Research Project: Improvement of Barley and Oat for Enhanced Productivity, Quality, and Stress Resistance

Location: Small Grains and Potato Germplasm Research

2023 Annual Report

Objectives
This project intends to produce improved barley and oat germplasm, and new information and techniques to facilitate increased efficiencies. The objectives below will be the specific focus for the next five years: Objective 1: Develop barley and oat germplasm with increased yield, better quality, and superior or novel resistances to biotic and abiotic stresses. • Subobjective 1A: Develop low protein barley lines suitable for all-malt brewing. • Subobjective 1B: Develop improved winter food barley varieties. • Subobjective 1C: Develop facultative malting barley. • Subobjective 1D: Develop barley varieties with improved Fusarium head blight resistance. Objective 2: Translate new, sequence-based information into breeder-friendly tools for crop improvement in barley and oats. • Subobjective 2A: Map Fusarium head blight (FHB) resistance and develop germplasm resistant to multiple diseases via marker-assisted selection. • Subobjective 2B: Map quantitative trait loci (QTL) from new sources of adult plant resistance to oat crown rust disease (OCR) and develop milling oat germplasm resistant to crown rust. Objective 3: Develop and implement novel biotechnological tools to produce barley germplasm with unique traits and enhance understanding of the genetic mechanisms underlying key traits. Subobjective 3A: Deliver a site-specific recombination (TAG) platform via Ds-mediated transposition, and demonstrate functionality for RMCE in barley. • Subobjective 3B: Construct and deliver Ds-bordered RNAi constructs that are transposition competent and that confer resistance to Fusarium head blight. • Subobjective 3C: Perform genetic analyses of seed total phosphorus and phytic acid in barley.

Approach
Objective 1: Productive varieties will be developed that are improved for agronomic performance, protein and beta-glucan contents, winter survival, and Fusarium head blight (FHB) resistance. Hybridization with generation advance in greenhouses, New Zealand, and by doubled haploids will be used for population development. Breeding efficiency will be enhanced by investigating the genetics of key traits to enable genomic selection and the development of novel selection schemes. Agronomic performance and FHB resistance will be assessed in multi-location field trials. Grain quality will be assessed by physical examination and chemical analysis of grain for malt quality, protein and beta-glucan contents, and mycotoxin content. Objective 2: Research will relate genetic sequence to disease resistance. Incorporating resistance to diseases that constrain oat and barley production outside of the Intermountain West will make Aberdeen germplasm more valuable. Since Idaho locations have low disease, direct selection for resistance is difficult. Indirect selection of sequence-based markers associated with resistance will combine good agronomic performance and grain quality with resistance to rusts and blotches. For diseases with established markers, development of new lines with specific markers will precede field screening in disease-prone sites outside of Idaho and in greenhouses using artificial inoculation. For other diseases, such as oat crown rust, screening in disease-prone sites will measure disease in multiple test lines, and statistical associations between specific sequences and resistances will identify and “map” new markers. Hybridization, generation advance, and genotypic and phenotypic screens will establish new populations from which lines that have improvements in disease resistance, yield, and quality will be selected. Objective 3: Research will develop tools for experimental genetic manipulations and knowledge of how phosphorus is stored in seeds. Phosphorus is a critical nutrient and a major water pollutant. The hypothesis that the gene lpa-M955 is responsible for reduced seed phosphorus will be investigated by investigating statistical associations between the gene and different levels of seed phosphorus as measured by chemical assays. The hypothesis that new mutations can be found that result in 25% less phosphorus but without negative impact on plant performance will be examined by finding low-phosphorus mutant seeds, and growing them and selecting healthy plants that will then be tested in greenhouses and fields. To facilitate future genetic engineering experiments to identify additional genes of importance, the hypothesis that causing test genes to “jump” (transpose) into specific locations will help answer genetic questions will be tested by attempts to move a test gene into a specially designed receiver site. To test the hypothesis that this process can be harnessed to produce a non-chemical method of controlling a fungus that produces toxins in crop seeds, transposition will deliver an antifungal gene, followed by greenhouse and growth chamber screening for the reduced ability of the fungus to grow and produce toxins.

Progress Report
This is the final report for project 2050-21000-034-000D. This work continues in new project 2050-21000-038-000D. For more information, see the new report. Progress was made towards the development of barley and oat germplasm with increased yield, better quality, and superior resistance to biotic and abiotic stress (Objective 1). Low protein lines suitable for brewing (Sub-objective 1A) were approved each year by the American Malting Barley Association (AMBA) for pilot-scale malting tests (2 to 5 lines per year). Lines were tested for up to three years. Based on the pilot-scale results, breeding lines were taken up by malting companies (1 to 2 lines per year) for industrial-scale testing which required up to 200 lbs. of seed. Although seed of several breeding lines was provided to commercial malting companies for further testing, none were taken up as varieties to contract for production. Spring (ARS10-82, ARS84-27, and ARS98-3) and winter (ARS431W, ARS669W, and ARS671W) elite malting barley breeding lines that met malt quality specifications but were not selected as varieties were released as germplasm to the Journal of Plant Registration. These will be available to public and private breeding programs for use as parents in cultivar development. To address the specific needs of the all-malt brewing industry, we worked with the Brewer’s Association to identify materials that have the best package of malting characteristics for their use. This work included screening elite lines for low protein content. Most of our lines had protein contents higher than desired, and new sources of the low protein trait were identified by screening lines from the National Small Grains Collection (NSGC) and using the top 20 lines as parents of new populations. The variety ‘Gemcraft’ was developed to meet specific needs of the craft brewing industry which has been increasing in use by local craft brewers. The high beta-glucan spring barley varieties ‘Kardia’ and ‘Goldenhart’ became available to growers to meet the food barley market class. A new variety, ‘Upspring’, was released as the first food barley with a winter growth habit (Sub-objective 1B). It was submitted to Plant Variety Protection and variety release applications via the USDA-ARS. A winter food barley will give growers greater management flexibility in that market class. We identified a new winter food barley elite line (12ARS777-2) which showed competitive yield potential and improved winter survival compared with the ‘Upspring’ variety, along with greatly improved beta glucan quality. This breeding line continues in field testing for evaluation of agronomic traits, and beta-glucan. The facultative growth habit, which enables either spring or fall planting, is a trait that was poorly represented in our elite germplasm (Sub-objective 1C). In FY19, we identified sources of this trait among breeding lines from other breeding programs and introduced them into our breeding program through crosses to our elite malting lines. Genetically stable progeny from these crosses were evaluated in both spring and winter performance nurseries to posit and verify a facultative growth habit. Facultative lines were advanced to agronomic performance and malting quality evaluations. Since the emergence of Fusarium head blight (FHB) in traditionally low-disease environments, industry concern has spurred additional investment in FHB research. To address the growing problem of FHB in the Intermountain West (Sub-objective 1D), a new screening nursery was established in Kimberly, Idaho, capable of evaluating both fall-planted and spring-planted germplasm. This collaboration with the University of Idaho doubled our FHB screening capacity. The screening of breeding material for resistance has now become routine and has identified useful levels of resistance among elite, high-performing breeding lines. Screening results were shared with stakeholders and some lines were requested by other researchers. A spring malting barley genomic selection training population was selected from the full breeding program and evaluated for FHB resistance over multiple years and locations. Molecular genetic markers associated with FHB resistance and deoxynivalenol (DON) mycotoxin levels were identified. Data on the training population was used to predict FHB response in all genotyped lines of the spring barley breeding program. Based on these predictions, the top 15 parents were selected for crosses to generate new combinations of FHB resistance. A new molecular screening technique to estimate fungal mycotoxin contamination was developed and tested. This technique shows promise for estimating mycotoxin concentrations more rapidly and cheaply than existing techniques and is now in use by several participants of the U.S. Wheat and Barley Scab Initiative. In support of Sub-objective 2A, two spring barley breeding lines were selected from the Aberdeen breeding program for their consistently low FHB severity and DON in screening nurseries over several years. Combining our efforts to map FHB resistance quantitative trait loci (QTL) with efforts to incorporate foliar disease resistance from other breeding programs, bi-parental mapping populations (95SR316A/Conlon, 95SR316A/ND Genesis, ND Genesis/2Ab08-X05M010-82 and 2Ab08-X05M010-82/Conlon) were created. As required for genetic mapping, the lines within these populations showed large differences in their level of resistance to FHB, especially at Fargo, North Dakota. These populations have been genotyped and we anticipate that characterization for FHB resistance will be completed in 2023. Analysis of this data will allow us to map and compare the number and locations of genes influencing FHB resistance in each population. Progeny with improved FHB resistance have already been identified in these populations. A winter growth habit can provide some innate protection against FHB, and characterization of the genetic resistance available in this type of barley is also desirable. After evaluating Aberdeen winter barley breeding lines over 3 years, lines with promising levels of FHB resistance were selected (06ARS789-34 and 10ARS523-3) and crossed with winter habit cultivars from other breeding programs (Wintmalt and Lightning). The progeny from these crosses are being advanced through self-pollination to create bi-parental mapping populations. Progress on research designed to increase resistance to oat crown rust (Sub-objective 2B) included the identification of oat accessions from the NSGC as donors of adult plant resistance. These are heirloom varieties from the US (CIav2272, and CIav3390) or South America (PI 237090), and landraces of Mediterranean or other European origin (PI140903, PI285583 and PI287296). These donor lines were crossed with an elite Aberdeen oat breeding line (HA08-03X21-1) to create a set of nested bi-parental populations. Characterization of these populations for crown rust resistance will begin in winter of 2023. We will map resistance contributed by each donor, compare resistance across donors for unique or repeating patterns, and identify resistant progeny for use in further breeding. In related work, we mapped the genomic locations of rust resistance genes already present in elite oat germplasm (Pc53, Pc54, Pc96 and Pg13). The donor line of Pc54 also exhibited an adult plant resistance to powdery mildew disease, so we mapped this as well. We found powdery mildew resistance to be conferred by a previously unmapped QTL (QPm.18) on chromosome 1A that may prove useful to breeders in areas where this disease causes damage. Molecular genetic markers linked to the mapped disease resistance genes were tested for their ability to correctly identify oat lines carrying those specific forms of resistance. Informative markers were used to characterize breeding lines in the International Oat Nursery for the probability that they carry rust resistance genes, and the information passed back to breeders. Development of a site-specific recombination (TAG) platform in barley was performed until the loss of the scientist leading this work (Sub-objective 3A). Barley Ds transposon tagged lines with distinct integration sites were evaluated against the Golden Promise genome sequence. The chromosome locations of Ds insertions were identified for 91 lines. Several barley plants were identified with potential “ideal” TAG-locus characteristics (that is, an isolated, single TAG locus in a homozygous state). The information on lines developed with Ds insertions at known genomic locations was shared with the research community and lines are available upon request. The work of site-specific recombination towards the goals of Sub-objective 3B was performed by the meristem transformation system described below. An RNAi construct (consisting of short, inverted repeats of a Fusarium gene) with potential to confer resistance to Fusarium head blight was created (Sub-objective 3B). Efforts to effect transformation using site-specific recombination were stymied by difficulties with the barley transformation process. To address in-house resource limitations that contributed to slow progress, we partnered with researchers at the University of Wisconsin who specialize in crop transformation. This collaboration resulted in the development and validation of an agrobacterium-mediated transformation method using excised barley embryos (shoot tips) as the explants. This method was used to successfully transform ‘Gemcraft’ embryos with vectors containing the RNAi Tri6 construct. Eleven transformed events were generated and confirmed with the molecular primers specifically amplifying the selection marker genes. The number of copies inserted into each plant was determined and five were identified to each carry a single copy. Evaluation of FHB response is ongoing to determine if expression of the FusariumTri6 fragment will reduce the accumulation of DON mycotoxin in infected plants.

Accomplishments
1. Elite facultative barley germplasm that could be planted in the fall or spring. Barley with a facultative growth habit can be planted in the spring to fit with common crop rotation systems or in the fall to maximize water use efficiency. Either planting schedule would allow for fall harvest of grain. Twelve facultative barley lines have been identified by ARS researchers in Aberdeen, Idaho, that have potential for variety development or commercial use based on their good agronomic performance and malting quality profile.

Review Publications
Sheng, H., Esvelt Klos, K.L., Murray, T.D. 2023. Seed infection rate, but not pathogen titer, positively correlates with disease index of Cephalosporium stripe in winter wheat. Phytopathology. 113(3):436-447. https://doi.org/10.1094/PHYTO-06-22-0211-R.
Babar, M.A., Harrison, S.A., Blount, A., Barnett, R.D., Johnson, J., Mergoum, M., Mailhot, D.J., Murphy, J.P., Mason, R.E., Ibrahim, A., Sutton, R., Simoneaux, B., Boyles, R., Stancil, B., Marshall, D., Fountain, M., Esvelt Klos, K.L., Khan, M., Wallau, M., Jordan, H.G. 2023. ‘FLLA11019-8’: A new dual-purpose facultative oat cultivar for grain and forage production in the southern United States. Journal of Plant Registrations. 17(2):238-246. https://doi.org/10.1002/plr2.20272.