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
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 towards the development of improved germplasm and cultivars (Objectives 1A and 1B) continued in FY 2017. Food barley germplasm that has exceptionally high levels of the heart-healthy beta-glucan fiber, previously identified within a mutagenized population, received patent protection. Plant scale tests for three winter malting barley lines and three spring malting barley lines were conducted for a second year. Before reaching this final stage of advanced industry testing, all lines showed successful agronomic performance and acceptable quality parameters in industry sponsored pilot scale tests, so the expectation is that one or more of these lines will be released as a variety following continued good performance in the plant scale tests. Another spring malting barley line, 2Ab08-X04M278-35, and a winter malting barley line, 05ARS561-208, performed well during pilot testing and have been qualified to be tested in plant scale tests. To facilitate plant scale testing, 3000 to 5000 pounds of seed of each variety were produced. To better deliver useful products to the marketplace, and to promote the use of materials produced by this project, we have forwarded data and seed of the new high beta-glucan barley ‘Kardia’ to several potential users, one of whom has decided to use ‘Kardia’ and a replacement for the older variety, ‘Salute’. To address the specific needs of the all-malt brewing industry, we have worked with the Brewer’s Association to identify materials that have the best package of malting characteristics for their use. For this use, lower grain protein than that used by the adjunct malting industry is preferred, but very little low protein germplasm exist in our program. Accordingly, a screen was conducted of breeding materials from various sources, including the National Small Grains Collection, and 70 low protein lines were identified and incorporated into the breeding program. To address the growing problem of Fusarium head blight in the Intermountain West, screening for resistance was conducted for a third year. The results suggest that several lines have resistance to this disease and to the mycotoxin (deoxynivalenol) produced by Fusarium, and these lines are expected to be useful either as new, resistant varieties or as parents of improved lines. Progress has been made towards improving barley for resistance to stripe rust. In work relevant to Objective 1C, the molecular genetic marker assays developed in the last fiscal year were used to identify carriers of Grannenlose Zweizeilige (GZ)-derived barley stripe rust resistance (BSR) alleles in an advanced backcross population made using 95SR316A as the recurrent parent. The markers are tightly linked to two quantitative trait loci (QTL) on chromosome 4H and to one each on chromosomes 6H and 7H. GZ has the potential to contribute to its progeny these unique seedling-stage resistance alleles, while 95SR316A is an elite line developed by this project with good malting quality and adult plant BSR resistance. Lines carrying all parentally-derived combinations of alleles at the four seedling resistance QTL were created and will be evaluated in the field at multiple locations for their relative reactions to BSR infection. These data will be collected by cooperators, including cooperators funded by non-assistance type cooperative agreements at the University of California-Davis (58-5366-4-012) and at Oregon State University (58-2050-6-005). One or more of these lines may combine the agronomic characteristics of 95SR316A with disease resistance equal to, or greater than, GZ. Lines derived from crossing 95SR316A and Lenetah (susceptible to BSR) were genotyped for thousands of markers across the genome, as were lines derived from a cross between 95SR316A and GZ. This genotype data will be combined with multi-year data on the reactions of these lines to BSR in the field to identify genomic regions contributing to adult plant resistance. Molecular markers flanking these regions will be developed (Objective 2A) and added to those already available for seedling resistance. Adult plant resistance is often more difficult for pathogens to overcome through adaptation, providing a lower level of resistance with greater long-term stability. By combining the adult plant resistance from 95SR316A with the seedling resistance from GZ, we hope to extend the period of effectiveness of the GZ-derived resistance. Populations designed to validate molecular markers linked to oat crown rust (OCR) resistance (Objective 2A) were advanced during this fiscal year to the point where they are ready to be evaluated for crown rust reaction in the greenhouse and field. Genotyping these populations will allow us to validate the previously mapped QTL. In addition, by crossing progeny, we have developed a population that utilizes 94197A1-9-2-2-2-5, CDC Boyer, TAM O-301 and MN841801 as parents to combine multiple genes for OCR resistance. Using molecular markers developed by this project, we characterized the lines in this multi-parent population. Seed from the selected lines is being increased for OCR resistance testing at multiple field locations. With this step, we can 1) provide a second level of validation for molecular markers linked to OCR resistance, 2) evaluate the effectiveness of these genes alone and in combination, and 3) identify lines from this population with potential to contribute crown rust resistance to the Aberdeen breeding program. To develop molecular markers for genes contributing to malting quality characteristics (Objective 2B) we have created four backcross-derived populations designed to test the potential for QTL identified in the cultivar Stellar to contribute to malting quality in the Aberdeen breeding program. These crosses have placed two targeted combinations of Stellar-derived QTL, onto two different Aberdeen breeding program genetic backgrounds that were chosen to provide genetic and phenotypic contrasts. All four subsequent populations are currently in the BC2F2 generation. Markers will be used to select lines carrying the desired QTL for further evaluation. The development of transgenic barley lines resistant to Fusarium head blight (FHB) using Ds-based or recombination-mediated cassette exchange (RMCE) (Objectives 3A and 3B) moved forward. The approach utilizes a phenomenon called RNA interference, which involves the suppression of fungal development via the expression of gene constructs that produce double stranded (ds) RNA with sequence identical to critical Fusarium genes. Using a fungal system that permits rapid assessment of potentially useful gene constructs, particular portions of the Fusarium TRI6 gene have been targeted by dsRNA and greatly downregulated. This resulted in the suppression of the TRI6-controlled pathway that produces the poisonous mycotoxin, deoxynivalenol. Deoxynivalenol contamination is one of the worst aspects of FHB, as it can render the grain unfit for human or animal consumption. Work is in progress to introduce into barley various gene constructs targeting TRI6, and plants encoding TRI6-specific dsRNA may be resistant to mycotoxin accumulation when infected with Fusarium. In addition to addressing the practical problem of resistance to FHB, this work involves better methods of transgene delivery. One aspect of the delivery system we are developing involves adding short terminal sequences to the dsRNA gene constructs that enable them to move, or transpose, from one location to another. This results in a two-step transformation system. In step 1, transgenes are delivered either by biolistics or by Agrobacterium, both of which are standard techniques used for plant transformation. In step 2, the resulting transgenic plants are hybridized with another transgenic plant that encodes a transposase enzyme, which causes sequences bordered by the short terminal sequences activity to move (transpose) to new locations. There are two advantages of this system: 1) transposition results typically in a single, intact copy of the gene construct being delivered to an areas that supports good gene expression; and 2) the transposed gene is relocated away from the original site of insertion, and therefore away from problems such as duplicated or inverted sequences, bacterial vector DNA, and unwanted selectable markers. RMCE offers similar advantages, but has the added advantage of enabling the incorporation of gene constructs in a particular, pre-selected place. To work, a single-copy RMCE “platform” transgene must be delivered to an area that supports good gene expression. These platforms include specific sequence motifs that enable exchange-based incorporation of transgenes that encode a trait of interest (such as encoding resistance to FHB). To deliver them as single, intact copies to suitable areas of the genome, we have made them competent for delivery via transposition. In 2017, we were able to identify individuals where the platform has transposed, and now we are seeking plants that have only the platform and which do not contain sequences left over from the original site of insertion.
1. Patent awarded for high beta-glucan barley lines. Beta-glucan is a component of grain fiber that has been associated with cardiovascular health and is especially prevalent in barley and oat. Beta-glucan can be taken in by direct consumption of grain (oatmeal, pearled barley) or it can be purified and added to various processed foods to increase their healthfulness. For this purpose, the higher the beta-glucan content, the better. A population of mutagenized barley seed was screened and lines with very high beta-glucan content were identified. These lines have uniquely high levels of this important compound, and in recognition of their exceptional value for future breeding, they have been awarded the patent #9,681,620: Barley Mutant Lines Having Grain with Ultra-High ß-Glucan.
Rashid, A., Baldwin, T.T., Gines, M., Bregitzer, P.P., Esvelt Klos, K.L. 2016. A high-throughput RNA extraction for sprouted single-seed malting barley (Hordeum vulgare L.) rich in polysaccharides. Plants. 6(1):1. doi: 10.3390/plants6010001.
Hu, G., Evans, C.P., Satterfield, K.L., Ellberg, S., Marshall, J., Obert, D. 2016. Registration of ‘Kardia’, a Two-Rowed Spring Food Barley. Journal of Plant Registrations. 10:213-216.
Esvelt Klos, K.L., Yimer, B.A., Babiker, E.M., Beattie, A.D., Bonman, J.M., Carson, M.L., Chong, J., Harrison, S.A., Ibrahim, A.H., Kolb, F.L., McCartney, C.A., McMullen, M., Mitchell Fetch, J., Mohammadi, M., Murphy, J.P., Tinker, N.A. 2017. Genome-wide association mapping of crown rust resistance in oat elite germplasm. The Plant Genome. doi: 10.3835/plantgenome2016.10.0107.