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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

2015 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 Objectives 1A and 1B included significant advancement towards variety release of both spring and winter barley lines. Three spring and three winter 2-row malting lines were advanced to plant scale malting tests, the final step of determining whether they meet industry standards. Each of these lines has some combination of improved agronomic traits or malting quality. These lines have shown favorable malting characteristics in two years of pilot scale malting tests. The winter lines are particularly noteworthy, as winter barleys with malting quality are a recent innovation from this project, and there are only two previously-released winter malting cultivars. Two cultivars were released, Sawtooth (hulless) and Harriman (hulled), both low-phytate feed varieties that enhance mineral nutrition and reduce phosphorus discharge into the environment when consumed by non-ruminant animals. Compared to the previously-released hulled, low-phytate variety Herald, Harriman has higher grain yield, higher test weight, higher inorganic P, and lower phytate P. Compared to the previously released low-phytate variety Clearwater, Sawtooth has higher grain yield and higher test weight. In addition, another hulled line for food use is in the process of being released. It has a significant increase (30-40%) in ß-glucan content, and similar grain yield, compared to the widely-used cultivar Salute. Barley bred specifically for "all-malt" brewing is a growing market segment relating to Objectives 1A and 1B. All-malt brewing requires a different malting quality profile than conventional methods and in recent years we have identified both spring and winter lines with characteristics that fit this profile. Three lines were identified and in 2015 a ton of each line was produced to facilitate further industry testing. The results of these tests will provide guidance on decisions on future releases and hybridization plans. Improving disease resistance in oat and barley will maintain the economic and environmental viability of these crops. We made progress in several areas. For Objective 1C, lines were developed from crosses to two barley stripe rust (BSR)-resistant lines, Grannenlose Zweizeilige (GZ) and 95SR316A. GZ is a poorly-adapted accession held in the National Small Grains Collection that displays seedling resistance to a wide spectrum of BSR races; 95SR316A is an elite germplasm line developed by this project that has adult plant resistance. Progeny of Lenetah/95SR316A (14 lines) and of Lenetah/GZ (five lines), selected in 2014 based on visual evaluations, were evaluated in a preliminary yield nursery and will be advanced pending further evaluation this winter. Four lines derived from the cross 95SR316A/Charles have been advanced to yield trials at multiple Idaho locations. These lines represent the first introduction of BSR resistance into the winter malting barley program. Linking resistance phenotypes to molecular markers to enable marker-based selection is the focus of Objective 2A. Progress was made with respect to both barley stripe rust and oat crown rust. We focused on single-nucleotide-polymorphism (SNP) markers. The Lenetah/GZ population, consisting of 153 recombinant inbred lines, was screened for BSR at flowering at three field sites (Davis, California; Corvallis, Oregon; and Mt. Vernon, Washington) in 2015. Preliminary analysis of the field data verified seedling resistance data that revealed a major GZ-derived resistance allele located on chromosome 4H, and minor alleles on 4H and 6H. These results will be used to develop markers to assist breeding for BSR resistance. The inclusion of the Davis and Corvallis sites via Non-Assistance Cooperative Agreements has been important to the success of this project, because in 2014 and 2015 the occurrence of leaf rust at the USDA-ARS-run Mt. Vernon site compromised the BSR data. Lenetah, GZ, and 95SR316A were inoculated with BSR at the flag leaf stage in greenhouse tests. The greenhouse screening method gave comparable results to field screening and may provide a more efficient and controlled way to measure high temperature adult plant resistance in barley. Lenetah was very susceptible, and the GZ and 95SR316A were resistant and moderately resistant, respectively. Seedling resistance to BSR was investigated also in lines other than GZ. Charles barley exhibited an intermediate level of seedling resistance to barley stripe rust race PSH-54 whereas the breeding line 95SR316A was very susceptible at the seedling stage. During the past year barley stripe rust screening began on the ‘Charles’/95SR316A population, which consisted of 188 lines that have been genotyped with the barley SNP chip. One experiment was rated and showed a clear distinction between susceptible and resistant lines. A second experiment is underway that will be completed by the end of the summer. Resistance to oat crown rust was investigated in the oat varieties Provena and Boyer, and the oat breeding line 94197A1-9-2-2-2-2-5, with the objective of developing effective greenhouse assays that clearly differentiate the crown rust resistance reactions in the parents. Greenhouse tests were conducted using a race of the crown rust pathogen to which the parents showed differential reactions. These tests were successful in that the expected levels of resistance were seen in the three parents. Now that an effective and efficient greenhouse assay is available, recombinant inbred lines from these populations will be tested, and the results from these tests used to identify SNP markers that will be useful for marker-assisted selection. Several crosses were made between two oat lines, BGS-32xMN841801 and OT-62. F2 plants from these crosses will be used to validate previously published SNP markers for crown rust resistance in Boyer, 94197A1-9-2-2-2-2-5, MN841801, Ogle, and TAM O-301. The ultimate objective of this work is to increase the durability of crown rust resistance by combining the resistance genes from these sources into a common genetic background. Marker assisted selection for complex characteristics such as malting quality and agronomic performance (Objective 2B) will require a comprehensive understanding of genetic variability in the elite germplasm used at Aberdeen. Genome-wide association analysis of malting quality characteristics was performed using 156 elite Aberdeen breeding lines. Six regions of the barley genome were identified as important to determining differences among Aberdeen breeding lines for malting quality, while other regions of the genome showed striking similarity among these lines. Using this information, we prioritized a region of chromosome 6H to target for incorporation of additional variation from the malting variety Stellar for use in the Aberdeen breeding program. Aberdeen lines with a range of contrasting malting quality characteristics were identified and have been crossed with lines containing the Stellar-derived loci. Markers within the genes that contribute to complex traits such as oat beta-glucan level 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. In oat, a barrier to this approach is the lack of basic tools for sequence characterization. To address this need in support of Objective 2B, a Bacterial Artificial Chromosome (BAC) library containing fragments of the oat (Avena sativa) genome was created in 2015. A BAC library consists of thousands of yeast lines, each of which contains a small (approximately 1000 to 1500 base pair) segment of the oat genome. Any given genomic segment is expected to be present an average of five times within our library, allowing us to sequence and annotate specific segments of interest. Delivery of transgenes via transposon-mediated delivery and site-specific recombination will result in transgenic plants with better, more predictable characteristics (Objective 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. Progress this year included the production of additional plants containing the transposon TAG gene (required for site-specific recombination), and the development of populations in which the transposon TAG gene was mobilized via crosses to a plant expressing an enzyme that causes transposition (or movement from the original location to a new, better location). It is the process of transposition that enables the creation of "clean" transgenic plants that have the transgene in a desirable location that is free of vector and selectable marker sequences. To harness the utility of the improved systems, we plan to use them to address the problem of 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 demonstration of several Fusarium genes that, when downregulated, reduce mycotoxin production. The downregulation was enabled by exposure of the fungus to double-stranded RNA, and the results of these experiments are being used to design transgene constructs that will be used to transform barley. The advantage of this approach is that various transgene components can be tested first in Fusarium, which can be done in months, versus testing transgene components in barley, which takes several years.


Accomplishments
1. Mapping resistance to crown rust disease in oat. Crown rust is the most damaging disease of oat worldwide. Generating new oat cultivars with crown rust resistance is an economical method of managing the disease. ARS researchers in Aberdeen, Idaho, and St. Paul, Minnesota, in cooperation with scientists from Louisiana State University, used newly developed oat molecular markers called ‘single nucleotide polymorphisms’ or SNPs to genetically map resistance in two oat lines. Loci for resistance were discovered on three oat chromosomes. The SNPs associated with these loci will be useful as genetic markers to help plant breeders develop new crown rust resistant oat cultivars.

2. Release of low-phytate cultivars Harriman and Sawtooth. Phytate is the primary storage form of phosphorus in grain. It is poorly-digested by non-ruminant animals and can make minerals unavailable, leading to reduced nutritive value and environmentally-damaging release of phosphorus. Both cultivars were developed by ARS researchers at Aberdeen, Idaho, in collaboration with University of Idaho scientists, and they offer better agronomic performance and reductions in phytate compared to previously-released low-phytate barleys. The improved performance of these cultivars increases the commercial viability of low-phytate barley, thus enhancing its potential to contribute to the profitability and environmental sustainability of animal production.

3. Determination of genetic diversity for Russian wheat aphid (RWA) resistance in barley. The RWA is a serious pest of barley that can greatly reduce yield. ARS researchers at Aberdeen, Idaho, and Stillwater, Oklahoma, have produced many improved germplasm lines and cultivars resistant to RWA, but previous efforts to map resistance loci and describe the diversity among these lines were limited by poor marker technology. Using newly-available, molecular ‘single nucleotide polymorphisms’ or SNPs, these researchers produced a more accurate map of resistance loci. They identified specific groups of RWA-resistant germplasm based on genetic similarity, and identified four loci that can be used as molecular markers to select for resistance. This information will enable breeders to more efficiently produce RWA-resistant cultivars that have multiple sources of resistance.


Review Publications
Brown, R.H., Singh, J., Singh, S., Dahleen, L.S., Lemaux, P.S., Stein, N., Mascher, M., Bregitzer, P.P. 2015. Behavior of a synthetic Dissociation element in barley: A novel tool for genetic studies and for breeding transgenic barley. Molecular Breeding. 35:85.
Hu, G., Burton, C.S., Hong, Z. 2014. Molecular and chemical characterization of a new waxy allele in barley (Hordeum vulgare L.). Cereal Chemistry. 91(5):438-444.
Schneider, A.B., Nava, I.C., Herve, C.B., Islamovic, E., Limberger, E., Jackson, E.W., Delatorre, C.A. 2015. Chromosome-anchored QTL conferring aluminum tolerance in hexaploid oat. Molecular Breeding. 35:121. doi: 10.1007/s11032-015-0315-4.
Babiker, E.M., Gordon, T.C., Obert, D.E., Jackson, E.W., Harrison, S.A., Chao, S., Carson, M.L., Bonman, J.M. 2015. Quantitative trait loci from two genotypes of oat (Avena sativa L.) conditioning resistance to Puccinia coronata. Phytopathology. 105(2):239-245.
Ladics, G.S., Bartholomaeus, A., Bregitzer, P.P., Doerrer, N.G., Gray, A., Holzhauser, T., Jordan, M., Keese, P., Kok, E., Macdonald, P., Parrott, W., Privalle, L., Raybould, A., Rhee, S., Rice, E., Romeis, J., Vaughn, J., Wal, J., Glenn, K. 2015. Genetic basis and detection of unintended effects in genetically modified crop plants. Transgenic Research. 24:587-603.
Raboy, V., Peterson, K.L., Jackson, C., Marshall, J., Hu, G., Saneoka, H., Bregitzer, P.P. 2015. A substantial fraction of barley (Hordeum vulgare L.) low phytic acid mutations have little or no effect on yield across diverse production environments. PLANTS. 4(2):225-239. doi: 10.3390/plants4020225.
Dahleen, L.S., Bregitzer, P.P., Mornhinweg, D.W., Esvelt Klos, K.L. 2015. Genetic diversity for Russian wheat aphid resistance as determined by genome-wide association mapping and inheritance in progeny. Crop Science. 55(5):1925-1933.