PROJECT PLAN PROSPECTUS

Genetic variation for nitrogen uptake in spring barley

Yadeta Anbessa^1*, Patricia Juskiw¹, Allen Good² and James Helm¹. Field Crop Development Centre, Alberta Agriculture and Rural Development, Lacombe, AB; Department of Biological Sciences, University of Alberta, Edmonton, AB

*Corresponding Author: (403) 782-8028, yadeta.kabeta@gov.ab.ca

Increased nitrogen uptake and utilization efficiencies are desirable to reduce the negative effect of excessive N on the environment and cut the need for expensive N fertilizer. As part of a venture to establish a baseline for the nitrogen use efficiency (NUE) of barley, we assessed the N uptake and biomass accumulation patterns of ten Canadian spring barley cultivars during the 2007 and 2008 seasons in the field at Lacombe, AB. Plant samples were taken from a 0.21 m² area (three replicates) at apex1, anthesis and maturity stages. For each plot sample, plants were separated into leaves, stems and heads; dried; and weighed. Then the tissues were sent for N content determination. Analysis of variance revealed significant differences among cultivars in leaf, stem and head dry weights. Also, significant differences were found among cultivars in tissue N contents. Subsequently, the total tissue N at physiological maturity varied widely among cultivars ranging from 22 to 37 g m^-2. This range in tissue N translated into differences in NUE that indicates that there is a potential for genetic improvement in NUE of spring barley.

Exploiting historical malting quality data from the Western Canadian Cooperative Two-row Barley trials using association mapping

Aaron D. Beattie¹*, Brian G. Rossnagel¹ and Michael J. Edney²

¹Crop Development Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8

²Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada R3C 3G8

*Corresponding author: 306-966-8349, aaron.beattie@usask.ca

Association mapping (AM) is a method to detect significant correlations between genotypic and phenotypic data using linkage disequilibrium (or the non-random association between alleles). Unlike QTL analysis, AM does not require production of a structured, experimental population, but can be used with unrelated genotypes (or for which the relationships between genotypes are taken into account). AM can exploit the larger number of segregating loci in such populations and typically allows more traits to be studied at once since there is more phenotypic variability. In addition, historical phenotypic data (such as that typically collected during collaborative testing) can be analyzed. A mixed-model approach was used to study seven malting quality traits in 91 barley genotypes from the 1994-2006 Western Canadian Co-operative Two-Row Barley trials. These genotypes represent elite malting quality genotypes from eight breeding programs, including the currently most popular Canadian malting varieties (AC Metcalfe, CDC Copeland, CDC Kendall, Harrington, Newdale) and newer varieties such as Calder, CDC Select, CDC Aurora Nijo, CDC Reserve and CDC Meredith. DNA was extracted from the 91 genotypes and sent for DArT (Diversity Array Technology) whole genome genotyping (Triticarte Pty. Ltd., Yarralumla, Australia) which identified >500 high quality markers. Analysis of the population using unweighted pair-group method (UPGMA) clustering (based on the Dice similarity coefficient) demonstrated genotypes from the same breeding program tended to group together, but a significant amount of exchange between programs was evident. This was also indicated by AMOVA which determined that only 16% of the total genetic variation was distributed between the breeding programs while most (84%) was distributed within the breeding programs. Between two and four significant loci were associated with each of the seven malting quality traits, including some regions previously known to be associated with malting quality (e.g. the lower distal region on chromosome 5H). The population will next be genotyped using the Barley CAP BOPA1 SNP markers to identify candidate genes associated with these traits.

Illuminating the Biofuel Potential of Barley Straw

Victoria Carollo Blake* and Thomas K. Blake

Department of Plant Sciences and Plant Pathology,MontanaStateUniversity, Bozeman,MT

Corresponding author: (406) 994-7229, vblake@montana.edu

As our need for biofuels to supplement petroleum-based fuels increases, so will our need for feedstocks for ethanol production. Barley straw, which to date has been an `agricultural waste' product in most industrialized barley growing regions, should be a prime candidate for ethanol production. The USDA Barley World Core Collection is a 1,917 member collection of Hordeum spp. representing barley accessions from six continents and provides a wealth of genetic diversity. We have screened this collection for three years for in rumen dry matter digestibility (%DMD) and found barley straw to vary from 34% to 79% digestible. Further analyses of the most digestible lines have revealed that a select few of these accessions contain a significant soluble carbohydrate concentration in the forage and straw. These soluble carbohydrates are ready solubilized in 37ºC water and could potentially enable the `leftover' straw from a barley crop to be efficiently used in ethanol production. Association analysis with Illumina genotyping data from 378 of the WCC lines and %DMD values have identified potential QTL for the high DMD trait. Several of the high %DMD lines have already been entered into the MSU barley breeding program, and fortunately, lines that provided the highest levels of soluble carbohydrates are already advanced in the breeding program. Characteristics of these lines and their carbohydrate fraction will be discussed.

Barley Boulevard: A New Barley Information Portal on GrainGenes

Victoria Carollo Blake¹*, David E. Matthews², Gerard L. Lazo³, Olin D. Anderson³

¹ Dept. of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717

² USDA-ARS/Dept. of Plant Breeding and Genetics, Cornell University, 409 Bradfield Hall, Ithaca, NY 14853

³ USDA-ARS Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710

Corresponding author: (406) 994-7229, vblake@montana.edu

GrainGenes, the international database and web site for the Triticeae and Avena (http://wheat.pw.usda.gov), has been a leading repository of information on genetics and genomics since 1992. As the wealth of information within the database and web site grows, so does our need to continue to organize the data. Last year, GrainGenes launched Avena Avenue (http://wheat.pw.usda.gov/GG2/Avena) as a shortcut to oat data on GrainGenes and elsewhere. This portal was very well received by the oat community and now the barley community has a similar tool. Barley Boulevard (http://wheat.pw.usda.gov/GG2/Barley) has now been launched as a web page to organize barley-related QuickQueries, external barley web resources, barley-related meetings, newsletters, publications and barley web sites of general interest. An overview of this new portal as well as an update on new barley data in GrainGenes will be presented.

The Journey: From a Breeder's Cross to a Malting Barley Variety.

A. D. Budde* and K. P. Smith. USDA ARS Cereal Crops Research Unit, 502 Walnut Street, Madison, WI 53726 and Dept. of Agronomy and Plant Genetics, University of Minnesota - St. Paul.

*Corresponding author 608-262-4483 (ph), 608-890-0302 (Fax), allen.budde@ars.usda.gov

The process of generating a barley variety takes at least eight to ten years from the time the cross is made until a new variety is released. Each year 50 to 100 crosses are made each one producing hundreds of progeny. Years later after cycles of inbreeding, selection, and evaluation for numerous traits a couple of these progeny will make it into the AMBA pilot malting evaluation program and possibly to plant scale brewing tests before being released as a variety. The variety “Rasmusson” has recently made this journey and is used to illustrate how a line is evaluated as a potential malting barley. A timeline with quality data documenting progress for advancing this line to malting barley status is presented.

A Comparison of Barley Malt Amylolytic Enzyme Activities and Malt Sugar Concentrations

Stanley H. Duke*, Department of Agronomy, University of Wisconsin, Madison, WI; and Cynthia A. Henson, United States Department of Agriculture-Agricultural Research Service, Cereal Crops Research Unit, Madison, WI, and Department of Agronomy, University of Wisconsin, Madison, WI

*Corresponding author: 608-262-6527, shduke@wisc.edu

This study was conducted to test the hypothesis that barley malt α-amylase activity would correlate better with malt sugar concentrations than the activities of β-amylase, or limit dextrinase. Seeds of four two-row and four six-row North American elite barley cultivars were steeped and germinated in a micromalter for 6 days. At 24h intervals throughout germination, green malt was removed and kilned. Malts were assayed for individual amylolytic activities and malt sugars were extracted and assayed. Increases in malt α- and β-amylase and limit dextrinase activities were greatest between day 1 and day 2 of germination. Over all days of germination, for all cultivars combined, malt α-amylase activities correlated much better with total sugar concentrations (r=0.830, P<0.0001) than β-amylase activities (r=0.665, P<0.0001), and somewhat better than limit dextrinase activities (r=0.785, P<0.0001). Correlations of individual sugar concentrations (glucose, maltose, sucrose, fructose, and the maltodextrins maltotriose through maltoheptaose) for all cultivars combined over all days of germination were greater with α-amylase activities than with β-amylase or limit dextrinase activities (e.g. glucose and maltose r value, respectively: α-amylase, r=0.872, P<0.0001, r=0.763, P<0.0001; β-amylase, P=0.587, P<0.0001, r=0.679, P<0.0001; and limit dextrinase, r=0.806, P<0.0001, r=0.733, P<0.0001). Over all days of germination, individual cultivar malt α-amylase activities correlated better with total sugar concentrations (low to high, r=0.738, P<0.0001 [Harrington] to r=0.925, P<0.0001 [Legacy]) than β-amylase activities (low to high, r=0.446, P=0.064 [Harrington] to r=0.891, P<0.0001 [Legacy]) or limit dextrinase activities (low to high, r=0.577, P=0.012 [Harrington] to r=0.867, P<0.0001 [Lacey]) . Correlations of individual sugar concentrations for individual cultivars over all days of germination, for the most part, were greater with α-amylase activities than with β-amylase or limit dextrinase activities (e. g. low to high r values for glucose: α-amylase, r=0.791, P<0.0001 [Garnet] to r=0.901, P<0.0001 [B1202]; β-amylase, r=0.619. P=0.0008 [B1202] to r=0.849, P<0.0001 [Garnet]; limit dextrinase, r=0.763, P=0.0002 [Harrington] to r=0.890, P<0.0001 [Merit]). Overall, malt α-amylase activity correlated better with sugar production during malting and a short period of mashing than other malt amylolytic enzymes, supporting the tested hypothesis.

A Comparison of Barley Malt Quality Measurements and Malt Sugar Concentrations

Stanley H. Duke*, Department of Agronomy, University of Wisconsin, WI and Cynthia A. Henson, USDA-ARS, Cereal Crops Research Unit, Madison, WI, and Department of Agronomy, University of Wisconsin, WI

*Corresponding author: 608-262-6527, shduke@wisc.edu

This study was conducted to test two hypotheses: (1) that malt osmolyte concentration (OC) values would be better correlated with malt sugar concentrations than malt extract (ME) values and (2) that malt α-amylase activity (α-AA) would be better correlated with malt sugar concentrations than diastatic power (DP). Seeds of four two-row and four six-row barley genotypes were steeped and germinated in a micromalter for 6 days. At intervals of 24 hr throughout germination, green malt was removed and kilned and then assayed for ME, OC, DP, α-AA, and sugar concentrations. Sugars were extracted from milled malt in H₂O at 70°C for 30 min. Except for cv. Harrington, total sugars increased throughout the entire 6 days of germination regime in two-row genotypes but either declined or plateaued after 4 days of germination in the six-row genotypes. Over all days of germination for all genotypes combined, OC correlated much better than ME with total sugar concentrations (OC, r=0.867, P<0.0001; ME, r=0.589, P<0.0001), strongly supporting the first hypothesis. When correlating individual sugar concentrations with ME and OC for all days of germination for all genotypes combined, OC also correlated much better than ME with glucose, maltose, sucrose, fructose, and the maltodextrins maltotriose through maltoheptaose [e.g. low to high r values for OC, r=0.642 (fructose) to r=0.924 and 0.928 (glucose and maltotetratose, respectively), P<0.0001; low to high r values for ME, r=0.282 (fructose) to r=0.723 (glucose), P=0.0524 to <0.0001], strongly supporting the first hypothesis. The increases in OC over six days (48 and 58%, respectively for 2- and 6-row) were reflected by increases in total malt sugars (48 and 66%, respectively for 2- and 6-row). In contrast, increases in ME over the same period (3.8 and 3.7%, respectively for 2- and 6-row) were far lower than those for total sugars or ME. OC reflects the total number of hydrolytic cleavages of oligosaccharides, whereas ME only reflects hydrolytic gain (the insertion of H₂O for each oligosaccharide cleavage), which is slight in magnitude compared to increases in osmolarity. For all genotypes combined, α-AA correlated slightly better than DP with total sugar concentrations over all days of germination (α-AA, r=0.743, P<0.0001; DP, r=0.711, P<0.0001), supporting the second hypothesis. When correlating individual sugar concentrations with α-AA and DP for all days of germination for all genotypes combined, α-AA also correlated better than DP with most sugar concentrations [e.g. low to high r values for α-AA, r=0.517 (fructose) to r=0.900 (glucose), P=0.0 002 to <0.0001; low to high r values for DP, r=0.412 (fructose) to r=0.792 (maltotetraose), P=0.0037 to <0.0001], supporting the second hypothesis. Overall, malt OC correlated better with malt sugar concentrations than ME, DP, or α-AA, indicating that OC best predicts starch hydrolysis during malting and a subsequent short mashing period.

Osmolyte Concentration: A New Method for Determining Malt Quality

*Corresponding author: 608-262-6527, shduke@wisc.edu

The primary ASBC, IoB, and EBC methods for measuring malt quality, although frequently modified, have basically remained the same for decades and in some cases centuries. Recent studies have found that malt osmolyte concentrations (OC) are excellent predictors of malt extract (ME), diastatic power (DP), and ASBC method α-amylase activity (α-AA). Also, OC has been found to correlate much better with malt sugars than ME, DP, ASBC α-AA, Megazyme Ceralpha method α-AA, α-amylase activity, or limit dextrinase activity. These observations would indicate that OC better tracks the degradation of all soluble polymers such as starch, proteins, and β-glucans that are important to maltsters and brewers. The intrinsic nature of malt OC is that it measures the total molar concentration of a solution and not the mass of material in solution as does ME. This characteristic of OC lends it to be more discriminating as to the composition of a malt or wort. For instance, the hydrolytic cleavage of 1 mol of maltopentaose in solution to glucose would result in a 500% increase in OC, but only a ca. 9.1% increase in ME. The increase in ME is strictly due to hydrolytic gain resulting from the insertion of a hydroxyl group and hydrogen from H₂O with each hydrolytic cleavage of glucose from maltopentaose, whereas the increase in OC is due to the increase in osmolality of the solution due to cleavage of all α-(1,4) bonds of maltopentaose. The simplicity, rapidity, cost effectiveness, and accuracy of the OC method will be discussed.

Barley homogeneity: Measurement and importance for malting

Michael J. Edney*, Aaron MacLeod, Steve Symons, Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Canada, John O'Donovan, Kelly Turkington, and George Clayton, Lacombe Research Station, Agriculture and Agri-Food Canada, Lacombe, Canada

*Corresponding author: (204) 983-8854, michael.edney@grainscanada.gc.ca

Maltsters require homogeneous barley to achieve a top quality malt. Barley with uniform kernel characteristics is easier to process and should produce more evenly modified malt that will help avoid potential problems in the brewery. Despite the importance of homogeneity, standard malt and barley quality analyses are based on a bulk sample which overlooks variation among kernels. The Single Kernel Characterization System (SKCS) and Near Infrared Spectroscopy are two technologies that can be used to quantify the homogeneity of barley. Germination Index is a simple test that can be used to measure evenness of germination. The uniformity of malt modification can be measured using standard EBC methods for Friability and Calcofluor homogeneity.

These techniques were used to evaluate the quality of barley grown under a range of agronomic conditions. Analysis by SKCS indicated a significant decrease in the variation of kernel size with higher seeding rate. By examining the near infrared spectra acquired using a common grain analyzer it was also possible to measure differences in the homogeneity of barley. Near infrared instrumentation capable of processing single kernels offers the opportunity to measure variation in protein content among individual grains, replacing the more traditional, but much more laborious, method of single kernel combustion nitrogen analysis.

Variation in the distribution of size, protein content, and germinative vigour of individual barley kernels affected the consistency of water uptake during malting leading to significant differences in uniformity of modification in finished malt as measured with the Calcofluor staining method.

Positive impacts of increased homogeneity on malt quality were clearly demonstrated with decreased levels of wort beta-glucan, and improvements in common modification indicators such as friability and Kolbach Index. With several analytical methods available, homogeneity is a criteria that should not be ignored when seeking a barley that will give maximum performance in the malthouse.

The Impact of qGPC6H on Malting Quality, Grain Composition and Agronomic Performance

A. Fischer, G. Fischer, D. Parrott, A. Budde, V.C. Blake, T.K. Blake*

Dept. of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717

*Corresponding author: 406-994-5055, blake@montana.edu

One of barley's more remarkable characteristics is its ability to tolerate variation in seed composition. Selection for increased percentage of one grain component (e.g. protein) can be accomplished by reduction in the percentage of another component (e.g. starch). Starch is the largest contributor to malt extract, and its negative correlation with grain protein percentage is widely appreciated. We previously identified a barley gene, qGPC6H, in which allelic variation significantly impacted grain protein percentage. Similar variation was observed in wheat, and the gene responsible for this variation was recently cloned. This barley version of this gene, HvNAM-1, is a transcription factor that has significant impact on the initiation of plant senescence. In barley, the allele derived from the variety `Karl' results in delayed plant senescence with no measurable impact on flowering date. This allele also results in reduced grain protein percentage. We backcrossed the `Karl' low grain protein percentage allele into the 2-rowed barley variety `Lewis', producing four independently-derived backcross (BC4) lines. We also backcrossed the `Lewis' high grain protein percentage allele into Karl, producing four independently-derived backcross lines. Dryland and irrigated replicated yield trials were performed in 2006 and 2007 with these lines. We measured grain composition from our trials in 2006 and malting quality from our trials in 2007. In this report we demonstrate that transfer of the `Karl' allele into the `Lewis' genetic background results in plants with higher grain yield, higher grain starch content and higher malt extract. Correspondingly, when the `Lewis' allele is backcrossed into the Karl background, grain yield and starch percentage are reduced, as is malt extract.

Net blotch quantitative trait loci - their practical application in barley breeding

Tajinder S. Grewal, Brian G. Rossnagel* and Graham J. Scoles. Crop Development Centre/Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8

*Corresponding author: 306-966-4976, brian.rossnagel@usask.ca

Net blotch, caused by Pyrenophora teres Drechs., is an important worldwide foliar barley disease. Resistant cultivars are the most economic and eco-friendly control method. Quantitative trait loci (QTL) associated with net blotch resistance were mapped in a doubled-haploid barley population (CDC Dolly/TR251) using diversity arrays technology (DArT®) markers. A major net-form net blotch (NFNB) seedling resistance QTL, designated Rpt6, was mapped to chromosome 6H for isolates WRS858 and WRS1607. QRpt6 was associated with adult-plant resistance in 2005 and 2006 field trials. A seedling resistance QTL (QRpts4) for the spot-form net blotch (SFNB) isolate WRS857 was detected on chromosome 4H as was a significant QTL (QRpt7) on chromosome 7H. Three QTL (QRpt6, QRpts4, QRpt7) were associated with resistance to both net blotch forms and lines with one or more of these demonstrated improved resistance. Simple sequence repeat (SSR) markers tightly linked to QRpt6 and QRpts4 were identified and validated in an unrelated barley population (MEH#486/Harrington). QRpt6 was further validated for seedling and adult-plant NFNB resistance in another doubled-haploid population (CDC Bold/TR251). Since the major 6H QTL, QRpt6, may provide adequate NFNB field resistance in western Canada the practical application plan for the Crop Development Centre barley breeding program is to routinely utilize MMAS for QRpt6 followed by normal field screening in net blotch field nurseries to pick up the additional resistance provided by the other QTL.

Using genetics to advance breeding: donning the winter CAP

Hayes, P.M.*, A. Corey, A. Cuesta-Marcos, T. Filichkina, P. Szűcs, and J. VonZitzewitz, Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA

* Corresponding author: 541-737-5878. Patrick.m.hayes@oregonstate.edu

The winter hardiness of cereal crops is associated with low temperature tolerance, vernalization, and photoperiod sensitivity. Understanding the genetics of these traits, using agronomically relevant germplasm, will provide new opportunities for sustainable and productive barley production. Via the Barley CAP, we have created new generations of germplasm, obtained massively parallel data sets, and are anticipating new insights into the ancestral and derived conditions of our crop. These insights will, optimistically, achieve the Barley CAP goal of using fundamental genetics tools to leverage marker assisted selection for variety development.

Nitrogen effects on low-protein and semidwarf genotypes for malting barley production in western North Dakota

M.R. Hochhalter, R.D. Horsley*, and P.B. Schwarz, Department of Plant Sciences, North Dakota State University, Fargo, ND 58105; and R.J. Goos, School of Natural Resources, North Dakota State University, Fargo, ND 58105

*Corresponding author: 701-231-8142, richard.horsley@ndsu.edu

Malting barley production has moved from the eastern part of North Dakota to the western part of the state in recent years mainly due to the accumulation of high levels of the mycotoxin deoxynivalenol (DON) produced by the fungus Fusarium graminearum Schwabe. When grown under dryland conditions of western North Dakota, current malt barley cultivars typically have excessive grain protein when fertilized according to NDSU Extension guidelines. When grown under irrigation in western North Dakota and eastern Montana, these same cultivars tend to lodge. Barley cultivars specifically adapted for production in western North Dakota are being developed as part of the North Dakota State University (NDSU) Western Malting Barley Project. Improved cultivars for dryland production will have gene(s) that reduce protein up to two percentage units as compared to current cultivars. New cultivars for irrigated production will have the semi-dwarf plant height so they are less prone to lodge. In addition, lines with the low-protein and semi-dwarf traits are being developed to determine if yields can be significantly increased under irrigation. It is thought that increasing N fertilizer of genotypes with the semi-dwarf and low-protein characters would allow for maximizing of yield, without the usual consequences of excessive lodging and grain protein. Research is being conducted to: 1) determine if genotypes with the low-protein character have better agronomic performance and malt quality than current cultivars under dryland conditions when different N fertilizer rates are applied pre-plant and 2) determine if genotypes with the semi-dwarf, low-protein, or both characters have better agronomic performance and malt quality than current cultivars under irrigated conditions when different N fertilizer rates are applied pre-plant. This research is being conducted under dryland and irrigated conditions. Twenty-five barley genotypes, including Robust, Stellar-ND, Drummond, Lacey, Legacy, Tradition, and Conlon; six conventional-height genotypes with the low-protein character; six semi-dwarf genotypes with the conventional-protein character; and six semi-dwarf genotypes with the low-protein character are being evaluated. Each of these genotypes is being subjected to four different fertility regimens and all pertinent agronomic and malt quality data are collected. In addition, In addition, biomass samples will be collected to determine the amount of residual N in straw and grain to determine how the low-protein lines differ from conventional protein lines with regards to N translocation in the plants. Dryland experiments were conducted in 2005 and 2006, and irrigated experiments were conducted in 2006 and 2007. Results from this research will be presented.

Evaluation of European barley genotypes for adaptation in the Northern Great Plains of the United States.

R.D. Horsley* and P.B. Schwarz, Department of Plant Sciences, North Dakota State University, Fargo, ND 58105; and S.M. Neate, Department of Plant Pathology, North Dakota State University, Fargo, ND 58105

*Corresponding author: 701-231-8142, richard.horsley@ndsu.edu

Since 2006 the North Dakota State University (NDSU) barley improvement program has been evaluating two-rowed lines from the breeding companies Saatzucht Breun and Saatzucht Ackerman in Germany, and Sejet in Denmark. The collaboration between our program and the ones in Europe has been facilitated by BayWa AG, located in Munich, Germany. The purpose of this collaboration is to identify European malting barley lines adapted for production in North Dakota. Evaluation of lines has been done in dryland and irrigated yield trials in North Dakota and Sidney, MT that include Midwest USA six- and two-rowed malting barley cultivars as checks. European lines evaluated include the cultivars Scarlett, JB Flavour (8125 d7), JB Umbrella (7571 f13), JB Mary, Marnie, Isotta, Jenuva, Anaconda, Posada, Grace, Audrey, Marthe, Belana, and Ingmar; and experimental lines. In general, many of these lines are competitive in yield with the NDSU cultivar Conlon, but do suffer comparatively lower yield and fewer plump kernels than Conlon under droughty conditions. In comparison to Scarlett, a German cultivar that is currently on the American Malting Barley Association's (AMBA) list of recommended malting barley varieties, many of these lines have superior agronomic performance. European lines tested for the first time in our program are handled the same as NDSU developed entries in our Preliminary Yield Trial. They are evaluated at North Dakota two locations and are advanced for further testing in subsequent years if they have satisfactory agronomic performance, disease resistance, and malt quality. Based on agreement with our European partners and the AMBA, lines with acceptable performance after three years of testing by our program will be entered in the AMBA's Pilot Scale Evaluation Program. Results from the yield trial, malt quality, and disease resistance testing of the European lines and selected checks since 2006 will be reported.

Understanding the genetics of barley β-glucan and diastatic power: ARS Aberdeen’s initial steps toward marker-assisted selection in barley

Eric Jackson*, Don Obert, Juliet Windes, Gongshe Hu, Phil Bregitzer, Gerald Lazo, Harold Bockelman, and Mike Bonman.

*Corresponding author (208) 397-4162 x 123, eric.jackson@ars.usda.gov

Two key objectives in the USDA ARS Aberdeen’s barley breeding program are improving β-glucan content in feed barleys and diastatic power, defined as known enzymatic activity, in malt barleys. Although studies have been done to investigate the genetics of barley beta glucan, the populations used (‘Steptoe/Morex’, ‘Harrington/TR206’, and ‘Blenheim’/E224/3) were developed with malting quality in mind. Utilization of these populations could have resulted in the identification of only allele(s)/allele combinations contributing to moderate levels of β-glucan. In response to this potential problem, our objective was to develop a new mapping population derived from two six-rowed hulless lines: ‘Azhul’, the primary source of high β-glucan in most commercial cultivars and breeding programs, and ‘Falcon’, a well adapted variety to Idaho with moderate levels of β-glucan. The Falcon/Azhul (FA) population will be used to study the genetics and environmental effects on barley β-glucan over several location-years in North America. We have developed a F_6:8 recombinant inbred line (RIL) population mapped with approximately 490 DArT and 100 previously developed SSR markers. In addition, we have added approximately 50 new EST-based SSR markers derived from the same contigs used for BOPA1 SNP development. Mapping results and preliminary QTL analysis using β-glucan measurement from seed grown in Yuma, AZ and Aberdeen, ID will be reported.

A major impediment to improving malt quality in barley is the large number of traits involved. For this reason, our objective is to identify and study key traits affecting areas of malting quality which are lacking in the Aberdeen breeding program. The first area targeted was low enzymatic activity known as diastatic power (DP). To study this area without compromising other key traits, we developed a mapping population from a cross of 01Ab8219, an elite six-rowed line from the Aberdeen breeding program, and ‘Stellar’, a six-rowed malting barley cultivar developed by the North Dakota AES. 01AB8219 has a suitable malting profile, except for low DP, while Stellar has a suitable malting profile, with significantly higher levels of DP than 01Ab8219. We have developed F₆ RILs which are being mapped with SSR markers and being increased for multi-location testing of F_6:8 RILs in 2009. Mapping results, multilocation testing plans, and new approaches to measure expression of the two alpha-amylase enzymes (Amy1and Amy2), the two beta-amylase enzymes (Bmy1 and Bym2), and the alpha-glucosidase enzyme (Agl) will be discussed.

We are also pursuing a third strategy to identify and mine novel alleles associated with β-glucan and DP by intensively genotyping 376 accessions from a subset of the NSGC barley core collection. To develop the population, single plant selections have been made from each accession and the resulting selections are being genotyped with 992 DArT and 48 SSR markers. Seed from each line is also being increased for multi-location testing. Genetic analysis of population structure will be presented and strategies for association mapping and allele mining from the collection will be discussed.

Stripe rust of barley and wheat in central Alberta

K. Kumar^1*, K. Xi¹, T. K. Turkington², X.M. Chen³, D. Salmon¹, J. Helm¹, P. Juskiw¹and J. Nyachiro¹.

¹Field Crop Development Centre, Alberta Agriculture and Food, 6000 C and E Trail, Lacombe, AB T4L 1W1 Canada, ²Agriculure and Agri-Food Canada, Lacombe Research Centre, 6000 C and E Trail Lacombe, AB T4L 1W1 Canada, ³USDA-ARS, 361 Johnson Hall, Washington State University, P.O. Box 646430, Pullman, WA 99164-6430 USA

*Corresponding author: 403-782-8880, krishan.kumar@gov.ab.ca

Spring barley is typically grown on over 4 million acres annually in Alberta and accounts for approximately 45% of the total barley acreage in western Canada. Stripe rust caused by Puccinia striiformis was observed both on barley and wheat in central Alberta, with disease severity and occurrence location being variable between the two cereal hosts. Studies are being undertaken to compare the epidemiology of stripe rust between barley and wheat. In the field experiment of the 2007 season, a trace level of stripe rust on a susceptible barley line was present compared with 50 - 90% stripe rust severity on susceptible spring wheat cultivars. Likewise, no stripe rust was observed in the barley differentials but substantial levels of this disease was present in the wheat differentials grown in the field plots. In the final assessment for the 2008 season, susceptible barley lines showed 80 - 95% stripe rust severity compared with 40 - 70% severity for a susceptible spring wheat cultivar. In the same season, however, the barley differentials showed a lower stripe rust severity than wheat differentials grown in the field. The P. striiformis isolates collected are being inoculated onto the differential sets under controlled environmental conditions to differentiate between P. striiformis f.sp. hordei and P. striiformis f.sp. tritici. These isolates are also being analyzed for differentiation using the simple sequence repeats procedure. The results in the identification of forma specialis will be discussed in relation to the development of cereal stripe rust in central Alberta.
Low phytate barley: The effect upon amino acid utilization during wort fermentation

Dennis E Langrell*, Michael J Edney, Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Canada, William G Legge, Brandon Research Station, Agriculture and Agri-Foods Canada, Brandon, Canada and Brian G Rossnagel, Crop Development Centre, University of Saskatchewan, Saskatoon, Canada

*Corresponding author: (204) 983-6154, dennis.langrell@grainscanada.gc.ca

Zinc is known in the brewing industry for its stimulative effect upon fermentation rate, based upon its function as a cofactor for several enzymes involved in carbohydrate metabolism. It is also essential for non-yeast enzymes involved in nitrogen metabolism such as L-glutamate dehydrogenase and carboxypeptidase. Studies have shown that higher levels of zinc can increase utilization of certain amino acids during fermentation resulting in a better attenuated wort. Zinc levels are naturally elevated in worts made from low-phytate barley malt as less mineral is chelated by phytate and removed with the spent grain.

Effects of the low phytate trait on wort zinc levels and the use of amino acids during fermentation was investigated with eight samples from two separate low phytate populations, one based on AC Metcalfe and the second on Harrington. The AC Metcalfe sample set included the two parents, AC Metcalfe and a low phytate line, along with two bulked segregates from the progeny, one bulk with normal phytate characteristics and the other with reduced phytate characteristics. The second set contained the parent Harrington and three low phytate lines. Malted samples were brewed with the Canadian Malting Barley Technical Centre's microbrewery and fermented in tall cylinder (1.5 L). Free amino acids in wort and fermented wort were measured with the Waters® UPLC system and the AccuTag Ultra® Amino Acid Analysis application. Zinc and magnesium levels were also monitored in worts.

The low phytate samples showed acceptable malt quality but quality did vary among the samples tested. All the low-phytate lines had significantly higher levels of zinc and magnesium which resulted in better fermentabilities. Low-phytate worts from the AC Metcalfe cross used more amino acids than the normal phytate worts. There was a similar tendency with the Harrington low-phytate lines but results were not as consistent.

A new high-throughput screening method for measuring lipoxygenase in barley seed.

Yin Li and Paul Schwarz*. Department of Plant Sciences, North Dakota State University. Fargo, North Dakota 58105-5051.

*Corresponding author Paul Schwarz 701-231-7732 Paul.Schwarz@ndsu.edu

Lipoxygenase is regarded as an undesirable enzyme that affects the flavor stability of beer, due to the enzymatic oxidation of linoleic acid to yield trans-2-nonenal by LOX pathway. Elimination of lipoxygenase from barley and malt will therefore greatly reduce the formation of trans-2-nonenal, resulting in significantly increasing the flavor stability of beer. Barley breeders are trying to identify barley germplasm that lacks of lipoxygenase. The major obstacle is to set up a fast and reliable lipoxygenase activity assay method. However, lipoxygenase was very difficult to achieve high-throughput screening as the reaction products (lipid hydroperoxides) are extremely unstable. In this research work, we established a new high-throughput screening method for lipoxygenase activity assay, which was based on the determination of lipid hydroperoxides by ferrous oxidation-xylenol orange (FOX) assay. The new method is able to measure over 100 samples per day. We feel it will be of great interest to the barley breeders, maltsters, and brewers.

Study of the effects of Pre-harvest Sprouting on the Storability and

Malting quality of AC Metcalfe, CDC Kendall and CDC Copeland Barleys

Yueshu Li*, Rob McCaig, Aleks Egi, Ken Sawatzky and Deye Tian, Canadian Malting Barley Technical Centre, 1365-303 Main Street, Winnipeg, Manitoba, Canada R3C 3G7

*Corresponding author: 204-984-0561, yli@cmbtc.com

Variations in germination and malting behaviors of AC Metcalfe, CDC Kendall and CDC Copeland barley samples during storage were examined at CMBTC. These barley samples have suffered different degrees of pre-harvest sprouting damage (PHSD) at harvest, and were stored under three different storage conditions during the course of this study. During storage PHSD barley samples showed significant changes in germination energy and water sensitivity than the sound barley (0% PHSD). The higher the degree of PHSD the more variations in germination energy were recorded. Although all the barley samples exhibited gradually increase in water sensitivity with storage duration, the samples with a higher degree of PHSD exhibited more changes than the samples with a lower degree of PHSD. Barley samples kept at room temperature showed more variations than the samples kept at a lower storage temperature. Varietal difference in germination energy, water sensitivity and their response to the storage condition were also observed. In the micromalting trials, at steep barley samples with higher degrees of PHSD showed a higher water uptake rate and lower chitting rates than the sound barley samples. Variations in the chitting rate showed a similar pattern as germination energy, barley samples with a high degree of PHSD and being stored at a higher temperature exhibited lower chitting rates. Some varietal differences in water uptake and chitting variations were also recorded. Micromalting trial results suggested that germination energy, water sensitivity, chitting rate, friability, enzyme level and beta-glucan content were all sensitive to PHSD and storage conditions. Changes in malt quality could be related to characteristics of the samples prior to storage, subsequent changes in barley quality during storage, and the interactions between pre-storage barley quality and storage conditions. Both barley pre-harvest condition and storage conditions largely determine the changes in germination and overall maltability, as well as the resultant malt quality of the barley.

Nutritional and functional properties of whole grain barley bagels

C.Lukie*, L. Malcolmson, A. Sarkar, and E. Sopiwnyk. Canadian International Grains Institute, 1000-303 Main St., Winnipeg, MB, R3C 3G7, CANADA.

Corresponding Author: C. Lukie 204-984-1063 clukie@cigi.ca,

Interest in incorporating whole grains into the diet of North American consumers has increased considerably due to reported health benefits associated with their consumption. Barley is an underutilized grain in food applications despite the recent health claim that is permitted for foods containing barley and beta glucan from barley sources. Hulless barley varieties have been developed which have an advantage in milling since the hull is naturally removed prior to milling. This study was undertaken to determine the functionality of whole grain barley flour made from the Canadian hulless barley variety CDC McGwire in a bagel application. Varying levels of whole grain barley flour were blended with wheat flour to determine dough and end product quality. Water absorption, volume, firmness, crumb structure and flavor were assessed. Nutritional analysis was also determined based on reported nutrient values. Results indicated that it was possible to create a whole grain barley bagel with good end-product quality, and that a suitable blend of whole grain barley and wheat achieved the soluble fiber health claim.

Screening for bird cherry-oat aphid resistance in barley

Do Mornhinweg USDA-ARS, 1301 N. Western, Stillwater, OK 74075 and Harold Bockelman, USDA-ARS, PO Box 386, 1691 S. 2700 W, Aberdeen, ID 83210

*Corresponding author: 405-624-4141, Do.Mornhinweg@ars.usda.gov

Bird cherry-oat aphid (BCOA), Rhopalosiphum padi (L.), has been reported to cause yield loss in small grains both through its role as an efficient vector of the PAV strain of Barley yellow dwarf virus (BYDV) and by actual feeding damage to winter and spring small grains by aviruliferous BCOAs. Barley accessions have been reported to have BCOA resistance based on the antibiotic effect of seedlings on the aphids. Whether this antibiosis translates to resistance in terms of grain yield has yet to be shown. Screening for BCOA resistance at the seedling stage has been difficult due to lack of visual symptoms on seedlings infested with BCOA using traditional greenhouse screening methods. In an attempt to develop a seedling screening technique for BCOA many variables were evaluated including flat type, soil type, infestation date, infestation rate, and screening conditions, (temperature and day length). In 2005, 78 barleys, reported to be antibiotic to BCOA, were screened with aviruliferous BCOA using traditional seedling screening methods under high temperature and long days and compared to non-infested controls. Seedlings were rated on a newly defined visual scale of 1 to 7 (1= resistant and 7= dead). Potential resistant and susceptible checks were identified. A replicated (2X) screening of a 960 accession subset of the Barley Core Collection was conducted using this technique. Seedlings from 284 accessions survived screening and were transplanted to pots in the greenhouse. Based on seedling survival percentages, the rating scale appeared to be valid. Seedlings from the surviving accessions were screened again in 2007 and an identical set of non-infested controls was grown. Surviving seedlings and their respective non-infested controls were rated, rescued, and transplanted to pots side by side in the greenhouse. Plant height, yield and yield components were measured to validate the rating scale.

An overview of the barley coordinated agricultural project

Gary J. Muehlbauer, Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN55108.

(612) 625-6228 muehl003@umn.edu

Barley geneticists and breeders have been successful in identifying hundreds of QTL that explain variation for yield, disease resistance, malting quality, and other agronomic traits. However, various reasons preclude the use of these QTL within breeding programs. The overall goal of the barley CAP is to identify QTL within breeding program germplasm and directly utilize the QTL in breeding efforts. The barley coordinated agricultural project (CAP; www.barleycap.org) is a community-based project focused on integrating genomics into barley breeding programs. Twenty-nine scientists from 19 institutions are participating in the project. An international collaboration composed of the Scottish Crop Research Institute, the German Institute of Plant Genetics and Crop Plant Research, NSF funding to Timothy Close (University of California, Riverside), and the barley CAP developed a 3,000 marker SNP genetic map and genotyping platform. The barley CAP QTL discovery platform is based on ten U.S. barley-breeding programs each submitting 96 F₄ or more advanced breeding lines each year of the project. Thus, over the four years of the project 3,840 lines will be submitted. The allele at approximately 3,000 SNP loci and phenotypic data on 40 traits will be collected on the breeding lines. All genotype and phenotype data will be stored in The Hordeum Toolbox database. The combined genotype and trait data provide the opportunity to use association genetics approaches to identify loci that control important traits. Results of some of the early association genetics analysis are promising.

Variation, causes, and significance of grain hardness in barley for food

Sindhu Nair, Byung-Kee Baik, and Steve Ullrich*, Dept. of Crop and Soil Sciences,WashingtonStateUniversity,Pullman,WA99164-6420.

* Corresponding author: 509-335-4936, Ullrich@wsu.edu

There is increasing interest in nutritional benefits of consuming barley food products, but experience in breeding of appropriate barley cultivars and their processing for food uses is limited. Kernel hardness (KH) of barley may influence post-harvest handling; pearling, rolling, and milling; flour particle size distribution; and end use product quality. There is limited understanding of genotypic variation and environmental influence on barley KH, the biochemical and physical basis of KH, and its significance in food processing. The objectives of this study were to determine the effects of genotype (G) and environment (E) on barley KH and the associations of KH with other kernel and food processing traits. De-hulled grains of 959 diverse breeding lines contributed by ten breeding programs in the USA as part of the USDA-funded Barley CAP were evaluated for KH using the single kernel characterization system (SKCS). Overall, KH varied from 30 to 92 units. Hulled and hulless types ranged from 30 to 92 and 42 to 91, respectively. Mean KH was 71 for winter and 62 for spring types. Overall mean kernel weight (KW) and kernel diameter (KD) ranged from 25 to 54 mg and 1.7 to 2.9 mm, respectively. Hulled lines exhibited wider variation in KW and KD than hulless lines. Spring and winter types were similar in KD distribution. The proportion of hull, determined by 80 sec abrasion, ranged from 10 to 21%. G and E effects on KH were determined using 14 lines grown with 3 replications at 12 locations. G, E, and G x E were significant with genotype playing the greatest role. Ten barley lines of varying KH (30 - 91) were selected to determine associations of KH with other kernel and food processing traits. KH showed no association with kernel weight, diameter, density, crease dimensions, protein and β-glucan contents, water absorption, and cooked barley texture. KH negatively correlated with pearling rate (r = -0.87***) and grain vitreousness (r = -0.82**) as measured by kernel brightness (L*). KH positively correlated with flour particles >106 µm (r = 0.93***) and percent starch damage (r = 0.93***).

Waxy Barley: N Applications for Yield, Beta-glucan, and Protein

O. Steven Norberg*, Oregon State University, Malheur County Extension Office; Brad Brown, University of Idaho, SW Idaho Research & Extension Center, Parma; Clint Shock, Oregon State, Univ., Malheur Experiment Station; Andrew Ross, Pat Hayes, and Juan Rey, Oregon State University, Crop and Soil Science Dept. Corvallis, OR.

*Corresponding author 541-881-1417, steve.norberg@oregonstate.edu

Nitrogen fertilizer (N) management was evaluated for growing irrigated waxy barley for higher protein and beta-glucan soluble fiber content. A local company has proposed the building of a barley fractionation plant to capitalize on these value-added traits. Salute and Merlin, two spring waxy barley cultivars, were fall-planted to compare yield and quality under different N treatments applied in late winter and at heading and to evaluate ethephon to reduce lodging. Fairly normal winter weather in the Treasure Valley reduced stands by 60 to 70% in 2006 and required replanting in the spring of 2007. In 2006, yield of Merlin and Salute were not different and averaged 5.4 Mg ha^-1. In 2007, Merlin yielded 6.6 Mg ha^-1 whereas, Salute only yielded 4.5 Mg ha^-1 and lodged heavily. In 2007, Salute had 30% lodging even where no late winter nitrogen was applied and lodging increased to 98% with only 67 kg ha^-1 of N. In 2007, ethephon at the full labeled rate increased Salute yield by 1.3 Mg ha^-1 and reduced lodging by 28%. Grain protein was influenced by year, variety, N rate, N at heading and ethephon application. Grain protein was 2.2 percent higher in 2006 than 2007. Merlin contained 0.8 percent more protein than Salute. Increasing the N rate from 0 to 180 kg ha^-1,increased grain protein from 10.0 to 12.9 percent. The application of 45 kg ha^-1 N at heading consistently increased protein 1 percent. Ethephon decreased grain protein content by 0.4 percent. Flag leaf N at heading was strongly related to grain protein (R²= 0.79). Ethephon influence on beta-glucan was inconsistent over years. Averaged over years and varieties, 45 kg ha^-1 N at heading increased the beta-glucan from 5.4 to 5.7%.

Prediction of Deoxynivalenol (DON) content in grain using Near Infrared Reflectance Spectroscopy (NIRS).

Lori Oatway*, James Helm and Kequan Xi. Alberta Agriculture and Rural Development, Field Crop Development Centre, 5030 - 50^th Street, Lacombe, AB Canada.

*Corresponding author: 403-782-8048, lori.oatway@gov.ab.ca

As long as current agricultural practices cannot prevent Fusarium infection in small grains, deoxynivalenol (DON) will continue to be a critical issue in animal and human health. The purpose of this project was to determine if near infrared reflectance spectroscopy (NIRS) can predict DON concentrations in cereal grain. First, the NIRS absorption spectrum of DON was isolated and used to develop a baseline calibration using specific wavelength ranges and standard samples developed from commercial flour and pure DON. The second part of this project transferred the baseline calibration to naturally-infected samples of ground wheat and barley. The best equation developed had a coefficient of determination (R²) of 0.90, standard error of calibration (SEC) of 0.64 ppm, and standard error of cross validation (SECV) of 0.72 ppm. The equations developed in this study demonstrate that NIRS can effectively predict DON content in wheat and barley.

Association mapping of malt quality QTL in the U of M barley breeding program: Detection, validation, and breeding

C.A. Powers^1*, Paul Schwarz², Yin Li ², K.P. Smith¹. 1-Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul MN 55108. 2- Department of Plant Sciences, Harris 201,North Dakota State University, Fargo ND 58105.

*Corresponding author: 612-625-3151, power134@umn.edu

Recently published association mapping studies have focused on germplasm collections, designed populations or varieties entered in national and regional trials. Association mapping within a breeding program has the potential to accelerate the translation of newly acquired genetic information to application in breeding. We have used 182 lines from the University of Minnesota barley breeding program to conduct association mapping for an array of malting quality traits. Preliminary results detected 13 QTL for eight traits (malt extract, alpha-amylase activity, diastatic power, residual beta-glucanase activity, wort protein, wort color, barley protein, and percent plump). Locations of the QTL were on chromosomes 2H, 4H, 5H, and 6H. Two QTL regions were associated with more than one trait. Validation using near-isogenic lines (NIL) has been initiated using Midwest breeding lines that are heterozygous for target QTL. To further characterize QTL, we assessed the pattern of linkage disequilibrium (LD) at the target QTL, identified haplotype blocks, and estimated the effects of QTL alleles. For example, we detected one QTL for barley protein on chromosome 4H that is contained within a 2 cM block of LD that includes nine markers. When considering only the seven markers significantly associated with protein there are four haplotypes. The least frequent haplotype (1.9%) contains the desirable allele that reduces grain protein. We are now in a position to use marker assisted selection to increase the frequency of this and other useful alleles in our breeding program to develop improved malting quality varieties.

Distribution and Diversity of Russian Wheat Aphid (Homoptera: Aphididae) Biotypes in North America

Gary J. Puterka* and Dolores W. Mornhinweg, USDA Agricultural Research Service, Plant Science Research Laboratory, Stillwater, OK 74075

*Corresponding author: 405-533-1050, gary.puterka@ars.usda.gov

Wheat with Russian wheat aphid (RWA) resistance based on the Dn4 gene has been important in managing RWA since 1994. Currently, there are eight biotypes (RWA1 - RWA5) of this aphid that have been described based on their ability to differentially damage RWA resistance genes in wheat. RWA2, RWA4 and RWA5 are of great concern because they can kill wheat with Dn4 resistance. In 2005, 365 RWA clone colonies were made from collections taken from 98 fields of wheat or barley in Oklahoma, Texas, New Mexico, Colorado, Kansas, Nebraska, and Wyoming to determine their biotypic status. The biotype of each clone was determined through its ability to differentially damage four resistant and two susceptible wheat entries in two phases of screening. Only two biotypes, RWA1 and RWA2, were identified in this study. The biotype composition across all collection sites was 27.2% RWA1 and 72.8% RWA2. RWA biotype frequency by state indicated that RWA2 was the predominant biotype and composed 73 - 95% of the biotype complex in Texas, Oklahoma, Colorado and Wyoming. Our study indicated that RWA2 is widely distributed and has rapidly dominated the biotype complex in wheat and barley within its primary range from Texas to Wyoming. Wheat with the Dn4 resistance gene will have little value in managing RWA in the United States, based on the predominance of RWA2. However, the two main sources of resistance in barley, STARS 9301B and 9577B, remain resistant to the eight known RWA biotypes.

Fluorescence microplate readers as an alternative to flow injection analysis for determination of wort beta-glucan.

Mark R. Schmitt* and Allen D. Budde. USDA Agricultural Research Service, Cereal Crops Research Unit, 502 Walnut Street, Madison, WI 53726. USA.

*Corresponding author (608) 262-4480 mark.schmitt@ars.usda.gov

Wort beta-glucan concentration is a critical malting quality parameter used to identify and avoid potential brewhouse filtration problems. ASBC method Wort-18 is widely used in malt analysis laboratories and brewhouses to measure wort beta-glucan. However, the chemistry underlying the method is suitable for other instrumentation, such as fluorescence microplate readers. Fluorescent microplate readers are significantly less expensive than commercial FIA systems and are widely available in research laboratories. The microplate implementation of the Calcofluor/beta-glucan method presented here is simple to execute, and offers several advantages due to its high throughput, flexibility, and low sample volume requirement. The latter consideration makes it well suited to analysis of worts from limited-availability samples, such as those from early-generation breeding lines.

Let's get small: Miniaturized malt quality analysis for fun and profit.

Mark R. Schmitt* and Allen D. Budde. USDA ARS Cereal Crops Research Unit, 502 Walnut Street, Madison, WI 53726.

*Corresponding author (608) 262-4480 mark.schmitt@ars.usda.gov

Most common laboratory-scale micromalting and malt analysis procedures are indeed micro-scaled compared to commercial-scale malting (the CCRU standard 170 g malting is approximately 10⁶ times smaller than a 6,000 bu Saladin box production malting run). While these amounts of grain are insignificant for a malthouse or brewery where railcar quantities are normally handled, the quantities needed for the micromalting and standard analysis procedures may be problematic for both research and breeding programs. The requirement for >100 g of seed for malting/analysis effectively precludes testing for important malting quality attributes until relatively late in a breeding program, even though meeting malting quality standards is one of the most critical selection criteria. This means that many lines with inferior malting quality must be carried through several generations before sufficient grain is available for even a first round of malt QA testing that would identify those lines with malting quality shortcomings, so that they could be eliminated from the breeding program. Similarly, it becomes much more difficult to analyze mapping and other special populations if seed increases are needed to produce the >100 gm of seed needed for standard QA tests for each line of interest. Fortunately, it has recently become possible to adapt traditional micromalting and malt analysis procedures to much smaller scales, reducing the quantities of grain needed to generate a malt quality profile similar to those from traditional methods. The availability of the reduced-quantity malt quality methods have greatly simplified analysis of genetically interesting populations and could facilitate earlier-generation analysis of malting quality in breeding programs when fully implemented. In addition, several labs globally have adapted a number of related research procedures to small-scale/high-throughput formats, facilitating studies on basic research questions relating to modification of barley carbohydrate, protein, and cell wall reserves. Examples of the modified malting, mashing, and analytical procedures will be presented.

Barley and oat grain and malt beta-glucan content measured by Calcofluor fluorescence in a microplate assay.

Mark R. Schmitt* and Mitchell Wise. USDA Agricultural Research Service, 502 Walnut Street, Madison, WI 53726.

*Corresponding author (608) 262-4480 mark.schmitt@ars.usda.gov

Beta-glucan levels in grains, particularly barley and oats, are receiving increased interest in part due their recognized benefits to human health. While a number of methods to determine grain beta-glucan levels are available, each suffers from significant drawbacks for routine implementation. Adaptation of a Calcofluor fluorescence method to detection by a microplate fluorimeter results in a simple mechanism for measuring beta-glucan, using inexpensive reagents, and affordable, readily available instrumentation.

Survey of Barley Producers in Idaho, Montana and North Dakota

Paul Schwarz. Institute of Barley and Malt Sciences. North Dakota State University. Fargo, ND 58105

*Corresponding author: 701-231-7732, Paul.Schwarz@ndsu.edu

The supply of domestic malting barley in the USA is a serious concern, and in fact, 2006 production was the lowest since 1933. In order to address satisfaction and concerns with malting barley as a crop, the Institute of Barley and Malt Sciences conducted a survey of 5000 barley growers in Idaho, Montana, and North Dakota. The rate of response to the survey was 32, 29, and 26% in Idaho, Montana, and North Dakota respectively. Slightly more than 1400 responses were received. The twelve question survey focused on production/economic issues and informational needs. Production/economic questions focused on the past 10 years, and targeted factors influencing a producer's decision to grow barley, the satisfaction or rank of barley relative to other crops produced, and rate of barley acceptance . Informational questions addressed the type of information that is of value to barley producers and their current or desired sources of the information.

Association mapping of Fusarium head blight QTL using contemporary barley breeding germplasm

Kevin P. Smith¹^*, Jon Massman¹, Blake Cooper², Rich Horsley³, Stephen Neate³, Ruth Dill-Macky⁴, Shiaoman Chao⁵, Yanhong Dong⁴, and Paul Schwarz³

¹University of Minnesota, Department of Agronomy and Plant Genetics

²Busch Agricultural Resources, Inc.

³North Dakota State University, Department of Plant Sciences

⁴University of Minnesota, Department of Plant Pathology

⁵USDA-ARS Biosciences Research Lab, Fargo, ND

*Corresponding author: 612 624-1211, email: smith376@umn.edu

The serious impact of Fusarium head blight (FHB) on barley production in the Midwest has prompted a substantial research effort to map quantitative trait loci (QTL) that could be exploited in marker assisted breeding. Previously described resistance QTL (identified in wide, bi-parental mapping studies) are often associated with negative agronomic traits such as late heading and taller plants, thus reducing their overall utility. As part of the USDA-funded Barley Coordinated Agricultural Project (CAP), we are using association mapping to identify QTL segregating in contemporary breeding lines which should be free of such associations. 768 breeding lines from four barley breeding programs were each evaluated for resistance in four experiments in 2006 and 2007. The lines were planted in a randomized complete block design with two replications, inoculated, and mist irrigated to encourage disease development. Each line was genotyped using 1,536 single nucleotide polymorphism (SNP) markers, and QTL were mapped using a mixed-model approach. Phenotypic variation among lines for disease severity was significant (p<0.0001) , but skewed toward resistant. Linkage disequilibrium extended more than 10cM on average, indicating association mapping should be feasible with the available marker density. Population structure was accounted for using molecular marker and pedigree data. Multiple QTL with generally small effects (R² < 5%) were identified. Fewer QTL were identified in analyses using only two-rowed breeding lines compared to six-rowed lines. Overall eight QTL for DON and four QTL FHB were reproducibly identified. These loci should be useful targets for immediate implementation of marker assisted selection to improve disease resistance.

Progress towards predictive models for Fusarium Head Blight and DON in barley.

J. Stein*, K. Bondalapati, L. Osborne - Plant Science Department, South Dakota State University, Brookings, SD; S. Neate - Department of Plant Pathology, North Dakota State University, Fargo, ND, and C. Hollingsworth -University of Minnesota Research and Outreach Center, Crookston, MN.

* Corresponding author, Ph: (605) 688-5540, E-mail: jeff.stein@sdstate.edu

Fusarium head blight (FHB), caused by the fungus Gibberella zeae (anamorph: Fusarium graminearum), continues to be a serious problem for barley producers in the U.S. Northern Great Plains and elsewhere. G. zeae can cause direct economic loss through a reduction in grain yield and it also produces mycotoxins that can impact the marketability of a crop, e.g. deoxynivalenol (DON). Management of FHB is accomplished with agronomic practices that limit in-field inoculum (e.g. rotation) and through the application of fungicides. The timing of application can be critical and therefore a need exists for a risk-advisory system that growers could use to make management decisions. The objective of this research was to develop model(s) for such a system that predict FHB and/or DON based on weather conditions. Varieties of regionally adapted barley (both 2- and 6-row types) were grown at multiple locations in the Northern Great Plains during the 2005-8 growing seasons. Crop stage was monitored regularly and no additional inoculum was applied. The incidence and severity of FHB was measured and environmental variables were recorded. Correlation analysis was conducted to identify the variables that were associated with high disease and/or DON events and then logistic regression was used to develop predictive models.

Simple weather variables that explained general trends (e.g. mean hourly temperature) tended to have the highest correlation coefficients and were most predictive of high FHB/DON instances. In general, high levels of disease and DON occurred at a location when the mean hourly temperature and relatively humidity were both greater than 22C and 75%, respectively, for the 9 days prior to full head emergence. A preliminary model was developed that combined these variables. This model had true positive and negative rates of ~90% when tested with the 2005-7 data sets and was able to predict low disease in all of the 2008South Dakota testing locations. Further analysis of model accuracy is ongoing.

Identification of a putative new barley leaf rust resistance gene

Yongliang Sun and Stephen M. Neate*

Department of Plant Pathology, North Dakota State University, Fargo USA

*Corresponding Author: 701-231-7078, stephen.neate@ndsu.edu

Resistance is the preferred means of controlling leaf rust of barley caused by Puccinia hordei G. Otth. However, changing virulence in P. hordei has rendered ineffective many of the known resistance genes. In this study, isolates `Race 8', `90-3', '90-15', `89-3', and `Neth 202' of P. hordei were used to differentiate resistance genes in 82 selected barley lines. Putative new resistance was identified in barley line `C2-02-134-2-2' and separated from Rph15 also known to be in that line. Based on disease reaction when challenged with isolate `Race 8', the F₂population of a cross between `C2-02-134-2-2' and a susceptible line `ZA47', segregated into a 15:1 resistant to susceptible ratio (χ²=0.853) indicating the presence of more than one gene. In the F₂ generation, the Rph15 phenotype (00;) separated from a second resistance gene phenotype (0;12-). To isolate the gene which gave the (0;12-) phenotype from Rph15, the 10 F₂ plants bearing the (0;12-) phenotype were transplanted and selfed, and the F_2:3families were screened for homogeneity of disease reaction. To determine inheritance and to undertake gene mapping using diversity array technology (DArT) a new cross was made between `Bowman' and a plant from a homogeneous family that showed the (0;12-) phenotype. The putative new gene was mapped on barley chromosome 7H and when inoculated with a selected set of P. hordei races showed different phenotypic reactions from Rph3 and Rph19 known to be on the same chromosome. Allelism studies with Rph3 and Rph19 are in progress.

Barley Feed Quality - Is It Consistent Across Species?

Mary Lou Swift*¹, James Helm¹, Tim McAllister², Fred Silversides³, and Ruurd Zijlstra⁴. ¹Field Crop Development Center, Alberta Agriculture and Rural Development, 5030-50^th Street, Lacombe, Alberta, Canada. T4L1W8. ² Research Centre, Agriculture and Agri-food Canada, Lethbridge, Alberta. T1J 4B1. ³Pacific Agri-food Research Centre, Agriculture and Agri-food Canada, POB 1000, Agassiz, British Columbia, Canada. V0M 1A0; ⁴Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5.

*Corresponding Author: 403-782-8693; Mary-lou.Swift@gov.ab.ca

Barley grain is primarily included in the diet of farm animals as a source of digestible energy. Regardless of species fed, there is variation in the feeding value of barley due to genetic and environmental factors. However, there is little information published in the scientific literature comparing the feeding value for different livestock classes of a standard set of barley samples. The Field Crop Development Centre together with university, government and industry collaborators conducted a three-year study to collect data regarding the nutritional value of barley for poultry, ruminants and swine. Fifty samples of hulled barley were evaluated for ruminants using in-vitro gas production, for swine using in-vitro 3-stage enzymatic digestion, and for poultry using an animal bioassay. Data was entered into JMP Version 4 (SAS Institute Inc.) and correlation coefficients (r) calculated to ascertain the relationships between the various measurements of feed value for ruminant and swine and for swine and poultry.

The best relationship between ruminant and swine, as determined by highest r value, was that of total ruminal volatile fatty acid production (24h) and swine DE content (r=0.33). The relationship between ruminal 24 h starch disappearance and swine DE was 0.12 and between protein disappearance and DE was 0.18. Relationships between swine DE values and poultry AME values were dependent on whether exogenous enzyme had been added to the diet. Without enzyme, the relationship was negative in nature (r= -0.21). With the addition of enzyme a positive relationship (r = 0.14) was found. However, the relationship between swine DE content and poultry performance was negative in nature, regardless of enzyme addition. For example, the relationship between swine DE and feed intake was -0.51 without enzyme and -0.22 with enzyme. Similarly, r values for swine DE and average daily gain of broilers was -0.47 and -0.09, without and with enzyme, respectively.

In summary, data from this study would indicate that selection of a hulled barley variety based on higher swine DE content may result in little or no improvement in the feeding value of that barley for the ruminant animal. In addition, the selection of the same hulled barley could well result in decreased broiler performance in terms of feed intake and average daily gain. This analysis would indicate that the design of plant breeding programs to enhance the feeding value of hulled barley should incorporate variety evaluation across species. Further study of the genetic linkages of these feed quality traits is needed.

Expression analysis of ethylene biosynthesis and receptor genes from barley embryo and tissue culture

Neerja Tyagi and Lynn S. Dahleen*. Plant Sciences Dept., North Dakota State University and USDA Agricultural Research Service, Cereal Crops Research Unit, 1307 18th St N, Fargo, ND 58105

*Corresponding author: 701-239-1384, lynn.dahleen@ars.usda.gov

Ethylene affects regeneration of green plants from barley tissue culture. With the availability of the HarvEST barley database and barley GeneChip, genome-wide expression studies have focused on differential development between Morex and Golden Promise at various stages of plant growth. The data from these studies are available from the Barley Gene Atlas and provide an excellent source for datamining genes of interest. We used ethylene biosynthesis genes 1-aminocyclopropane-l-carboxylic acid synthase (ACS) and ACC oxidase (ACO), and ethylene receptor 1 (ETR1) as search criteria and selected the respective unigenes and their probes from HarvEST. The probes were used to examine the expression results from embryo, coleoptile and radicle tissue in the Barley Gene Atlas. The consensus sequences from a set of same unigenes as above were used to design primers that were tested on RNA (cDNA) extracted from Morex and Golden Promise at various tissue culture stages. The results from datamining and cDNA experiments were compared. The different probes from same unigene gave variable expressions in the two cultivars. The cDNA experiment indicated that ACS-6923 was constitutively expressed in both cultivars, while ACS-13017 and ACS-1108 showed differential expression. Similar results were seen for ACO. The unigenes ACO-1780 and ACO-1786 tested in Morex were differentially expressed in different stages of tissue culture. In the Barley Atlas experiment, ACO-1781 was the most differentially expressed ACO between the cultivars. The expression of ACO-1784 varied considerably between tissues; 38% higher in Morex embryos, 90% higher in Morex coleoptiles, but almost three times higher in Golden Promise radicle tissue. cDNA expression of ETR1 was not studied and the data from Barley Gene Atlas indicates constitutive expression. The dataming expression studies confirm our findings but also show some interesting differences as the same unigenes are represented by different probes and more than one probe exists for some unigenes. In most cases the expression in Morex was higher than Golden Promise, sometimes by as much as 90%. This variation in expression could contribute to the differential performance of these cultivars in tissue culture and can be used to modify the expression of these genes to achieve higher regeneration.

Greenhouse and Field Demonstration of Microbial Suppression of Fusarium Head Blight in Barley.

J.E. Van Cauwenberge*¹, Schisler, D.A.¹, Cooper, D.², Smith, K. P.³ ¹Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, Peoria, Il. ²Busch Agricultural Resources, Inc., Fort Collins, Co. ³Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Mn.

^*Corresponding author: (309) 681-6011, jim.vancauwenberge@ars.usda.gov

Fusarium head blight (FHB) is a disease of wheat and barley responsible for extensive losses in quality and yield. The primary causative agent for FHB is Gibberella zeae (anamorph = Fusarium graminearum) which initially infects spikes to produce dark brown water soaked spots on glumes of florets and ultimately can cause thin blighted kernels, reduced test weights, and reduced grain quality. To determine the feasibility of reducing FHB on barley using microbial antagonists, seven strains of gram positive bacteria and yeasts previously described as suppressive to FHB on wheat were examined. After production in liquid culture, antagonists were sprayed onto barley heads at anthesis in both greenhouse and field trials. Lacey and Tradition, six-rowed malting varieties with high yield, good lodging resistance and kernel plumpness, were used in all trials. In greenhouse studies, suspensions of Gibberella zeae conidia were applied onto heads immediately after antagonist suspensions. In field trials Gibberella zeae was grown on cracked corn then broadcast over the test plot two weeks prior to anthesis and application of microbial treatments. In at least one set of duplicated greenhouse experiments, five microbial strains reduced FHB by 25-84% on Tradition and four strains reduced FHB by 52-71% on Lacey. In field trials, four microbial strains reduced FHB by 10-23% on Tradition while one strain reduced FHB by 17% on Lacey. Strain efficacy in the greenhouse was not always predictive of field performance. Further research to evaluate the potential of including microbial antagonists in the integrated management of FHB on barley is justified.

β-Amylase activity and thermostability in wild and cultivated barleys with different Bmy1 intron III alleles

Marcus A. Vinje¹, Stanley H. Duke¹, and Cynthia A. Henson^{1, 2*}

¹University of Wisconsin-Madison, Department of Agronomy, ²USDA-ARS, Cereal Crops Research Unit

^*Corresponding author: 608-262-0377, Cynthia.henson@ars.usda.gov

The third intron of barley (Hordeum vulgare L.) β-amylase 1 (Bmy1) is extremely polymorphic. The use of specific insertion/deletions (indels), in the third intron as selective markers for cultivar development, has been suggested based on associations with β-amylase activity and thermostability. The third intron of Bmy1 in 40 barley accessions was sequenced and four alleles (Bmy1.a, Bmy1.b, Bmy1.c, and Bmy1.d) were identified based on indels of 126-bp, 38-bp, 11-bp, and 21-bp. β-Amylase activity and thermostability were assayed in 22 North American cultivars important to the malting and brewing community and 12 wild barley accessions, which are a rich source of genetic diversity. The cultivars were found to have the Bmy1.a and Bmy1.b alleles with β-amylase activity ranges of 946-1723 and 1443-2146 U/g flour, respectively, and β-amylase thermostability ranges of 4.1-36 and 22.5-26.3% residual activity, respectively. There were significantly different activities among cultivars carrying either allele, according to Fisher's least significant difference (LSD) analysis. Cultivars carrying the Bmy1.a allele also had significantly different thermostabilities. The wild barleys were found to carry Bmy1.a, Bmy1.b, and Bmy1.c alleles with β-amylase activity ranges of 668-1124, 908-1554, and 628-1629 U/g flour, respectively, and β-amylase thermostabilities of 26.6-33.3, 20.4-27.7, and 23.2-49.1% residual activity, respectively. LSD analysis revealed significantly different activities in accessions carrying any of the intron alleles and significantly different thermostabilities in accessions carrying the Bmy1.b or Bmy1.c alleles. These data suggest that the use of the Bmy1 intron III alleles as a marker to predict and select for β-amylase activity and/or thermostability is unreliable.

Differential RNA expression of two barley β-amylase genes (Bmy1 and Bmy2) in developing grains and their association with β-amylase activity

Marcus A. Vinje¹, David K. Willis³, Stanley H. Duke¹, and Cynthia A. Henson^{1, 2*}

¹University of Wisconsin-Madison, Department of Agronomy, ²USDA-ARS, Cereal Crops Research Unit, ³USDA-ARS, Vegetable Crops Research Unit

^*Corresponding author: 608-262-0377, Cynthia.henson@ars.usda.gov

RNA expression from the barley β-amylase1 (Bmy1) gene was determined during seed development in four genotypes (Legacy, Harrington, Ashqelon, and PI 296897). The Bmy1 transcript amount in Legacy and Harrington was not significantly different at 17, 19, or 21 days after anthesis (DAA). Ashqelon Bmy1 expression is around three-fold higher than Legacy and Harrington at 17, 19, and 21 DAA. The Bmy1 expression in PI 296897 is around 40-fold lower than Legacy and Harrington at 17 and 21 DAA and around 24-fold lower than Legacy and Harrington at 19 DAA. PI 296897 had over 100-fold lower Bmy1 RNA levels at 17 and 21 DAA and 76-fold lower Bmy1 expression at 19 DAA than Ashqelon. β-Amylase activity was measured to determine if the expression levels were comparable to activity. Harrington had 2.9, 2.6, and 2.1-fold lower activity at 17, 19, and 21 DAA than Legacy even though their Bmy1 transcript levels were the same. The β-amylase activity levels of Legacy and Harrington in mature grain were the same. The peak expression of Bmy1 in Harrington was not ascertained because the Bmy1 expression at 19 DAA was statistically the same as the Bmy1 expression at 21 DAA. The discrepancy between β-amylase activity in Harrington at 21 DAA and at maturity indicated that Bmy1 transcript levels may peak later or Bmy1 is expressed over a longer time period than occurs in Legacy and/or other genotypes. Ashqelon had three-fold higher Bmy1 expression than Legacy and Harrington and had higher β-amylase activity. PI 296897 had higher β-amylase activity at all developmental time points and at maturity than Legacy and Harrington despite having between 24 and 44-fold lower Bmy1 transcript levels. This discrepancy led us to determine the RNA levels of Bmy2, which is known to be expressed in early seed development. Bmy2 expression is very low in Legacy and Harrington at 17, 19, and 21 DAA indicating that it probably is not contributing significantly to the total β-amylase activity. Bmy2 transcript levels in Ashqelon were not significantly different from Legacy or Harrington. Bmy1 expression seems to be the major contributor to β-amylase activity in the time points measured for Legacy, Harrington, and Ashqelon. PI 296897 has between 3 and 11-fold higher Bmy2 expression at 17, 19, and 21 DAA than the other three genotypes. These data lead us to propose that the expression of Bmy2 is contributing more β-amylase activity in PI 296897 by being expressed later in seed development.

Agronomic practices to improve winter hardiness of two-row winter malt barley `Charles'.

Juliet M. Windes^1* and Don E. Obert². ¹University of Idaho, Idaho Falls, ID 83402. ²USDA-ARS, Aberdeen, ID, 83210.

*Corresponding author: 208-529-8376, jwindes@uidaho.edu

Yield improvement of quality malt varieties is a priority goal for barley breeders. Winter varieties offer a substantial improvement on yield potential compared to spring types, and the release of `Charles', the first two-row winter malt variety, could mean increased yields with less irrigation than spring types. Limiting the adoption of Charles is its susceptibility to winter kill. Planting methods were investigated to improve winter survival. Four replications of Charles were planted in a split-split plot design with `Eight-Twelve,' a winter feed barley with better winter hardiness than Charles. Both varieties were planted in four-row plots, either drilled conventionally or planted in a 4-inch deep furrow created by shanks placed ahead of double disks. Randomized within planting method were six seeding rates, varying from 200,000 seeds/A to 1.25 million seeds/A. Plots were planted late, October 11, 2007, to increase potential for winter stress. Plots were rated twice for spring stand, and data were collected for yield, test weight, plumps and protein. Results show significant differences in yield between varieties (P=0.0002), planting method (P<0.0001), and planting rate (P<0.0001). There were significant differences in early stand between varieties (P<0.0001), planting method (P<0.0001), and planting rate (P<0.0001), as well as in late stand between varieties (P=0.0005), planting method (P<0.0001), and planting rate (P<0.0001). Stand and yield of both varieties increased significantly when planted in deep furrows. Averaged over variety and planting rate, conventionally drilled barley yielded 58 bu/A and deep furrow-planted barley yielded 113 bu/A, and stand increased from 11 to 66%, respectively. With both methods, significant increases in yield did not occur above the 400,000 seed/A planting rate. Over all planting rates, Charles yielded 7 bu/A when conventionally drilled, and 84 bu/A when furrow-planted, while Eight-Twelve yielded 109 bu/A and 141 bu/A, respectively. Significant improvements in yield and stand can be achieved by deep-furrow planting, but not as much or not at all by increasing seeding rate.

Tocol Content of Barley CAP germplasm, Preliminary Results

Mitchell L. Wise, USDA Agricultural Research Service, Cereal Crops Research Unit, 502 Walnut St., Madison, WI 53726

608-262-4292, mitchell.wise@ars.usda.gov

Tocochromanols (tocols) is the collective term for vitamin E active constituents found primarily in plants. These compounds have potent anti-oxidant properties and are a recommended nutrient for human health. Although the precise benefits of vitamin E are not known, diets deficient in vitamin E appear to be associated with atherosclerosis. Moreover, the tocotrienols, tocols incorporating a polyunsaturated isoprene chain, are known to reduce cholesterol levels in several experimental models, including humans. Barley provides a moderate source for tocols compared to other grains such as wheat and rye, however, they are relatively rich in the tocotrienols and they produce all eight naturally occurring tocol congeners. Moreover, from the few published accounts on barley tocol production, there appears to be significant genotypic variation. The present study presents preliminary data from analysis of tocol composition of the first two years of Barley Coordinated Agriculture Project (Barley CAP).

Validation of molecular markers for scald resistance in two-row barley.

Zantinge* J.L., Juskiw, P., Hartman, Z., and Xi K. Field Crop Development Centre, Alberta Agriculture and Food, 5030 - 50th Street, Lacombe, AB., T4L 1W8, Canada.

*Corresponding author (403)782-8692 jennifer.zantinge@gov.ab.ca.

www1.agric.gov.ab.ca/app21/rtw/selsubj.jsp

Scald (Rhynchosporium secalis) of barley is prevalent in central Alberta, Canada and causes considerable yield and quality losses. Scald can rapidly change in pathotype composition and frequency, thereby making it difficult to develop durable scald resistance in barley. For the past fifteen years in our two-row barley-breeding program at the Field Crop Development Centre, we have been trying to incorporate durable or multi-gene resistance to scald into lines with good malting quality. It has proven to be illusive. Previous studies have however, shown that the cultivar Seebe, a two-row feed barley, carries durable genetic resistance (released in 1992 it still gets resistant ratings in field evaluations), however, barley breeders have found this resistance difficult to transfer into new barley lines with good malting quality. Molecular marker analysis has identified that the SSR markers linked to scald resistance from Seebe represent three putative QTLs. These QTLs for scald resistance were identified to be on chromosomes 3HL, 4H (centomeric region) and 5HL. This study was under-taken to validate the utilization of these molecular markers for scald resistance; as well as, investigate the relationship between the scald resistance from Seebe and malting quality. While Seebe has proven to have durable resistance, it does not modify well and has high beta-glucan levels in the malt. Four crosses were made between Seebe and lines with malting quality. Lines from these four crosses were advanced to the F5 using single seed descent. In 2006, up to one hundred lines from each cross were assessed for seedling resistance to a common R. secalis strain isolated from the susceptible cultivar Harrington and markers for the Seebe scald resistance genes. The F6 generation of these lines and up to an additional 100 lines per cross were grown out in 2007 for marking of Seebe scald resistance genes, assessment of field resistance to scald, and malting quality determination using NIRS. Results from these two years of study showing the relationship of seedling and field response to R. secalis, Seebe scald resistance markers, and malting quality will be presented.

QTL Mapping and Molecular Marker Development for Seed Dormancy in Spring Barley.

J.L. Zantinge*, J Nyachiro, S. Xue, J. H. Helm, P. E. Juskiw and D. Salmon. Field Crop Development Centre, Alberta Agriculture, Food and Rural Development, 5030 - 50 Street, Lacombe, AB T4L 1W8

*Corresponding author (403)782-8692 jennifer.zantinge@gov.ab.ca

Wet field conditions just prior to harvest can cause pre-harvest sprouting in barley (Hordeum vulgare) resulting in significant economic losses especially in barley genotypes with low seed dormancy. On-the-other-hand, too much dormancy can cause inconsistent germination, creating problems in the malt house or during crop seeding. Seed dormancy is defined as the failure of viable kernels to germinate under optimum conditions of moisture, oxygen, and temperature. Selection for sprouting resistance in a barley-breeding program is challenging because the dormancy trait is influenced by the environment, and is controlled by multiple genes, that are often linked to important quality traits. Utilizing molecular markers linked to dormancy would be one method of selecting for desirable levels of seed dormancy in barley without the problem of environmental effects on expression, and could be used to break up undesired linkage (repulsion) effects that reduce seed quality. The objective of this study was to identify, map and develop molecular markers linked to genes affecting dormancy from `Samson' barley. Several recombinant inbred lines (RILs) were developed by crossing `Samson' derived lines (high in dormancy) with `TR118' (a malt variety from Dr. B. Harvey,Saskatoon). Dormancy levels were determined using a weighted germination index (WGI) on this population of 239 RILs. This phenotyped population was subsequently analysed with SSR markers and several QTLs for dormancy were identified across the genome. Several of the QTLs identified in this population, confirmed the locations of previously identified QTLs found in other genetic populations. In addition we also identified two new QTLs for seed dormancy on chromosomes 2H and 4H.

Exploiting historical malting quality data from the Western Canadian Cooperative Two-row Barley trials using association mapping

Aaron D. Beattie¹, Brian G. Rossnagel¹ and Michael J. Edney²

¹Crop Development Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8

²Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada R3C 3G8

The potential of a barley genotype to produce good malt is evaluated for a range of traits such as alpha amylase, beta-glucan, diastatic power, fermentable extract, free amino nitrogen, germination energy and Kolbach index. These indicators are used in various ways to predict fermentability, the most valuable characteristic of malt. For example, Evans et al. (2005) determined that a combination of alpha-amylase, beta-amylase, limit dextrinase and heat stable beta-amylase correlated well with fermentability.

Malting quality traits are time consuming to measure and are assessed at the final stages in barley breeding programs on a limited number of selections. Identifying molecular markers associated with these traits would allow screening of larger number of genotypes earlier in the breeding cycle to ensure important malting-related loci are maintained. Malting analysis would then be valuable to help select the best remaining lines which may be segregating for loci affecting malting quality which have not yet been identified.

A number of studies have identified malting quality regions of the genome using QTL analysis. These studies have tended to use populations derived crosses between a good malting genotype and a poor one (such as a feed genotype). For example, the Steptoe x Morex (Hayes et. al. 1993) and Harrington x TR306 (Mather et. al. 1997) populations were used to identify QTLs across all seven chromosomes. Typical of QTL studies, poor mapping resolution results from the limited number of segregating markers in biparental crosses and further work is required to refine the location of trait-associated loci. For example, Gao et. al. (2004) fine mapped an important region near the telomere on the short arm of chromosome 4H which had been previously identified by Hayes et. al. (1993). These studies have done a good job identifying genomic regions that differentiate very good malting genotypes from poor, however its likely that most good malting barley selections within breeding programs already incorporate these positive alleles.

Rather than using a collection of genotypes which include a wide range of malting quality, this study used only elite malting quality barley genotypes from the Western Canadian Co-operative Two-Row Barley Trials to determine if novel loci could be identified which may differentiate good from very good malting genotypes. In addition, a greater degree of mapping resolution should be possible using the AM approach making subsequent molecular marker-assisted selection (MMAS) more accurate.

Material and Methods

Ninety-one two-row barley genotypes from the 1994-2006 Western Co-operative Two-Row Barley trials were collected from participating breeding programs or the Plant Gene Resources Centre of Canada – Agriculture and Agri-Food Canada (AAFC; Saskatoon, SK). These genotypes represent elite malting genotypes from eight different breeding programs, including the currently most popular malting varieties (AC Metcalfe, CDC Copeland, CDC Kendall, Harrington, Newdale) and newer varieties such as Calder, CDC Select and CDC Aurora Nijo, CDC Reserve and CDC Meredith. DNA was extracted and sent for DArT (Diversity Array Technology) whole genome genotyping. Malting quality data was collected by the Grain Research Lab (Canadian Grain Commission).

Population clustering was analyzed by unweighted pair-group method (UPGMA) clustering (based on the Dice similarity coefficient) using NTSYSpc v. 2.2 to provide a visual representation of the relatedness of the AM population. Genetic variation within and among breeding programs was evaluated by an analysis of molecular variance (AMOVA) and described using Wright’s fixation index (F_ST). Pairwise F_ST comparisons were made between the breeding programs to describe genetic diversity. Arlequin v. 3.11 was used for these calculations

. Kinship information (generated with SPAGeDi v. 1.2) and population structure (generated with Structure v. 2.2) was incorporated into a mixed-model for AM.

Significance levels were adjusted for multiple testing using the Bonferroni correction.

True loose smut (Ustilago nuda) resistance (controlled by a single gene) ratings collected on the 91 lines as part of the trials was analyzed to evaluate the robustness of the AM methods employed in the study.

Results and Discussion

Approximately 500 high quality, polymorphic DArT markers were used to analyze the AM population. Clustering demonstrated that genotypes from the same breeding program tended to group together, but a significant amount of exchange between programs was evident. Most of the Agricore United (AU)/Coors/Cargill genotypes grouped together while Crop Development Centre, University of Saskatchewan and AAFC, Brandon genotypes were intermingled. The Busch Agricultural Resources Inc. (BARI) and AAFC, Lethbridge genotypes clustered more strongly by breeding program (Figure 1). Consistent with the dendrogram, F_ST values were low between the CDC and AAFC Brandon genotypes and high between the AU/Coors/Cargill genotypes and most others (Table 1). AMOVA determined that 16.7% of the genetic variation was distributed between the breeding programs with 83.3% within, indicating a large pool of common shared alleles.

DArT markers bPb-1959 to bPb-6065 on chromosome 1H were highly correlated with true loose smut resistance (Table 2). These markers encompass the region previously identified with resistance (Eckstein et. al. 2002) and confirmed the validity of the AM methods.

Twenty genomic regions, predominantly located on chromosomes 2H, 5H and 7H, were significantly associated with the seven malting quality traits evaluated (Table 2). Some regions, such as the telomeric end on the long arm of chromosome 5H, have already been associated with malt quality. This raises the question as to whether these are the same alleles previously identified which differentiate good from poor malting genotypes, or if these are alleles which distinguish good from very good genotypes. Numerous potentially new loci which need to be validated were also identified (Table 2). These regions are being analyzed for candidate genes using rice synteny and barley CAP BOPA1 SNP markers which have been mapped and integrated with barley DArT markers using the Oregon Wolfe Barley population. The population will also be genotyped using the Barley CAP BOPA1 SNP markers to facilitate future candidate gene discovery. This study demonstrates the power of AM to extract information from large historic datasets and provides potential new markers for marker-assisted selection.

Eckstein PE, Krasichynska N, Voth D, Duncan S, Rossnagel BG, Scoles GJ (2002) Development of PCR-based markers for a gene (Un8) conferring true loose smut resistance in barley. Can. J. Plant Pathol. 24: 46-53.

Evans DE, Collins HM, Eglinton JK, Wilhelmson A (2005) Assessing the impact of the level of diastatic power enzymes and their thermostability on the hydrolysis of starch during wort production to predict malt fermentability. J. Am. Soc. Brew. Chem. 63: 185-198.

Gao W, Clancy JA, Han F, Jones BL, Budde A, Wesenberg DM, Kleinhofs A, Ullrich SE, North American Barley Genome Project (2004) Fine mapping of a malting-quality QTL complex near the chromosome 4H S telomere in barley. Theor. Appl. Genet. 109: 750-760.

Hayes PM, Liu BH, Knapp SJ, Chen F, Jones B, Blake T, Franckowiak J, Rasmusson D, Sorrells M, Ullrich SE, Wesenberg D, Kleinhofs A (1993) Quantitative trait locus effects and environmental interaction in a sample of North American barley germ plasm. Theor. Appl. Genet. 87: 392-340.

Mather DE, Tinker NA, LaBerge DE, Edney M, Jones BL, Rossnagel BG, Legge WG, Briggs KG, Irvine RB, Falk DE, Kasha KJ (1997) Regions of the genome that affect grain and malt quality in a North American two-row barley cross. Crop Sci. 37: 544-554.

Figure 1. Dendrogram produced by UPGMA clustering based on the Dice similarity coefficient. AU: Agricore United, BARI: Busch Agricultural Resources Inc., CDC: Crop Development Centre, AAFC: Agriculture and Agri-Food Canada, AAFRD: Alberta Agriculture, Food and Rural Development.

Table 1. Pairwise Wright’s fixation index (F_ST) values describing genetic diversity among the breeding programs.
	AU	Coors	Cargill	BARI	CDC	AAFC Brandon	AAFC Lethbridge	AAFRD Lacombe
AU	-	0.087	0.000	0.170	0.334	0.246	0.119	0.061
Coors	0.061	-	0.025	0.284	0.411	0.319	0.212	0.155
Cargill	0.380	0.378	-	0.255	0.333	0.256	0.135	0.071
BARI	0.000	0.001	0.000	-	0.291	0.185	0.116	0.107
CDC	0.001	0.000	0.001	0.000	-	0.035	0.196	0.141
AAFC Brandon	0.000	0.000	0.000	0.004		-	0.101	0.075
AAFC Lethbridge	0.021	0.011	0.023	0.002	0.000	0.000	-	0.027
AAFRD Lacombe	0.030	0.001	0.038	0.000	0.000	0.000	0.144	-
F_ST values above diagonal, P values below diagonal. AU: Agricore United, BARI: Busch Agricultural Resources Inc., CDC: Crop Development Centre, AAFC: Agriculture and Agri-Food Canada, AAFRD: Alberta Agriculture, Food and Rural Development.

Table 2. DArT markers associated with seven malting quality traits and true loose smut resistance.
Trait	Marker	Chromosome	Position (cM)	P Value
AA	bPb-7039	2H	71	1.4x10^-4
	bPb-9868 to bPb-7292	5H	184-190	4.8x10^-6 to 7.31x10^-11
	bPb-8084*	7H	5	4.2x10^-4
BG	bPb-6194*	2H	102	2.8x10^-4
	bPb-5766 to bPb-7292	5H	188-190	2.4x10^-5 to 3.7x10^-6
DP	bPb-0615 to bPb-5519	2H	11-16	1.4x10^-4 to 2.0x10^-5
	bPb-9868 to bPb-7292	5H	184-190	2.7x10^-5 to 3.6x10^-9
	bPb-6821 to bPb-5091*	7H	50	3.3x10^-4
	bPb-1737	7H	158	5.9x10^-5
FExt	bPb-3056 to bPb-6881*	2H	70	2.6x10^-5 to 2.7x10^-6
	bPb-0351	5H	21	1.7x10^-4
	bPb-5766 to bPb-7292	5H	188-190	3.8x10^-4
Friab	bPb-6821 to bPb-5091	7H	50	1.6x10^-4 to 9.4x10^-5
	bPb-3394*	7H	79	1.3x10^-4
	bPb-7781*	7H	103	8.0x10^-5
Prot	bPb-2055 to bPb-9414*	1H	13-16	2.4x10^-5 to 9.1x10^-6
	bPb-9632 to bPb-0050*	5H	31	3.8x10^-4 to 1.1x10^-4
	bPb-4219 to bPb-7952*	7H	74	2.3x10^-4 to 9.1x10^-5
SP	bPb-1986*	2H	147	1.0x10^-5
	bPb-4809 to bPb-7292	5H	186-190	1.4x10^-5 to 2.5x10^-14
LS	bPb-1959 to bPb-6065	1H	133-136	1.2x10^-5 to 9.4x10^-13
AA: alpha amylase, BG: beta-glucan, DP: diastatic power, FExt: fermentable extract, Friab: friability, Prot: protein, SP: soluble protein, LS: loose smut. * Potential new malt quality locus.

Net blotch quantitative trait loci – their practical application in barley breeding

Tajinder S. Grewal, Brian G. Rossnagel and Graham J. Scoles. Crop Development Centre/Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8

Barley net blotch, caused by Pyrenophora teres Drechs. [anamorph: Drechslera teres (Sacc.) Shoemaker] is an important foliar disease in Canada (Tekauz 1990) and elsewhere (Steffenson 1997). Two types of leaf symptoms occur: the net form (NFNB), caused by P. teres f. teres, which causes a dark brown reticulate venation pattern that sometimes turns chlorotic; and the spot form (SFNB), caused by P. teres f. maculata, which results in dark brown circular or elliptical spots accompanied by chlorosis of the surrounding leaf tissue (Khan and Tekauz 1982). The most effective and economic method to control this disease is the use of resistant cultivars; however, most commonly grown barley cultivars are susceptible to most isolates of P. teres (Tekauz 1990, 2000). The variability observed in P. teres and lack of lines resistant to all isolates suggests breeding for resistance should emphasize pyramiding resistance genes to develop broad-based durable resistance. Molecular markers allow breeders to rapidly introgress and pyramid resistant genes into elite lines.

Here we report the mapping of net blotch resistance quantitative trait loci (QTL) in a doubled-haploid (DH) barley population using Diversity Arrays Technology (DArT®) markers; identification of SSR markers linked to major QTL; validation in other barley populations and potential use in molecular-marker assisted selection (MMAS) in the Crop Development Centre (CDC) barley breeding program.

Materials and methods

One hundred and fifty DH lines from the cross CDC Dolly/TR251 were screened at the seedling stage with NFNB isolates WRS858 and WRS1607 and SFNB isolate WRS857 in the Phytotron at the University of Saskatchewan. For details on planting, inoculation and infection assessment see Grewal et al. (2008). The 150 DH lines were also screened at the adult-plant stage in the CDC NFNB field disease-screening nursery at Melfort, SK in 2005 and 2006. Analysis of variance was performed using PROC Mixed of SAS 9.1 (SAS Institute Inc. 2005). Least significant differences (LSD) for mean comparisons were calculated (P = 0.05) for each test. Heritability was calculated for each test using variance estimates derived from PROC Mixed. The phenotypic variance was σ²_DH
lines+ σ²_residual/replications.

DArT mapping and QTL analysis was as described in Grewal et al. (2008). “QRptt” indicates a QTL for resistance to P. teres f. teres (NFNB) and “QRptm” indicates a QTL for resistance to P. teres f. maculata (SFNB). “QRpt” indicates a QTL for resistance to P. teres, i.e., effective against both NFNB and SFNB. “QRpt” is followed by “s” or “a” if effective only at the seedling or adult-plant stage and followed by the barley chromosome onto which the QTL was mapped.

To determine the effect of individual QTL and their combinations on net blotch resistance, the mean infection response (IR) of all lines with the resistance allele for each QTL or their combinations was calculated and compared with the mean of lines having the susceptible allele for that particular QTL. T-tests (P = 0.05) were performed to determine statistical significance.

Simple sequence repeat (SSR) markers linked to major net blotch resistance QTL were identified and validated in other barley populations. One hundred and fifty DH lines from the CDC Bold/TR251 population were screened at the seedling stage with NFNB isolates WRS858 and WRS1607 and SFNB isolates WRS857 and LO233. This population was also screened at the adult plant stage in the CDC NFNB field disease-screening nursery at Melfort, SK in 2006, 2007 and 2008. Eighty-nine lines from this population were mapped using DArT, AFLP and SSR markers (Li et al. 2008). QTL analysis was performed as described in Grewal et al. (2008). Four populations (MEH#486/Harrington, McLeod/CDC Helgason, KXN/TLN 147/CDC Helgason, SB01045/TR02367) were screened with SSR markers HVM74 and HVM03 linked to QRpt6 and QRpts4, respectively. MMAS selected lines were screened at the seedling stage in the Phytotron and at the adult-plant stage in the field to determine the effectiveness of MMAS for net blotch screening.

Results and Discussion

A major NFNB seedling resistance QTL, designated QRpt6, was mapped to chromosome 6H for isolates WRS858 and WRS1607 (Fig. 1) in the CDC Dolly/TR251 population. QRpt6 was associated with adult-plant resistance in 2005 and 2006 field trials. These results confirm earlier reports of a major QTL or gene on chromosome 6H (Steffenson et al. 1996; Manninen et al. 2000; Cakir et al. 2003; Gupta et al. 2004; Friesen et al. 2006). Additional QTL for NFNB seedling resistance to the more virulent isolate WRS858 were identified on chromosomes 2H, 4H, and 5H. A seedling resistance QTL (QRpts4) for the SFNB isolate WRS857 was detected on chromosome 4H as was a significant QTL (QRpt7) on chromosome 7H. Three QTL (QRpt6, QRpts4, QRpt7) were associated with resistance to both net blotch forms and lines with one or more of these demonstrated improved resistance. SSR markers tightly linked to QRpt6 and QRpts4 were identified.

QRpt6 was further validated in the CDC Bold/TR251 population for seedling resistance to NFNB isolates WRS858 and WRS1607 and adult-plant resistance in 2006, 2007 and 2008 field trials. The major seedling and adult plant resistance QTL/gene on 6H in TR251 may be sufficient to provide adequate field resistance to NFNB and could be selected for based on Phytotron screening or using molecular markers. High heritability for different tests indicated that selection based on molecular markers is feasible.

Fig. 1 Multiple-QTL model (MQM) LOD scans of chromosomes where QTL were detected for NFNB resistance in the CDC Dolly/TR251 DH population. Vertical lines indicate significance threshold for each experiment, estimated from 1000 permutations. One QTL on 4H associated with NFNB (WRS858) resistance. A major QTL on 6H associated with seedling (WRS858, WRS1607) and adult-plant (Field 2005, field 2006) resistance.

Screening the parents of 22 barley populations with SSR markers linked to QRpt6 and QRpts4 identified several polymorphic populations, indicating these SSR markers could be used for practical MMAS in the CDC barley breeding program. Four populations were screened with SSR markers HVM74 and HVM03 to select lines resistant to both net blotch forms.

Screening selected barley lines from MEH#486/Harrington population at the seedling stage in the Phytotron and at the adult-plant stage in the field demonstrated that MMAS selected resistant lines showed significantly better resistance (Table 1). Selection based on marker HVM74 (QRpt6) alone showed better resistance than no marker and similar to selecting for the both markers, proving selection based on QRpt6 alone could select resistance to both net blotch forms. This was further confirmed in populations McLeod/CDC Helgason, KXN/TLN 147/CDC Helgason, SB01045/TR02367.

Table 1. Average infection response of lines from the MEH#486/Harrington population with or without major net blotch resistance QTL (QRpt6, QRpts4)

QTL/Test		NFNB				SFNB
	^bAllele	^cWRS858	Field 2006	Field 2007	Field 2008	^cWRS857	^d# of lines
QRpt6	R	4.0**	3.0**	2.7**	2.8**	5.7**	79
QRpt6	S	6.8	4.5	4.0	3.8	6.8	69
QRpts4	R	5.0	3.6	3.0*	3.2	6.0	62
QRpts4	S	5.6	3.8	3.6	3.4	6.4	83
QRpt6+QRpts4	R	3.4**	2.8**	2.4**	2.8**	5.4**	25
QRpt6+QRpts4	S	7.1	4.8	4.5	4.0	6.9	32

^b Resistance (R) or susceptible (S) allele at a particular QTL locus/loci.

^cPyrenophora teres isolate

^d Total number of lines with R or S allele at particular QTL.

*Significant (P <0.05) or **highly significant (P <0.01) using T-test at particular QTL or their combinations.

The results demonstrate that MMAS resistant lines had significantly lower infection than susceptible lines, indicating that MMAS for net blotch resistance is practical. Since the major 6H QTL, QRpt6, may provide adequate NFNB field resistance in western Canada, the practical application plan for the Crop Development Centre barley breeding program is to routinely utilize MMAS for QRpt6 followed by normal field screening in field net blotch nurseries to identify additional resistance provided by resistance genes associated with other QTL. Resources permitting the CDC program has implemented routine MMAS for QRpt6 and recommends this approach to other breeding programs.

Selected references

Cakir, M., S. Gupta, G.J. Platz, G.A. Ablett, R. Loughman, L.C. Embiri, D. Poulsen, C.D. Li, R.C.M. Lance, N.W. Galwey, M.G.K. Jones, and R. Appels. 2003. Mapping and validation of the genes for resistance to Pyrenophora teres f. teres in barley (Hordeum vulgare L.). Aust. J. Agric. Res. 54:1369-1377.

Grewal, T.S., B.G. Rossnagel, C.J. Pozniak, and G.J. Scoles. 2008. Mapping quantitative trait loci associated with barley net blotch resistance. Theor. Appl. Genet. 116:529-539.

Steffenson, B.J. 1997. Net blotch. In: Mather DE (ed) Compendium of barley diseases, 2nd edn. American Phytopathological Society, St. Paul, MN, pp 28–31.

Tekauz, A. 1990. Characterization and distribution of pathogenic variation in Pyrenophora teres f. teres and P. teres f. maculata from western Canada. Can. J. Plant Pathol. 12:141–148.