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ARS Home » Pacific West Area » Aberdeen, Idaho » Small Grains and Potato Germplasm Research » Research » Research Project #424532

Research Project: Genetic Improvement of Barley and Oats for Enhanced Quality and Biotic Stress Resistance

Location: Small Grains and Potato Germplasm Research

2016 Annual Report


Objectives
Objective 1: Identify and develop barley and oat germplasm with improved stress resistance (rust resistance, winter-hardiness) and enhanced end-use quality (malt, ß-glucan content). Subobjective 1A: Improve productivity and quality of barley and oat germplasm, with emphasis on malting quality (barley) and food quality (oat). Subobjective 1B: Develop winter malting cultivars with improved quality and winter survival. Subobjective 1C: Introgress resistance to barley stripe rust (BSR) into improved barley germplasm. Objective 2: Develop methods to facilitate accelerated breeding for adaptive traits and utilization of germplasm diversity in barley and oat. Subobjective 2A: Identify SNP markers linked to resistance genes for BSR and oat crown rust (OCR) for use in marker-assisted selection (MAS). Subobjective 2B: Validation of SNP markers linked to ß-glucan content, malt extract, and diastatic power. Objective 3: Develop novel biotechnological approaches for the production of genetically engineered barley and oat. Subobjective 3A: Develop Ds-based and/or recombination-mediated cassette exchange (RMCE) gene delivery systems. Subobjective 3B: Develop transgenic barley lines resistant to Fusarium infection and/or deoxynivalenol (DON) accumulation


Approach
The three objectives are separate but complementary. Objective 1: Germplasm improvement is based on modified pedigree breeding with single seed descent, multi-location in-house and collaborative test locations, off-site winter nurseries, and winter greenhouse facilities to minimize time for varietal development. Multiple breeding targets, corresponding to specific end uses, include: spring malting barley, spring barley, and oat for food and feed. The process is similar for barley and oat, except for the specific set of targeted characteristics. Procedures are based primarily on phenotypic measurements and laboratory analyses of grain quality. Markers are being developed for key traits to enable more efficient allelic selection for trait improvement and will be used as they are developed. Failure to make adequate progress will be corrected by incorporating alternative germplasm, increasing numbers of crosses, and by increasing test environment numbers and/or quality to increase the frequency of obtaining superior progeny. Objective 2: Based on the hypothesis that SNP markers closely linked to genes for OCR and BSR resistance genes, and for malting quality, will exist. OCR resistance will be assessed and mapped within five oat populations with markers from the oat 6K SNP iSelect Illumina array. BSR resistance will be assessed and mapped within four barley populations, and malting quality in two barley populations, with markers from the barley 9K SNP iSelect Illumina array. Phenotyping will be based on multi-year, multi-location field and/or greenhouse trials. Markers identified will be validated as useful by examination of allelic effects in alternative existing or newly-created populations. Multiple genomic databases will be examined to assist the identification of candidate genes underlying QTLs. Failure to identify useful QTLs or candidate gene may require development and testing of additional populations, novel markers, or analysis of new genomic resources as they become available. Objective 3: Based on the hypothesis that Ds-mediated transposition will produce plants with single-copy loci in regions suitable for high expression, the goal is to produce "clean" transgenic plants without with intact, single-copy transgenes free of extraneous DNA derived from bacterial vectors or selectable markers genes. This system utilizes two very short (~600 bp in total length) sequences derived from another food crop, maize, that when flanking other sequences can transpose--along with the intervening sequences--to new location. Transposition is controlled by introducing the relevant enzymatic activity via crosses to Ac transposase--expressing plants. Vectors will be constructed and introduced via Agrobacterium-mediated delivery. Research will concentrate on commercial cultivars, and genes with activity against Fusarium head blight or that suppress mycotoxin production will be introduced. Failure of proper gene expression is guarded against by using. The use of Ds transposition to deliver genes tends to promote good expression, and multiple candidate genes are available, some of which have imparted useful levels of resistance in preliminary work.


Progress Report
Developing improved cultivars is a continuous, incremental process that targets multiple characteristics that depend on end-use. Progress towards Subobjectives 1A and 1B included significant advancement towards variety release of both spring and winter barley lines selected for malting quality. Three spring and three winter two-rowed malting lines performed sufficiently in the first year of plant scale malting tests to merit testing for the second year. Plant scale testing is the final step of industry standards before approval for malting use, and is normally done for three years. The winter lines are particularly noteworthy, as winter barleys with malting quality are a recent innovation from this project, and only two previously-released winter malting cultivars exist. Previously selection for malt quality has been made for characteristics suited for large-scale production of traditional American lagers that use ‘adjunct’ sources of starch other than barley, such as rice. However, the proliferation of new breweries across the U.S. has resulted in a robust and growing market for barley bred specifically for "all-malt" brewing where no adjunct is used. All-malt brewing requires a different malting quality profile than adjunct brewing. To support this growing market and its stakeholders, selection for new lines is being made for traits necessary for all-malt brewing, especially for lines having low grain protein. Most current malting barley cultivars and breeding lines are too high in grain protein, a consequence of selection for high enzymatic levels, which are desired for adjunct brewing. Starting with 377 candidate lines from the National Small Grains Collection with low grain protein content, 110 have been selected based on agronomic adaptation and field performance for further agronomic evaluation, for malting quality, and for selection as parents of new populations. This work is financially supported by a stakeholder organization. In addition to finding novel sources of all-malt-associated quality traits, screens of current materials in the breeding program have identified two lines, 2Ab07-XM219-46 and 2Ab08-XMoio-65, that have potential utility for industry. These lines are being tested by a local industry cooperator. Barley for food use is an emerging market segment because of cardiovascular health benefits, particularly those associated with beta-glucan. Recurrent selection techniques are now routinely enabling the selection of lines with beta-glucan contents of 10-13%, and 16 lines are now being evaluated in the elite nursery (our most-advanced nursery). A new food cultivar, Kardia, was released this year with improved agronomic characteristics and beta-glucan content. Kardia is a hulled, two-rowed, spring barley with a grain beta-glucan content of 8.5%. Versus its most widely-grown competitor, Salute, Kardia has better yield potential and 40% more grain beta-glucan. Kardia is an ideal candidate to replace Salute. Improving disease resistance in oat and barley will maintain the economic and environmental viability of these crops. Progress was made in several areas. For Subobjective 1C, assays to detect single-nucleotide-polymorphisms (SNPs) flanking four barley stripe rust (BSR) resistance alleles (two on chromosome 4H, and one each on chromosomes 6H and 7H) were developed from the barley accession known as Grannenlose Zweizeilige (GZ). These SNP markers were used to assist in backcrossing the alleles into the improved genetic background of 95SR316A. 95SR316A is an elite germplasm line developed by this project that has adult plant resistance to BSR. Lines from these crosses should carry the GZ-derived resistance, but closely resemble the adapted parent in other respects. This work was facilitated by data obtained as a result of contributions made by collaborators under subordinate projects 58-5366-1-350 and 58-5366-4-012. Progress was made towards developing molecular markers linked to both BSR and oat crown rust (OCR) resistance (Subobjective 2A). Testing lines from a cross between Charles and 95SR316A for seedling resistance to BSR was started this year and added to information from previous field trials. Preliminary analyses indicate the presence of a resistance locus on chromosome 1H derived from Charles. Markers flanking this region will be evaluated in Aberdeen breeding lines derived from Charles for their ability to predict BSR resistance in the breeding program. Assays for SNPs flanking three loci for adult plant resistance to OCR were developed. These will be used to combine resistance to OCR from three sources. Crossing to create this population was completed. Assays for molecular markers linked to genes conferring seedling resistance to OCR have also been developed using published mapping data. Assays for markers linked to specific genes for OCR resistance were developed: Pc38, Pc48, Pc54, Pc68, Pc71 and Pc91. These assays are currently under evaluation for their ability to identify the presence of the resistance alleles in related and unrelated oat lines. In addition, we have crossed oat lines selected for use in mapping the locations of OCR seedling resistance genes within the oat genome. Markers within resistance genes are likely to be much more useful for marker-assisted selection than markers that are close to, but not in the gene. To develop such markers, sequence-level characterization of critical genes will be needed. To address this need in support of Subobjective 2B, we have identified 16 clones in the Oat Bacterial Artificial Chromosome (BAC) library that contain DNA fragments spanning the Pc58a gene for OCR resistance and its surrounding genome region. We are currently sequencing these prior to assembly and gene annotation. The detailed sequence information obtained will guide our efforts to identify SNPs within candidate genes. Marker assisted selection for such complex characteristics as malting quality is the focus of Subobjective 2B. Malting quality quantitative trait loci (QTL) identified in the cultivar Stellar were targeted for transfer to germplasm of the Aberdeen barley breeding program. Two lines from the cross Stellar X 01Ab8219 mapping population were selected as donor parents because they carried the target QTL. Three Aberdeen lines were selected on the basis of contrasting malting quality phenotypes to serve as recurrent parents. The Abs-14 X 02Ab1L08MTC15-70, Abs-14 X Merit, Abs-124 X 02Ab08X05X061-218, and Abs-124 X Merit populations have been backcrossed twice. Markers will be used to select lines carrying the desired QTL for further evaluation. Delivery of transgenes via transposon-mediated delivery and site-specific recombination will result in transgenic plants with better, more predictable characteristics (Subobjective 3A). Transposons are mobile pieces of DNA, and site-specific recombination can put transgenes in a specific, preselected and appropriate site. These two technologies can produce transgenic barley that contains no sequences derived from bacterial vectors (used for producing the transgenes) or selectable markers (used for separating non-transgenic from transgenic plants during their creation), and which has high and stable levels of transgene expression. This year, F2 populations from two independent transgenic events containing the transposon TAG gene (required for site-specific recombination) were screened via adapter-ligation mediated chromosome walking, and putatively-transposed TAG sites were identified. In many cases, such sites could not be verified, suggesting that unexpected behavior of this site was occurring. However, four F2 plants contained verifiable sites. One of these plants died, another produced 13 seeds, and the other two plants are reaching maturity and are expected to produce larger amounts of seed. The improved, transposon-based system of transgene delivery will be used to address Fusarium head blight (Objective 3, Subobjective 3B), a disease of wheat and barley that reduces yield and produces a potent mycotoxin. Progress this year included the conclusion of experiments in which transgene constructs were tested first in Fusarium before introduction into barley. The advantage of this approach is the speed with which various transgene components can be tested in Fusarium, which can be done in months, versus testing transgene components in barley, which takes several years. Constructs designed to express double-stranded RNA targeting the full-length Tri6 gene, which mediates expression of mycotoxins via expression of the gene Tri5, reduced the ability of Fusarium to infect wheat and barley and virtually eliminated mycotoxin production. Molecular analysis confirmed the reduced expression of Tri5, and the expected production of small RNAs mapping to the Tri6 gene, showing that the mechanism was RNA interference.


Accomplishments
1. Release of the new food barley cultivar, Kardia. Increasing the levels of grain beta-glucan in barley is desirable as it is associated with cardiovascular health. Barley bred and grown specifically for food use has therefore received more attention recent years. The food barley Kardia, developed by researchers in Aberdeen, Idaho, has 40% higher grain beta-glucan and better grain yield than the current industry standard cultivar, Salute. This combination of characteristics will increase the efficiency of producing food-grade, high-beta-glucan barley which will increase producer and processer profitability, and increase availability of nutritional food for consumers.

2. Release of the transposon-tagged barley population II. Studies of plants with mutated genes can tell geneticists the functions of such genes. Transposon tagging, a technique that causes mutations based on moving a mobile genetic element (a transposon) into genes, has been used to create a genetic resource for barley geneticists. ARS researchers in Aberdeen, Idaho have released a second set of 61 lines, each with a transposon insertion at a different location. Seed of each line is being released and deposited into the National Small Grains Collection, and data on the mutated sequences is being deposited in Genbank. These resources will be freely available to researchers worldwide for studying the function of key genes, and have contributed to the understanding of the control of the expression of the Cly1 gene - which controls flower opening, in barley.

3. Identification of markers for use in selection for barley stripe rust resistance. Barley stripe rust can cause severe losses in yield and quality of barley, but it does not reliably occur in the breeding nurseries used for selection of new lines. Therefore, breeding progress can more quickly and efficiently happen if molecular selection can be made regardless of whether the disease occurs in any particular year. ARS researchers in Aberdeen, Idaho, and Pullman, Washington, in collaboration with scientists at the University of California, Davis, California and Oregon State University, Corvallis, Oregon, made crosses with a resistant landrace held in the National Small Grains Collection, mapped four genetic loci associated with resistance, and identified molecular markers that can be used for selection in the absence of disease. This information will be useful for the development of superior, stripe rust resistant varieties.


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Review Publications
Bregitzer, P.P., Hu, G., Marshall, J.M., Raboy, V. 2016. Registration of Harriman low-phytate, hulled spring barley. Journal of Plant Registrations. 10:105-108.
Esvelt Klos, K.L., Huang, Y., Babiker, E.M., Beattie, A., Bekele, W.A., Bjornstad, A., Bonman, J.M., Carson, M.L., Chao, S., Gnanesh, B.N., Harrison, S.A., Howarth, C.J., Hu, G., Ibrahim, A., Islamovic, E., Jackson, E.W., Jannink, J., Kolb, F.L., Mcmullen, M.S., Fetch, J.M., Murphy, J., Obert, D.E., Ohm, H.W., Rines, H.W., Rossnagel, B., Schuleter, J.A., Wight, C.P., Yan, W., Tinker, N.A. 2016. Population genetics related to adaptation in elite oat germplasm. The Plant Genome. 9(2):1-12. doi: 10.3835/plantgenome2015.10.0103.
Esvelt Klos, K.L., Gordon, T.C., Bregitzer, P.P., Hayes, P., Chen, X., Bonman, J.M. 2016. Barley stripe rust resistance QTL: Development and validation of SNP markers for resistance to Puccinia striiformis f. sp. hordei. Phytopathology. doi: 10.1094/PHYTO-09-15-0225-R.
Winkler, L., Bonman, J.M., Chao, S., Yimer, B.A., Bockelman, H.E., Esvelt Klos, K.L. 2016. Population structure and genotype-phenotype associations in a collection of oat landraces and historic cultivars. Frontiers in Plant Science. doi: 10.3389/fpls.2016.01077.
Chaffin, A.S., Huang, Y., Smith, S., Bekele, W.A., Babiker, E.M., Gnanesh, B.N., Foresman, B.J., Blanchard, S.G., Jay, J.J., Reid, R.W., Wight, C.P., Chao, S., Oliver, R., Islamovic, E., Kolb, F.L., McCartney, C., Mitchell Fetch, J.W., Beattie, A.D., Bjornstad, A., Bonman, J.M., Langdon, T., Howarth, C.J., Brouwer, C.R., Jellen, E.N., Esvelt Klos, K.L., Poland, J., Hsieh, T., Brown, R., Jackson, E., Schlueter, J.A., Tinker, N.A. 2016. A consensus map in cultivated hexaploid oat reveals conserved grass synteny with substantial sub-genome rearrangement. The Plant Genome. 9(2). doi: 10.3835/plantgenome2015.10.0102.
Hu, G., Trupia, S., Ellberg, S. 2015. A promising low beta-glucan barley mutation of m351 for better bioethanol production use. BioEnergy Research. 8:1158-1164.