winter lines in the second year of AMBA pilot scale evaluation: 02Ab431 and 02Ab671, Those two 2-row winter malting barley lines showed better yield potential than Charles; 3). A 6-row winter malting line of 93Ab669 was in the first year of AMBA pilot testing. 93Ab669 has a great yield potential. Challenges in Aberdeen program Major challenge in our program is short of a helper. There was a full time helper who was working the seed preparation and cleaning. The helper was supported by USDA-NIFA Barley for Rural Development earmarked grant. The grant expired on August, 2012 and will not be renewed due to Congressional elimination of earmarks. Our basic funding is not sufficient to support the position. Development of Improved Malting Barley Varieties Tom Blake, Montana State University Improving yield and reliability while retaining Harrington-like quality and flavor is key to restoring the health of American malt barley production. Barley acreage declined from more than 12 million acres in 1986 to currently about 2 million acres. Three states, North Dakota, Idaho and Montana, account for about 80% of US barley acreage. If barley is to regain its historical position, it must be more competitive with its traditional rotation crops, winter and spring wheat. This will depend upon the deployment and utilization of more competitive barley varieties, and aggressive marketing efforts by the US malting and brewing industries. Hockett successfully entered commercial production in Montana in 2010. Hockett displaced Harrington in 2011 as the second most widely grown malt barley variety in Montana. MT010158 is in its final year of plant scale evaluation with the 2011 crop, and will likely enter commercial production following a large Foundation Seed increase in 2012. The most efficient, lowest cost method for increasing the rate of germplasm improvement is through enhanced germplasm exchange among capable barley improvement programs. In 2007, the MSU barley improvement program volunteered to perform replicated yield trials for the 8 sets of lines submitted to the USDA Barley CAP program from North Dakota, Utah, Washington, Montana, Minnesota, USDA-Aberdeen and Busch Ag. Resources. These 768 entries were subject to Illumina Barley OPA1 genotyping, and the 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 12 agronomic datasets from MSU and the genotype data sets produced by Dr. S. Chao at the USDA-NDSU small grains genotyping laboratory were made available to the barley community in January, 2008. Now data from field experiments spanning the US are available from 2007-2010 (www.triticeaetoolbox.org). This remarkable program is currently leveraging the investments made in each program for the benefit of all. We collaborate extensively with the Malteurop barley improvement program, and are again providing evaluation of early generation selections and advanced breeding lines. Those lines that demonstrated excellence in 2011 are being advanced to multi-location and statewide trials in 2012. Hockett successfully entered commercial production in Montana in 2010. Hockett was seeded to 125,000 acres in Montana in 2012, up from 60,000 acres in 2011. Improving yield and reliability while retaining Harrington-like quality and flavor is key to restoring the health of American malt barley production. Barley acreage rebounded in 2012 with 3.3 million seeded acres nationally. If barley is to regain its historical position, it must be more competitive with its traditional rotation crops, winter and spring wheat. Oilseeds and pulses are providing additional competition for acres across the Northern Plains. The most efficient, lowest cost method for increasing the rate of germplasm improvement is through enhanced germplasm exchange among capable barley improvement programs. In 2007, the MSU barley improvement program volunteered to perform replicated yield trials for the 8 sets of lines submitted to the USDA Barley CAP program from North Dakota, Utah, Washington, Montana, Minnesota, USDA-Aberdeen and Busch Ag. Resources. During the lifespan of the USDA-supported Barley CAP project, we tested 3070 spring barley lines in replicated, dryland and irrigated field trials. These lines were completely genotyped using the Illumina Golden Gate single nucleotide polymorphism detection system by Dr. S. Chao (USDA- NDSU). These datasets are available at www.triticeastoolbox.org . Over the past 18 months we have learned how to extract and distill the information contained in the data from these very large field experiments. When we compare the results of the agronomic trials with those of the analyses provided by the USDA-CCRU micromalting laboratory, we see that while gains can be made in field performance, the gains to be made in malting quality are substantially larger. I predict that over the next five years, lines will be advanced to pilot scale evaluation that will combine Metcalfe’s nearly ideal malt profile with more reliable dryland performance. We will make malting barley a more competitive alternative to its rotation crops. The growth of the craft brewing industry is now being followed by the development of craft malting. Small malting systems are now operating in Washington, Nevada, Texas and Massachusetts, and many more are being built. It is my hope that these small malting companies that will attempt to utilize locally-grown barley and specialty grains will provide a platform to evaluate and release malting barley varieties across much more of barley’s traditional growing regions, and that we can reclaim many of the acres our favorite crop has lost. Making the switch - accelerated development of two-row winter malting barley Pat Hayes, Oregon State University How this project will help meet AMBA’s mission and primary objective: Public breeding programs have an evolving and critical role in helping AMBA achieve its mission to provide the malting and brewing industries with an abundant supply of high quality malting barley. One important contribution, highlighted in the recent survey of research priorities for AMBA members, is the development of winter malting barley varieties. Oregon State University has historically focused on the development of winter and facultative six-rows by integrating QTL mapping, marker assisted selection, and fundamental research in low temperature physiology, quantitative disease resistance, and malting quality. In the interest of brevity, in the remainder of this proposal we will use “winter” to describe fall-sown barley, although the thrust of our efforts is on facultative. Given the malting and brewing industries increased preference for two- 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 13 row, the OSU program is shifting gears. Making the switch from six-row to two-row will require accelerated and efficient selection. Acceleration will be achieved via doubled haploid production and efficiency via genomic selection. Together, these approaches constitute doubled haploid genomic selection (DHGS). The DHGS program will leverage genotyping resources from the Triticeae Coordinated Agricultural Project (T- CAP) and contribute to national and international efforts to develop a more complete understanding of the genetics of barley growth, development, and malting quality. The research will address AMBA’s primary objective – ensuring that barley is a competitive crop – by incorporating malting quality into high yielding winter habit varieties that provide growers with a sustainable and economically viable cropping option. Making the switch does not mean abandoning the 25 years of effort invested in winter six-row. There is excellent germplasm in the pipeline that will be tested, advanced, and released. Accordingly, 2012 – 2013 will require simultaneous activity in two-row and six-row. Therefore, in addition to the recurring breeding program budget request we are making an additional special request to cover the costs of doubled haploid production and assessment. Three major issues are addressed: improving (1) malting quality, (2) winter hardiness, and (3) agronomic performance: Malting quality - We continually make progress in meeting AMBA malting quality expectations for winter six-row, as summarized in our 2011 AMBA Progress Report. For the past 17 years we focused exclusively on six-row winter malting barley, based on the stated preferences of our research sponsors. However, shifting interests and needs have changed this orientation to focus on winter two-row. This shift is in alignment with international interests and will simplify some of the challenges associated with six-row: kernel sizing and beta glucan. Winter hardiness - Oregon AMBA Pilot entries, and prospective entries, have winter hardiness levels comparable to the agronomic check (Eight-twelve). Our fundamental research efforts will allow us to do better. We have identified a new winter hardiness QTL in winter six-row lines developed by Stephen Baenziger at the University of Nebraska (Lincoln). We have embarked on an extensive association analysis of low temperature tolerance in a sample of ~ 1,000 accessions from around the world, supported by the T- CAP. These resources will allow us, via genomic selection, to rapidly and efficiently accumulate favorable alleles for this complex trait. Agronomic performance - Winter malting barley has impressive yield potential. In western states - where water is a principal limiting factor - dryland winter barley captures prevailing winter precipitation and irrigated winter barley uses less irrigation than irrigated spring barley. Winter barley is also harvested earlier than spring barley, which brings temporal diversity to the farm and reduces risk of weather-related damage at a single harvest date. Winter malting barley has potential for other regions of the country, including the Upper Midwest. We now have an integrated program with Dr. Kevin Smith, University of Minnesota. Goals and outputs - A malting barley breeding program has five principal components (1) parent selection; (2) crossing; (3) assessment and selection of cross progeny in preliminary, advanced, and regional trials; (4) assessment of selected lines in the AMBA pilot and plant scale programs; and (5) variety release. In our new winter two-row initiative we will take a new approach to the first three steps via DHGS. Our goals are to maximize efficiency and productivity of the entire process. One-year and long-term goals - Our one-year goal is to achieve excellence in the five steps of a breeding program, as enumerated in the preceding section. Details are presented in the Experimental Plan section of this proposal. Our long-term goals are to (i) ensure a steady stream of AMBA pilot and plant scale candidates and (ii) provide the AMBA barley breeding community with effective tools for molecular plant improvement. Our experience with DHGS will provide a useful template for all AMBA scientists and we envision providing doubled haploid production on a for-fee basis. 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 14 2012 was the year the Oregon program switched from six-row to two-row. Based on industry interest and directives, we just said “no” to lateral florets. We have not abandoned six-rows entirely – all advanced lines in the program with agronomic merit are included in the TCAP GWAS panels. Optimistically, one or more varieties may come from that pipeline. Nor have we abandoned winter barley. We are still all about winter barley, specifically facultative barley. Facultative barley has maximum cold tolerance and it can be planted at any time. If it is planted in the fall, it has the cold tolerance “wiring” to survive. If it is planted in the spring, the cold tolerance circuit is never “turned on” and therefore there is no metabolic cost to the plant. The switch to two-row would be inconceivable were it not for: accelerated generation time, high throughput genotyping, top shelf phenotyping, and innovative staff. We are achieving accelerated generation time via doubled haploid production: we are also offering our services to other breeding programs. Genotyping is afforded by the TCAP project, which allows us to leverage genetic resources for applied breeding objectives. We have established a pool of field equipment that allows us to effectively manage trials and process seed. Our staff has conceived new breeding methods (doubled haploid genomic selection), implemented improved field phenotyping and data handling procedures, and established doubled haploid laboratory and greenhouse delivery systems. Thanks to a diverse portfolio of support for winter two-row malting barley research (current total = $119,040), we are a vigorous public sector breeding program. Additional funding, however, will be necessary to sustain and expand our activities. We are deeply grateful to AMBA for long-term funding and for the additional one-time support in 2012 for launching the doubled haploid lab. AMBA funding lays an essential foundation that we build on with the support provided by Busch Agricultural Resources, Great Western Malting, and the grower commissions of Oregon and Idaho. Russian Wheat Aphid-Resistant Malting Barley Germplasm Enhancement and Evaluation Dolores Mornhinweg, USDA-ARS, Stillwater, OK Whenever insect pests impact grain yield of barley, Hordeum vulgare (L.), they affect malting quality. There are three yield limiting aphid pests of barley in the US, Russian wheat aphid, Diuraphis noxia (Kurdjumov), RWA; greenbug, Schizaphis graminum (Rondani), GB; and bird cherry-oat aphid, Rhopalosiphum padi (L.), BCOA. RWA is a pest in the malting barley production areas of the western US while BCOA is more widespread and common to all barley production areas. The major cause of yield loss with RWA feeding is head trapping which results in reduced fertility and a severe decrease in plumpness of surviving seed. Not only is less grain available for malt, but the quality of that grain is greatly decreased. When RWAs feed on susceptible plants, the new leaves do not unroll and aphids build up in high numbers inside the unrolled leaves where they are protected from contact insecticides as well as natural parasites and predators, wind and rain. In years of severe or early infestation, chemical control can only be accomplished with repeated applications of systemic insecticides. These chemicals are not only expensive to the grower; they could quite possibly end up in malt produced from treated fields. The solution to these problems is resistant varieties. 116 unadapted germplasm lines have been developed in previous years as a part of this project after screening the entire NSGC. Two lines (STARS 9301B and STARS 9577B) were officially released to breeders. Inheritance studies, also accomplished in previous years by this project, have given barley breeders valuable information on how to best utilize these germplasm lines in their breeding programs. Negative effects on yield and malting quality are often associated with the use of unadapted germplasm in a breeding program. A pre-breeding program was initiated to bring resistant genes from unadapted lines into good malting quality backgrounds adapted to all the barley growing areas of the US. Due to the common occurrence in aphid populations of biotype change, after which the aphid can damage previously resistant lines, all 116 resistant sources have been utilized in this pre-breeding program in hopes of producing germplasm with genetic diversity for resistance which could protect barley from any future aphid biotype 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 15 change. A total of 60 adapted RWA resistant germplasm lines have been released from this program. Thirty- six of these lines were in malting barley backgrounds (17 two-rowed and 19 six-rowed). Four feed barley cultivars have also been released. Since the introduction of RWA (RWA1) in 1986, 7 new biotypes (RWA2 – RWA8), have been identified and more are suspected. All 43 adapted spring germplasm lines were screened for resistance to RWA1- RWA5. Their resistance held. STARS 9301B and STARS 9577B have been reported to be resistant to RWA1 - RWA8 as well. It is critical that we stay informed on future biotype changes and continue to test released resistant lines/cultivars to these biotypes as soon as they are detected. All germplasm releases will be screened to new biotypes as they are identified. A set of 94 unadapted and adapted germplasm lines from this program are currently being screened to other world biotypes. The second phase of the pre-breeding program is to determine the inheritance and genetic diversity of resistance in released lines. Inheritance studies will give breeders information on how best to utilize these germplasm lines in their breeding programs. The genetic populations necessary for inheritance studies have been developed in previous years. Inheritance studies are labor intensive and time consuming limiting the number of studies to 1 or 2 within an acceptable screening environment any given year. An inheritance study on resistant source R017 was completed in 2011/2012. Out of the 16 inheritance studies completed so far in the project, this is the first single gene resistance for RWA found in barley and is the resistant source in STARS 0505B, a winter feed barley. STARS 0505B and its unadapted parent were the only 2 lines found to have resistance to the virulent Iranian biotype. Inheritance studies will again be the focus of research for 2012/2013. We propose to complete genetic studies on as many germplasm lines as time and environment will allow. Cooperative projects to identify genetic markers and map genes for RWA resistance in STARS 9301B and STARS 9577B have verified that genetic diversity for RWA resistance does exist in these 2 lines. Cooperative projects to identify genetic markers and map genes for RWA resistance in 6 more RWA- resistant lines are very near completion. In the future, marker analysis will be combined with traditional multiple allelism tests to determine genetic diversity and genetic control of RWA resistance in released germplasm. BCOA most often considered a crop pest because of its ability to vector yield devastating BYDV, has been reported to cause yield reduction of up to 50% from aphid feeding alone. BCOA is adapted to barley production conditions worldwide and can damage winter barley in the fall as well as winter and spring barley in the spring. Yield reductions are most often attributed to a reduction in seed number and seed weight and have been associated with BCOA damaged plants’ inability to withstand environmental stress including cold temperatures. BCOA feeding negatively impacts the root system as well as above ground growth and can reduce winter hardiness of cereals. With the new emphasis on developing winter malting barleys, resistance to BCOA could become more vital to malting barley production. Resistance to BCOA would be essential to protect malting barley from feeding damage, reduce BYDV incidence by a reduction in phloem feeding, and reduce the negative impact on winter hardiness. Application of established seedling screening techniques to identify BCOA resistance has not been successful because seedlings under standard conditions are asymptomatic. A screening technique has been developed by the USDA-ARS, Stillwater, in which seedlings show a varied response (from live to dead) to BCOA feeding. After years of testing a 1-4 rating scale has been defined and experiments conducted to validate both the technique and rating scale. Close to 900 lines were screened in the spring of 2012 to identify homozygous resistant lines which will be used in 2013 to validate the screening technique and rating scale. Once the screening technique is validated in the greenhouse, screening of the NSGC will begin. A selection of 166 most recent barley cultivars was screened in late summer of 2011 and is currently being screened again. Analysis is ongoing but preliminary results indicate large vulnerability of US barley to BCOA. The ultimate validation, planned for future years, would 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 16 be evaluation of grain yield and yield components of identified resistant lines under field infestation of BCOA. GB is more often a pest of winter feed barleys in the Southern Plains. Although GB research is conducted on this project with respect to barleys for feed and ethanol, as a rule it does not concern malting barleys. We have successfully identified homozygous BCOA-resistant lines and are currently harvesting seed. This seed will be used in an experiment in late spring 2013 to validate the screening technique and rating scale developed from this project. Once the validation is complete we will be able to screen all barleys in the NSGC as seedlings in the greenhouse to identify BCOA resistance. While spring infestations of BCOA can reduce grain yield, fall infestations can greatly affect winter survival of small grains which would compound the problem of reduced winterhardiness in future winter malting barleys. A preliminary screening of 166 of the most recent barley cultivars identified some resistance in spring barley cultivars Burton and Ishi and winter barley cultivars Tambar 500 and Charles. A set of 74 barleys resistant to US biotypes of RWA are currently being screened to other world biotypes of RWA by cooperators in France to ascertain the threat to US barley production by another introduction event such as the one which occurred in 1986. This is part of an association mapping project and the genes for resistance, if identified, will be mapped. We are continuing inheritance studies to provide breeders with information which will allow them to best utilize released RWA-resistant germplasm. Utilizing Marker Assisted selection for Malting Barley Improvement in Washington Kevin Murphy, Washington State University The overall goal of this project is to identify molecular markers that can be used to supportand enhance selection of malting quality traits in the malting barley breeding lines in the WSU two-row spring barley breeding program. Due to the genetic complexity of malting quality traits, and the expense involved in measuring malting quality parameters, the use of marker-assisted selection (MAS) is particularly valuable for malting barley improvement. Using molecular markers together with phenotypic data will speed up the overall breeding process from initial cross to eventual varietal release. Specifically, it will allow for more efficient tests for the presence/absence of relevant traits; 2) evaluate a larger number of genotypes in earlier generations; and, 3) enable selection during off-season cycles, thus streamlining the breeding process and increasing the rate of genetic gain. The specific objectives of this project are 1) to identify, optimize and validate the molecular markers that will allow for rapid, large-scale identification and scoring of traits of importance to malting barley production and quality improvement, and 2) to integrate several years of phenotypic data based on multi-location trials of a diverse set of cultivars and breeding lines with existing genotypic data. Specifically, these traits include malt extract, diastatic power, grain nitrogen, alpha amylase activity, grain protein content, β-glucanase activity (kilned), β-glucanase activity (green malt), kernel plumpness, β-glucan (barley), β-glucan (malt), test weight, kernel weight, soluble/total protein, fine coarse difference, extract viscosity and polyphenol oxidase activity. Other traits of interest include pre-harvest sprouting tolerance, herbicide tolerance, fermentability prediction, and analysis of -amylase, β-glucan, phenolic compounds, and/or phytates. Within the one-year funding period of this project, we have identified 48 single nucleotide polymorphism (SNP) loci as molecular markers to complement the field-based selection program which focuses on evaluations of malting and food quality traits. These SNP loci or markers were based on the linkage map of the Oregon Wolfe Barley Mapping Population, which has integrated hundreds of several kinds of molecular markers associated with malting quality12. This first set of SNP markers will be used to genotype an initial 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 17 set of 200 malting and food barley breeding lines. However, since this set of SNP markers had been identified from a linkage map generated from one mapping population, we need to know if these markers can be used for marker-assisted selection on our malting barley breeding lines. We anticipate that after this initial step, we will test and validate the robustness of these markers and use these to genotype 1920 breeding lines. Markers can be added to the initial set of SNP markers in response to the marker validation tests. We will also integrate several years of phenotypic data based on multi-location trials of a diverse set of cultivars and breeding lines with genotypic data to really fine-tune the set of markers we can use for MAS of malting barley. Within three years, we anticipate the establishment of a full set of molecular markers for genomic selection that will increase the speed and improve the overall quality of the malting barley lines in, and varieties released by, the WSU barley breeding program. These goals reflect a reasonable, outcome-based plan and should be attainable within the project timeline of one to three years. Methodology and Results: A set of 48 SNP loci/markers (Table 1) were identified using the Barley Integrated Map for malting quality (Szucs et al. 2009). This set of markers will be used to genotype 200 malting and food barley breeding lines (Table 2). In coordination with the USDA-ARS Western Regional Small Grains Genotyping Laboratory, the sequence file containing the SNP for each SNP locus was examined. Using these sequence files, the pre- amplification and extension oligonucleotides have been designed. The oligonucleotides will be ordered and paid for with funds from the T-CAP (Triticeae Coordinated Agricultural Project, around $1,500). The 200 lines will be grown and DNA extracted from the seedlings. The cost of the reagents for the genotyping assays will be covered with AMBA funds ($750). Update: Instead of 200 lines, we grew 192 lines to the seedling stage and extracted DNA from the leaves. We reduced the number of lines to 192 for ease of handling, since the plates used for the machines have 96 wells. This number of lines also worked better for the set of SNP markers we used, which was 48. The oligonucleotides were ordered and paid for with funds from the T-CAP ($1,500). The genotyping assay was conducted in the USDA-ARS Western Regional Small Grains Genotyping Laboratory, with the cost of the reagents covered by the AMBA funds ($750). Using the Sequenom® iPlex GoldTM Assay technology, we genotyped the 192 lines with the 48 SNP markers we have identified earlier (Szucs et al. 2009). At present, we have 18,480 data points. This data will be analyzed in the next couple of months, specifically for genetic similarity of the lines using principal component analysis and the software STRUCTURE, and correlations will be made between the genotype and phenotypic traits related to malting. Other Barley Research and Future Direction of Program Use MAS to augment field based selection of lines to be developed and released as feed, food and malting barley; coordinate efforts with T-CAP in developing markers for MAS Once the validity of the 48 SNP markers for malting traits have been established, we plan to use these markers on a larger set of barley lines and cultivars to conduct association mapping of malting traits. Breeding barley for imidazolinone resistance by a) continuing to backcross a ‘Bob’ herbicide resistant mutant field testing for resistance, yield, and other agronomic and quality traits and b) transferring the gene responsible for herbicide resistance to other barley cultivars adapted to WA Update: We crossed the ‘Bob’ mutant (resistant to imidazolinone) to two malting varieties, Radiant and Conrad, in order to transfer the herbicide resistance gene. We had 37 crosses between ‘Radiant’ and the ‘Bob’ mutant, 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 18 and we obtained 380 seeds. We also had 35 crosses between ‘Conrad’ and the ‘Bob’ mutant, and obtained 454 seeds from these crosses. All these F1 seeds have been sown in the greenhouse and the F2 seeds will be harvested and screened for herbicide resistance. Molecular markers are also being screened on 13 barley genotypes for polymorphism, and will be used to identify the chromosomal location of the herbicide resistance gene in barley. Study of the Malting Quality of New Barley Selections Stan Duke, University of Wisconsin The primary objective of the Malting Quality Analysis project at Cereal Crops Research Unit in Madison is the timely return of accurate malting quality data of barley submissions to state and federally funded barley breeders. This partnership allows them to select and advance promising lines with the release of improved malting barley varieties as the ultimate goal. Barley subsamples are received from cooperating breeders; they are characterized, micro-malted, and the subsequent malts are analyzed for quality using several ASBC methods. The data are tabulated and returned to our cooperators who can then assess the suitability of the lines for development of commercial malting varieties. A second major goal is the efficient support of AMBA’s testing program through barley characterization, malting, and analysis of Pilot Nursery, MVBN, WRSBN, and WMBT samples. We aim to provide complete data on these samples, ahead of the AMBA Spring TC Meeting. A final major goal is to support biochemical and genetic investigations on barley and malt, by supplying raw materials (barley and malt) or facilities access to interested researchers. A tertiary aim is the promotion of malting barley acreage through support to interested cooperative extension agents and growers. The current number of samples submitted for analysis from the 2011 crop year is 5250: 4809 breeders’ and 441special (AMBA nurseries / WMBT). As of March 30, 2012, we have malted nearly 3640 of these submissions, and analyzed nearly 3100. We expect similar numbers for the upcoming crop year and intend to generate the data for our stakeholders in the same type of timeframe as in recent years. We averted negative impacts on the timely return of malting quality data due to Federal budgeting issues, and a reduced staff since October 2011. With the support of AMBA, and a full QA staff in place from the start, we anticipate the 2012CY will rival the record setting efficiency of the 2010CY. The Malting Quality Analysis program at Cereal Crops Research Unit attained its major goal, for the 2011 Crop Year, of the efficient return of accurate barley and malt quality data to interested parties. Final tallies included characterization, malting and quality analysis of 5014 submissions from national barley breeders, characterization of 49 AMBA drill strip barley samples, characterization/malting/analysis of 77 AMBA Pilot Nursery samples, and the same for 78 Western Regional Spring Barley Nursery (WRSBN) and 104 Mississippi Valley Regional Spring Barley Nursery (MVBN) submissions, respectively. A report on the malting quality of the 2011 MVBN was written and posted to our website, and a report for the 2011 WRSBN is near completion. We received the first year of samples of the Winter Malting Barley Trial (WMBT), coordinated by Dr. Kevin Smith (U. of Minnesota), from 6 locations, and these 108 submissions were malted, analyzed, and data returned. In addition, we analyzed the quality of 16 (including ours) 2011 AMBA Plant-Pilot Collaborative malt samples (AC Metcalfe and Legacy), and returned data. The Malting Quality Analysis team is in much better shape, staffing-wise, than at this time, last year. Our AMBA-supported, UW Research Specialist, Andy Standish, attended LECO FP528 Nitrogen Determinator training in St. Joseph, Michigan, which he found quite instructive, and we now have both full time QA technicians fully proficient in LECO maintenance and troubleshooting. In addition, USDA-ARS CCRU Research Leader, Dr. Cynthia Henson, was able to convert the Federal position of our Assistant Maltster, Jordon Geurts, to a year-round position, from seasonal; this has greatly solidified our ability to maintain malting efficiency. 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 19 One potential quandary is the looming Federal Sequestration. If enacted, its consequences could affect our output. However, the Antaris FT-NIR might lessen such negative effects, as we’ve developed excellent calibrations for %moisture and %protein in barley and malt. Presently, this is a valuable back up for our reference Moisture (Lindberg Drying Oven) and Nitrogen (LECO Dumas Combustion) ASBC methods, which are the “gold standards”. However, if in a true budgetary, labor bind, the use of FT-NIR results for barley/malt moisture and protein data, could become viable, and help us continue. (Efforts toward additional calibrations such as hull-less barley/malt %moisture and %protein, and kernel hardness, are continuing, time-permitting). Barley breeder submissions from the 2012 Crop Year are arriving daily, and we’ve received around 1,400 to date. About 900 samples have been malted and quality data returned on nearly 400. We’re confident in our ability to maintain the pace of malting, analysis, and quality data return of recent years, baring any of the aforementioned potential Federal budgeting issues. Measurement of Hormones and Other Metabolites Associated with Dormancy and Pre-Harvest Sprouting in Barley Paul Schwarz, North Dakota State University Barley dormancy and pre-harvest sprouting (PHS) both can have significant impacts on malt and beer quality. The ideal situation is for barley not to sprout in the field, but to lose its dormancy soon after harvest. The result of barley improvement programs that have targeted increased levels of amylase enzymes, has been some lines that show significantly decreased dormancy, but also a predisposition to PHS. Research efforts on the genetics of dormancy have identified several quantitative trait loci (QTL) (Li et al 2003, Han et al 1996, Takeda 1996 and Ullrich et al. 2002). It is typically accepted that the hormone abscisic acid (ABA) is strongly involved in the mechanism of dormancy and that the gibberellin hormones are involved in germination of barley (King 1976, Koornneef et al. 1982, Karssen et al. 1983, Seo et al. 2006, Jacobsen et al. 2002, Finch-Savage and Leubner-Metzger 2006). However, the actual mechanisms that regulate seed germination are still largely unknown (He et al 2011). Difficulties in connecting the genetic data to metabolic data are due to a number of factors. First is the fact that, up to 15 different gibberellins (GA1, GA3, GA4…GA69) have been reported in germinating or developing grains (Jacobsen et al. 2002). Second is that the quantification of GAs has been difficult and extremely time-consuming. The measurement of the ABA and GA hormones in barley grains has typically been by GC-MS (Frank et al 2010, Okamoto et al. 2009,Widodo et al. 2009). However, the GC-MS methods require a significant amount of initial purification by HPLC, and these complex multi-step methods (Figures 1 and 2) are not amenable for the study of large numbers of samples, at various states of physiological maturity/dormancy. A relatively new alternative is to couple HPLC with quadrupole time-of-flight (QToF) MS. This type of instrument and associated software a permits the analysis of much more complex mixtures (including overlapping peaks), increases sensitivity, and provides more accurate mass analyses. Derivativization of compounds is not required for HPLC, and sample purification may not be required given the resolving power of the instrument. HPLC QToF MS has been recently used to profile hormones and to investigate dormancy in Macadamia integrifolia (Fletcher and Mader, 2007). The Wheat and Barley Cereal Chemistry group in the Department of Plant Sciences at NDSU recently purchased an Agilent HPLC QToF MS (≈$500,000). To our knowledge, this is the only such instrument in the USA that is devoted only to the study of cereal grains. We propose to apply this technology to the measurement of hormones and other metabolites associated with dormancy and pre-harvest sprouting in barley, as it fits very closely with Dr. Horsley’s work on the genetics of PHS. The technology, however, will also be of great value in other physiological studies, research on FHB and mycotoxins, and investigations on malt flavor. 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 20 A recent objective of the Cereal Chemistry group in the Department of Plant Sciences at NDSU (http://www.ag.ndsu.edu/plantsciences/research/cereal) is to expand work into the area of metabolomics. An Agilent HPLC Quadraplole Time of Flight Mass Spectrometer (QTOF MS) was purchased in late 2011($500,000) and installed in March 2013. This complements existing GC-MS capabilities. Focus will be on small molecules (metabolites) associated with various metabolic states, disease stress, or processing conditions. The GC-MS is best suited to the study of non-polar compounds, while the HPLC QTOF MS to more polar metabolites. Initial studies on barley were proposed to be the measurement of hormones and other metabolites (e.g. phenolics) associated with dormancy and pre-harvest sprouting in barley, as it fits very closely with Dr. Horsley’ s work on the genetics of pre-harvest sprouting (PHS). Work conducted in the spring and summer of 2012 involved the evaluation of GC-MS for the measurement of gibberellins (GAs). Both diazomethane and trimethylsilyl diazomethane were evaluated as methylation agents, under various reaction conditions. However, it was found that all conditions resulted in the production of artifacts. More specifically we believe the conditions of methylation resulted in the formation of tautomers and dimers. As such multiple peaks were observed for only GA3, greatly obscuring the interpretation of results. This is especially important when multiple GAs are present, as differences in the masses are minimal. Work on the use of HPLC QTOF MS for the measurement of GA3 and various phenolics began in the Fall of 2012. First efforts focused on the development of an extraction solvent system. Current efforts involve fine-tuning of the elution gradient (water and acetonitrile acidified with 0.1% formic acid). It appears that a linear gradient from 95% water to all acetonitrile gives the best peak resolution. To date we have detected and positively identified GA3 as well as vanillic, protocatechic, and syringic acids. Limits of detection are being investigated, and associated with this, is the need to use excised embryos vs. whole seeds. A method for excising embryos has been developed. Once all analytical conditions are established, the goal is to evaluate differences in the metabolites present in samples of Robust and Stander at various stages following physiological maturity. Actual analysis time will only involve a few weeks. However, the processing of data through the Mass Hunter and Mass Profiler Professional software will require several months. Current Mass Databases include largely mammalian metabolites, and few that are specific to plants. Investigations on Barley Diseases and Their Control Brian Steffenson, University of Minnesota Plant diseases are one of the most important constraints to barley (Hordeum vulgare) production and quality in the United States. Our Cereal Disease Resistance Project is part of the Minnesota Barley Improvement team led by Dr. Kevin Smith that develops six-rowed malting barley cultivars for the Midwestern United States and beyond. However, we also conduct disease evaluations for other barley breeders (both spring and winter programs) across the country. The primary mission of the Cereal Disease Resistance Project is the control of economically important barley diseases. For many diseases, this goal is best achieved through the development of cultivars with genetic resistance. Thus, the long-term goal of this project is to develop the knowledge base, resources, and germplasm for achieving durable disease resistance in malting barley cultivars. In conjunction with this goal, it is also essential to conduct barley disease surveys and monitor pathogen populations for new virulence types. Our specific objectives for 2012/13 are to: evaluate breeding lines for resistance to important diseases; survey commercial barley fields for diseases; collect pathogen isolates from barley cultivars and determine their virulence phenotype; increase, maintain, and distribute pathogen stocks for testing barley germplasm for resistance; and cooperate with other investigators conducting research on malting barley and barley diseases. Our research goals all directly address AMBA’s 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 21 primary objective of developing malting barley cultivars, broadly adapted for producers in North America, with improved agronomic and quality characters--in this case, the important trait of disease resistance. In turn, the deployment of disease resistant cultivars will help ensure that an adequate supply of high quality malting barley is available to the malting and brewing industry—the primary purpose of AMBA. All of the objectives listed above can be completed within a one year period. We anticipate no obstacles to fulfilling these goals. Our project works in association with Dr. Ruth Dill-Macky to ensure that all barley breeding lines and cultivars grown in the region have sufficient disease resistance. The emphasis, of course, changes with the current pathogen threats. For the past five years, our project has led the national and international effort to identify and incorporate resistance to African stem rust in barley germplasm. We have taken full advantage of our special BSL-3 Plant Pathology Containment Facility to screen materials at the seedling stage and the USDA-ARS-funded nurseries in Njoro Kenya or AMBA-funded ones in South Africa to screen materials at the adult plant stage. We have established genetic populations with African stem rust resistant materials and are mapping the genes through support from a USDA-ARS Specific Coop Agreement secured with assistance through Mike Davis. We are also emphasizing research on bacterial leaf streak, which has caused significant losses over the past decade. This disease became more prominent with the widespread use of fungicides, which adequately controlled the leaf spotting fungi, but left an open niche for the bacterium to infect the crop. In addition to these two diseases, we routinely screen for resistance to the important diseases of stem rust (domestic races), spot blotch, and net blotch. We evaluate materials primarily from the Minnesota Barley Improvement program; however, we also assist the Busch Agricultural Research Inc. program and Miller-Coors program in these evaluations. Our goal is to ensure all barley cultivars, regardless of their program of origin, are resistant to the major diseases in the region. A future emphasis in our program for 2012 will be to initiate crosses against a new virulent isolate of the spot blotch resistance, which was discovered in North Dakota. This isolate is virulent on all of the malting barley cultivars in the region and threatens the crop. Since the mid-1960s, we have been fortunate to have durable resistance to this disease. This project will be done in association with Shaobin Zhong at NDSU. I have been working closely with Kevin Smith on developing winter barley varieties for the region. This project has great potential for producers because winter barley often gives higher yields and better quality than spring types--and is more efficient in water and N usage. I am doing more than just the pathology work in this project. I am utilizing the variation present in the Wild Barley Diversity Collection to breed barley with greater low temperature tolerance. There is great promise in this material as some accessions from Central Asia have survived 4 Minnesota winters. I continue to conduct my annual barley surveys, collect isolates and store them for long term use. This information is critical for discovering potential new disease outbreaks as we have had numerous times over the past two decades. We have the largest collection of barley pathogens in the world and are a repository for individuals who wish to use them for detecting disease resistance. Also, we are a source of resistant germplasm for individuals interested in breeding for resistance. We continue to be actively involved in large cooperative project (BCAP and TCAP) that can greatly enhance our efforts to develop resistant barley cultivars for not only our region but nationally and internationally. I love working on barley and have contributed a substantial amount of my endowment funds (about ~$20,000 per annum) toward research on the crop. However, it is getting increasingly more difficult for me to keep a technician fully funded on barley. Winter Hardy Barley Germplasm Development P. Stephen Baenziger, University of Nebraska 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 22 The purpose of this research is to develop the most winterhardy winter barley germplasm in North America and to make it available to the U.S. barley community. As winter barley is becoming more important for malting, a second purpose is to increase the allele frequency of malting barley genes in winter barley. This germplasm will be used by winter malting barley breeders through the U.S. to enhance the reliability of winter malting barley production. The key aspect of this research will be the use of testing sites in Nebraska which have previously been shown to reliably select for winterhardy winter barley, wheat, and triticale. These locations are unique in that they consistently stress winter barley so that winter tender lines are lost, but are not so severe that every line is winterkilled. Winterhardiness sites for winter barley often are too mild for effective selection so that you keep everything or too severe so that you often lose everything. What is critical are sites that can differentiate lines that are winter tender from those that are winterhardy. For over 70 years, Nebraska has developed these sites and successfully created germplasm that has been used and studied by those most interested in developing winterhardy barley lines. This research by its nature is long-term. However, for the upcoming year we expect to continue our breeding effort to enhance our selection of lines with superior winterhardiness and malting quality. We will plant our F2 bulks at Lincoln, NE (a forgiving site for winter survival) to remove very winter tender lines and then plant the harvested F3 seed in bulks at Lincoln, NE and Mead, NE. We will grow our F6 and later generations experimental lines at Mead, Lincoln, and Sidney, NE. Mead NE is the site where we select for wheat winterhardiness and our hope will be to develop winter barley with winterhardiness similar to winter wheat. By replicating the F3 nursery and our F6 and later generations at Lincoln, we will ensure that we avoid total losses of breeding material except in the most severe years. Sidney, NE is our drought prone location and allows us to select lines with drought tolerance and winter hardiness. We began this breeding methodology with AMBA’s support in 2010, so it is currently too soon to know the success of this effort. Our main limitation will be variable climate which may affect our selections. To enhance the frequency of malting barley alleles, we will increase our number of crosses to winter and spring malting barley types with an emphasis on 2-row malting barley parents. In 2011-2012, we expanded our testing to include the Winter Malting Barley Trial (WMBT), in addition to the Uniform Winter Barley Winterhardiness, Uniform Winter Barley Yield Trial, and our routine breeding trials (elite nursery (BVT), preliminary nursery, and observation nursery). Both the WMBT and BVT were submitted to the USDA-ARS Cereal Crops Research Laboratory for malting quality. In Nebraska, the barley lines from Michael Goetz and those from Euromalt had better malting quality. The Nebraska winter barley lines were mediocre to very poor which was to be expected as they have never been selected for malting quality, but it was surprising to find even mediocre lines. These lines will be crossed to elite 2-row and 6- row malting lines. P-845 has been recommended for release. The pedigree of P-845 is Krasnodar K304/NE90701. It is a high yielding grain type that also has excellent forage potential and is adapted to central KS and south. It will be marketed by Paramount Seed Farms who have the exclusive rights to market tour lines globally. Based upon 2012 seed sales, winter barley seems to be making a comeback in the southern Great Plains where forage and grain for dairy cows is becoming more important as dairies are moving into this region due to cost savings and freedom to operate issues. Barley, with its water use efficiency and early season maturity thus escaping heat stress, compared to winter wheat, is an ideal drought tolerant crop for this region. It also adds crop diversity in the agroecological landscapes and choice for producers. Identification of Barley Metabolites that Influence Malt and Beer Quality Jessica Prenni, Colorado State University 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 23 Our recent investigations of barley and malt metabolite variation demonstrate a novel relationship between barley grain metabolite content and malting quality. Numerous barley metabolites have been identified that exhibit significant correlation with various malting traits related to beer quality. Based on these results, we hypothesize that (1) a panel of barley grain metabolites can be established to predict malting quality and (2) that these correlations will extend to beer quality traits such as flavor, aroma and stability. Thus, the long term goal of the proposed work is to establish a panel of metabolites predictive of both malting and beer quality which can be incorporated in barley breeding process. We will test our hypotheses in two Aims. First, metabolite profiles will be collected for barley grain from 36 2-row varieties grown in 2011 and another 36 2-row varieties grown in 2012. This data will be combined with our previous study (36 2-row varieties grown in 2009) and will provide three independent years for which malt quality and metabolite associations can be performed. From this analysis we will validate our previous barley grain metabolite-malting quality associations and develop a robust statistical prediction method. Secondly, correlation between barley metabolites and beer quality will be investigated by the analysis of eight barley varieties which were selected based on divergence in their metabolite profiles as observed in our previous work. Specifically, these eight select varieties will be grown, malted, and brewed, and subsequent metabolite profiling, malting quality assessment, and sensory analyses will be performed. The data will be interrogated to determine correlations between metabolite content and beer quality. The overall expected outcomes of this work are: (1) the development of a validated statistical model of barley metabolite content predictive of malting quality and (2) preliminary information on the correlation of metabolite content and beer quality. These goals can be met within the 1 year funding time frame. We anticipate no significant obstacles with the completion of the proposed work in Aim 1. Our selection of the varieties to be analyzed in Aim 2 is based on their divergence in metabolite profiles and the assumption that there is a correlation between barley metabolite content and beer quality. To our knowledge this has not been tested and warrants investigation. Due to practical limitations involved with growing, malting, and brewing we are limited to the investigation of 8 varieties, which is admittedly small replication for statistical rigor. However, success of this aim would lay the groundwork and provide justification for future, larger scale studies to validate our initial findings. Furthermore, while our study is limited to commercial and in- trail AB varieties we believe that the genomic and malting quality diversity represented in our population will allow for our model to be translated to other breeding populations. Future studies will be focused on evaluation of our methods and statistical model by expanding our study to include a broader population of non-AB varieties. Our aims for grant funded in Fiscal Year 2012 are to (1) develop and validate a statistical model to predict malting quality using barley and/or malt metabolite profiles and (2) evaluate the relationship between barley metabolites and beer quality. For Aim 1, we characterized 218 metabolites (out of 27,420 total molecular signatures) that correlated to at least 1 of 20 quality traits. The chemical classes included amines/amino acids, lipids, alkaloids, polyphenolics, purines, small peptides, and others (including many unknowns). The set of 218 metabolites was used to predict quantitative values for each of the 20 traits using a regression model. The metabolite set successfully estimated the rank of varieties for many traits (e.g. high vs. medium vs. low fine extract content). Future plans include extracting metabolites from barley and malt samples (2011 and 2012 growing season) to determine (i) which of the 218 metabolites follow similar trends in the 2011 and 2012 datasets and (ii) evaluate data from one year to predict another. We will extract and run the 2011 and 2012 samples later this year. 2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS EXECUTIVE SUMMARIES & RESEARCHER UPDATES Page 24 For Aim 2, current efforts are to coordinate malting and brewing with metabolite detection and beer quality measurements. For this study, select barley varieties will be malted and brewed, and samples will be set aside for metabolite and quality analysis from both fresh and stored beer. A diverse set of barley varieties was chosen to maximize variation in beer quality without requiring different malting and brewing parameters. We will then characterize barley and/or malt metabolites that correlate with downstream beer quality metrics using the same techniques as Aim 1.
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