Research Project: Genetic Improvement of Wheat and Barley for Environmental Resilience, Disease Resistance, and End-use Quality

Location: Wheat Health, Genetics, and Quality Research

2021 Annual Report

Objectives
The long-term objective of this project is to improve the resilience of wheat plants under environmental stress. Specifically, during the next five years we will focus on the following objectives. Objective 1. Genetically improve soft white winter and club wheat for environmental resilience, disease resistance, and end-use quality. Subobjective 1A: Develop and release club (Triticum aestivum ssp. compactum) wheat cultivars with resistance to major regional diseases and adaptation to diverse environments in the western U.S. Subobjective 1B: Select breeding lines with better end-use quality and high Falling Numbers (FN) due to preharvest sprouting (PHS) and late maturity alpha-amylase (LMA) resistance. Subobjective 1C: Select soft white wheat breeding lines using indirect selection based on high throughput phenotyping (HTP) targeted to specific combinations of climate variables. Objective 2. Identify genetic resources and introgress multiple genes for resistance to stripe rust and to soil borne diseases into wheat germplasm. Subobjective 2A: Identify novel genetic resources with resistance to stripe rust and soil borne disease and identify loci controlling this resistance. Subobjective 2B: Introgress novel sources of resistance to stripe rust and soil borne disease from landraces into adapted wheat germplasm. Subobjective 2C: Conduct collaborative pre-breeding to introgress disease resistance from multiple germplasm accessions into adapted germplasm. Objective 3. Develop, evaluate, and use genotyping technologies and sequence information to increase knowledge of basic genetic processes controlling environmental resilience, disease resistance, and end-use quality in wheat and barley. Subobjective 3A: Identify genetic and molecular mechanisms that regulate response to low temperatures. Subobjective 3B: Identify genetic and molecular mechanisms controlling seed dormancy, germination, and resistance to preharvest sprouting. Subobjective 3C: Identify genetic and molecular mechanisms causing late-maturity alpha amylase expression during grain development. Subobjective 3D: Identify genetic mechanisms for resistance to disease. Objective 4. Incorporate genomic data in wheat and barley selection strategies by collaborating with regional breeding programs. Subobjective 4A: Develop molecular methods for use in genome wide association (GWAS), genomic selection, and transcriptomic strategies to evaluate wheat and barley germplasm. Subobjective 4B: Develop bioinformatic pipelines to facilitate use of genomic data in wheat and barley improvement. Subobjective 4C: Provide genomic and phenotypic data to Western Regional and U.S. Wheat and Barley improvement programs.

Approach
Subobjective 1A: Doubled haploids, genomic selection, and high throughput phenotying are used to increase gains from selection targeted to dry or high rainfall environments in the USDA-ARS club wheat breeding program. Subobjective 1B: Selection for preharvest sprouting and late maturity alpha-amylase resistance based on phenotypic and genotypic data identifes wheat breeding lines with stable high falling number. Tools and tests are developed to detect low falling number wheat, and to distinguish between late maturity alpha amylase and preharvest sprouting. Controlled environment and field-based screening systems are optimized. Plant genetic, biochemical and physiological components associated with low falling numbers are investigated, including the protein biochemistry of alpha amylase, and hydrolytic enzymes expressed during wheat grain development and gation. Subobjective 1C: Genomic selection, high throughput phenotyping and meta-environmental analylsis are used to increase the accuracy breeding program data. Genome estimated breeding values are calculated for soft white and club wheat. Subobjectives 2A and 2B: Dominant male sterility, marker-assisted selection and phenotypic selection are used to incorporate new sources of resistance to stripe rust and to soil borne disease into adapted backcross populations of wheat.Subobjective 2C: F4 bulk populations are developed and selected for adult plant resistance to stripe rust in collaboration with U.S. wheat breeders, followed by selection for agronomic traits and re-evaluation for resistance. Subobjective 3A: Genes, identified from expression studies that contribute to low temperature tolerance, are combined to increase the level of low temperature tolerance in wheat. Subobjective 3B: Preharvest sprouting resistance is increased when mutant alleles associated with altered hormone sensitivity are combined to provide increased seed dormancy. Markers linked to emergence traits are developed. Subobjective 3C: A genome wide association study for resistance to late maturity alpha amylase is conducted, near isogenic lines differing for susceptibility loci are developed and breeding populations are screened in collaboration with wheat breeders. Subobjective 3D: The functional gene for stripe rust resistance is identified using a knock out of that resistance in an EMS-mutagenized population. Subobjective 4A: Targeted amplicon sequencing of at least 1500 known informative markers that are important for selection in western U.S. breeding programs is used to genotype breeding lines. Subobjective 4B: Software tools are developed to apply genomic data to crop improvement. Subobjective 4C: Genomic and phenomic data are provided to public and private sector participants in the Western Regional Cooperative Nurseries and the Western Regional Small Grains Genotyping Laboratory.

Progress Report
This report documents progress for project 2090-21000-033-00D, entitled “Genetic Improvement of Wheat and Barley for Environmental Resilience, Disease Resistance, and End-use Quality”, which started in March 2018. Progress was made on all four objectives, which fall under National Program 301, Component 1, Crop Genetic Improvement, and Component 3, Crop Biological and Molecular Processes. Under Sub-objective 1A, ARS researchers in Pullman, Washington, made significant progress in developing new cultivars of club wheat. The new club wheat cultivar, ‘Cameo’ (previously identified as ARS09X492-6CBW) was proposed for release. Breeder seed was produced in collaboration with the Washington Crop Improvement Association and the line was entered into multi-state variety trials. There is strong interest in Cameo from local wheat growers and seed growers. Cameo is targeted to northern Idaho, southeastern Washington, and northeastern Oregon. The year 2021 was the third year of the Club wheat technical collaboration with the Japanese Flour Millers Association, the Washington Grain Commission, the ARS, and Washington State University. The purpose of the collaboration is to evaluate new club wheat breeding lines and ensure that selections are acceptable for end-use quality. All new club wheat breeding lines evaluated in 2021 are meeting expectations for the Japanese market. This collaboration directly enhances the ability of ARS scientists to achieve the technical quality expectations this important export market. Under Sub-objective 1B, ARS researchers in Pullman, Washington, made significant progress to select wheat breeding lines with better end-use quality and high falling numbers, an important gauge of wheat grain quality. Low falling numbers are caused by two physiological responses to the environment. Preharvest sprouting is due to rain at physiological maturity that causes the mature grain to germinate on the spike prior to harvest. Resistance to preharvest sprouting was evaluated in wheat breeding lines and segregating populations using spike wetting tests. Low falling numbers are also caused by production of the enzyme alpha amylase during grain filling. Resistance to this late-maturity alpha-amylase (LMA) was evaluated in multiple wheat cultivars and genotypes by inducing alpha amylase during grain fill using a cold treatment. The rankings of genotype response to these screens for the two causes of low falling number were provided to wheat breeders and producers in the Pacific Northwest. We discovered that the loci for resistance to late maturity alpha amylase in North American germplasm are similar to those previously identified in Australian germplasm which facilitates marker assisted selection for that trait. For Sub-objective 1C, ARS researchers in Pullman, Washington, collaborated with scientists at Washington State University to collect high throughput image data on multiple field nurseries and they are developing the data management and analysis methods for this data. Significant progress was also made in Sub-objective 2A. ARS researchers in Pullman, Washington, evaluated over 1800 breeding lines from 19 different public and private breeding programs located throughout the United States at two locations in Washington and provided resistance data in July to all cooperators. Resistance was also rated on all major cooperative nurseries. Stripe rust disease occurred throughout the midwestern, southern and western United States in 2021 and collaborators in Louisiana and Indiana discovered a high degree of correlation between the rankings of breeding lines rated in Eastern Washington and in the rest of the United States. Stripe rust is an annual occurrence in Pullman, Washington, and the data that is provided to the rest of the United States has relevance to stripe rust breeding efforts throughout the country. For Sub-objective 2B, ARS researchers in Pullman, Washington, discovered specific loci associated with high protein and protein stability in the ‘D-genome Nested Association Mapping (DNAM) hard winter wheat population. They also developed a recombinant inbred population segregating for resistance to cereal cyst nematode with resistance derived from a D-genome parent used in the DNAM population. This is a novel source of resistance to cereal cyst nematode. For Sub-objective 2C, ARS researchers in Pullman, Washington, incorporated multiple genes for adult plant resistance to stripe rust into breeding lines targeted to regions in the United States where stripe rust is a threat to production. Selected lines that had been evaluated in 2019 and 2020 were returned in the fall of 2020 to wheat breeders in Colorado, Oklahoma, Georgia, Illinois, and Nebraska. Lines that were resistant in 2020 were planted again in the field for increase and selected lines were returned to cooperators in 2021. For Sub-objective 3A, ARS researchers in Pullman, Washington, identified a set of molecular markers that can increase winter survival in wheat. They also conducted RNA-seq on samples of Norstar wheat that had been advanced using either cold or photoperiod to enhance flowering. A small set of differentially expressed genes was identified. Follow up work in this area will investigate these genes in different backgrounds. For Sub-objective 3B, ARS researchers in Pullman, Washington, made progress in identifying the mechanisms controlling seed dormancy, germination, and resistance to preharvest sprouting. A perfect marker was identified for the Enhanced Response to ABA-8 (ERA8) allele which provides increased sensitivity to the dormancy hormone Abscisic Acid (ABA). This marker was used to select for the allele in several wheat breeding populations to develop improved germplasm with resistance to preharvest sprouting. Selections were made in the field in 2021. Progress was made on Sub-objective 3C in identifying the genetic and molecular mechanisms causing late-maturity alpha-amylase expression during grain development in spring and in winter wheat. ARS researchers in Pullman, Washington, screened two biparental populations for the late-maturity alpha-amylase phenotype in order to determine the genetic architecture of the trait in adapted germplasm. Progeny from crosses between resistant and susceptible parents were advanced to develop near isogenic lines differing for specific susceptibility loci so that the effect of each locus can be determined. These populations are now in the F-2 stage of development. For Sub-objective 4A, ARS researchers in Pullman, Washington, conducted exome capture, (sequencing of the protein coding genes in the wheat genome) on set of wheat breeding lines and cultivars contributed by regional breeders and distributed the results. For Sub-objective 4B, ARS researchers in Pullman, Washington, developed a pipeline that enhances the efficiency of the genotype by multiplexed sequencing (GMS) approach to genotyping for wheat and barley that provides increased numbers of data points per sequencing lane, identifies segregation for major known loci of importance to wheat breeders, and simplifies the post processing of sequencing data. This technology was used to genotype several barley and wheat populations. Signatures of selection were identified using iHS, Rsb, and xpEHH loci in four regional spring, four regional winter, five market class, four state spring, and nine state winter populations. Multiple candidate regions or signals for positive selection in state, regional, and market class populations. Further validation of these candidate regions in specific breeding programs may lead to identification of set of loci that can be selected to restore population-specific adaptation without multiple backcrossing which will facilitate the use of novel alleles from germplasm and landraces. For Sub-objective 4C, ARS researchers in Pullman, Washington, coordinated the Western Regional Cooperative Nurseries which were grown at multiple locations in the Pacific Northwest. The nurseries were sent for disease screening for leaf and stem rust at the Cereal Disease laboratory and in Kenya, Africa. Data was reported on the unit website and shared via email as soon as it was available. ARS scientists in Pullman, Washington, assayed molecular markers of importance to regional public and private sector breeders on the Western Regional Nurseries. These services aid regional breeders in maintaining the efficiency and high quality of their breeding programs so that productive wheat cultivars are available for farmers to grow.

Accomplishments
1. Molecular marker developed to select for the mutant ERA8 (Enhanced Response to ABA8) gene. Wheat grain undergoes preharvest sprouting or germination on the mother plant when rain occurs before harvest, resulting in substantial discounts for farmers due to low falling numbers from elevated grain alpha-amylase. There is a major need for new wheat cultivars with resistance to low falling numbers. ARS researchers at Pullman, Washington, and collaborators at Washington State University and John Innes Centre, performed a bulked segregant analysis of exome-DNA-sequence data that mapped ERA8, a new mutant allele in wheat that improves resistance to low falling numbers, to a specific single nucleotide change in the TaMKK3-A sequence. This perfect DNA marker for ERA8 is being used by wheat breeders to select for increased preharvest sprouting tolerance to avoid farmers’ risk of discounts due to low falling number test results.

2. Alternative method of emergence from deep sowing identified in reduced height wheat varieties. The commercial product Release-LC uses the plant hormone gibberellic acid (GA) to improve yield by stimulating grain germination and efficient seedling emergence in farmers’ fields, but also caused a problem because crown root systems were pushed out of the soil putting seedlings at risk of drying out. There was a need to explain why this chemical caused this problem because the manufacturer thought this problem could not occur in wheat lines carrying the GA-insensitive Rht-1 (reduced height) dwarfing genes. ARS researchers in Pullman, Washington, along with Washington State University collaborators, characterized a collection of wheat lines that varied for Rht-1 alleles and found that some GA-insensitive Rht-1 lines could respond to GA with excessive seedling elongation, especially if they had been selected for better emergence. These results provided farmers with information about how to use GA to enhance germination and identified an alternative method to develop wheat with better emergence.

3. Wide-spread late maturity alpha-amylase (LMA) susceptibility is a likely cause of low falling numbers in North American wheat. Farmers lose money when their wheat grain is discounted due to low falling numbers caused by the starch-degrading enzyme alpha-amylase in the grain and the two causes of low falling numbers are difficult to differentiate, but have different genetic control. While U.S. breeding programs focus selection for resistance to preharvest sprouting, they did not know the extent or genetic control of late maturity alpha amylase in North American germplasm. ARS researchers in Pullman, Washington, in collaboration with collaborators at Washington State University, evaluated varieties from 10 North American spring wheat breeding programs and found that most of the late maturity alpha-amylase (LMA) susceptibility mapped to the same loci as LMA in Australian wheat. US wheat breeders now know that selection for LMA resistance is critical to improve falling numbers and have molecular markers to use for this selection.

4. Genotyping by multiplexed sequencing (GMS) method developed to identify multiple known genes in barley. Barley breeders and geneticists need cost effective and efficient methods to identify multiple known genes in breeding populations. Although genotyping methods based on resequencing and analysis of individual genes are popular, there is a need for a cost-effective genotyping platform that can simultaneously identify multiple known genes. ARS researchers at Pullman, Washington, in collaboration with U.S. barley breeders, developed and tested a new genotyping by multiplexed sequencing (GMS) method combined with a data analysis pipeline that can accurately identify alleles at 267 genes of interest to barley breeders. This platform is being used by barley breeders to conduct genetic analysis and select for winter tolerance, improved malting quality, and agronomic traits in barley.

5. Changes in genetic diversity in wheat over time examined to determine if diversity remained for major economic traits. Crop breeders need genetic diversity to improve agronomic traits and to select for pest resistance. Due to long term selection for high grain yield and adaptation, genetic diversity can be lost and breeding progress reduced. ARS researchers at Pullman, Washington, examined population structure and changes in genomic-level and agroecosystem-level genetic diversity of Pacific Northwest (PNW) wheat over the past 120 years. Long-term shifts in gene diversity were not detected, but fluctuations were significant within market classes and within a subset of the most widely grown spring and winter varieties. At the agroecosystem level, diversity has been on a rising trend since the 1990s as the dominance of acreages by a few varieties has become less common. Cultivation of multiple market classes and periodic incorporation of new germplasm by breeding programs have been able to maintain useful levels of genetic diversity in PNW wheat over time indicating that breeding progress will continue.

Review Publications
Hagerty, C., Lutcher, L., Mclaughlin, K.R., Hayes, P., Garland Campbell, K.A., Paulitz, T.C., Graebner, R.C., Kroese, D.R. 2021. Reaction of winter wheat and barley cultivars to Fusarium pseudograminearum inoculated fields, 2018 and 2019. . Agrosystems, Geosciences & Environment. 4(2). Article e20173. https://doi.org/10.1002/agg2.20173.
Horgan, A.M., Garland Campbell, K.A., Carter, A.H., Steber, C.M. 2021. The three-way interaction of varietal emergence capabilities, rht semi-dwarfing alleles, and GA3 seed application from deep planted wheat (Triticum aestivum L). Agrosystems, Geosciences & Environment. 4(1). Article e20144. https://doi.org/10.1002/agg2.20144.
Sexton, T.M., Steber, C.M., Cousins, A.B. 2021. Leaf temperature impacts whole plant water use efficiency independent of changes in leaf level water use efficiency. Journal of Plant Physiology. 258-259. Article 153357. https://doi.org/10.1016/j.jplph.2020.153357.
Liu, L., Yuan, C., Wang, M., See, D.R., Chen, X. 2020. Mapping quantitative trait loci for high level resistance to stripe rust in spring wheat PI 197734 using a doubled haploid population and genotyping by multiplexed sequencing. Frontiers in Plant Science. 11. Article 596962. https://doi.org/10.3389/fpls.2020.596962.
Parajuli, A., Yu, L., Peel, M., See, D.R., Wager, S., Norberg, S., Zhang, Z. 2021. Self-incompatibility, inbreeding depression, and potential to develop inbred lines in alfalfa. In: Yu LX., Kole C., editors. The Alfalfa Genome. Compendium of Plant Genomes. Springer. Cham, Switzerland. p.255-269. https://doi.org/10.1007/978-3-030-74466-3_15.
Brandt, K.M., Chen, X., Tabima, J.F., See, D.R., Zemetra, R.S. 2021. QTL analysis of adult plant resistance to stripe rust in a winter wheat recombinant inbred population. Plants. 10(3). Article 572. https://doi.org/10.3390/plants10030572.
Nazarov, T., Chen, X., Carter, A., See, D.R. 2021. Fine mapping of high-temperature adult-plant resistance to stripe rust in wheat cultivar Louise. Journal of Plant Protection Research. 60(2):126-133. https://doi.org/10.24425/jppr.2020.132213.
Garland Campbell, K.A., Allan, R., Burke, A., Chen, X., DeMacon, P., Higginbotham, R., Engle, D., Klarquist, E., Mundt, C., Murray, T., Morris, C.F., See, D.R., Wen, N. 2021. Registration of “ARS Crescent” soft white winter club wheat. Journal of Plant Registrations. 15(3):515-526. https://doi.org/10.1002/plr2.20135.
Garland Campbell, K.A., Carter, A.H., Allan, R., Chen, X., Steber, C.M., DeMacon, P., Esser, A., Higginbotham, R., Engle, D., Klarquist, E., Morris, C.F., Mundt, C., Murray, T., See, D.R., Wen, N. 2021. Registration of Castella soft white winter club wheat. Journal of Plant Registrations. 15(3):504-514. https://doi.org/10.1002/plr2.20132.
Liu, T., Bai, Q., Wang, M., Li, Y., Wan, A., See, D.R., Xia, C., Chen, X. 2021. Genotyping Puccinia striiformis f. sp. tritici isolates with SSR and SP-SNP markers reveals dynamics of the wheat stripe rust pathogen in the United States from 1968 to 2009 and identifies avirulence associated markers. Phytopathology. https://doi.org/10.1094/PHYTO-01-21-0010-R.
Bai, Q., Wan, A., Wang, M., See, D.R., Chen, X. 2021. Population diversity, dynamics, and differentiation of wheat stripe rust pathogen Puccinia striiformis f. sp. tritici from 2010 to 2017 and comparison with 1968 to 2009 in the United States. Frontiers in Microbiology. 12. Article 696835. https://doi.org/10.3389/fmicb.2021.696835.
Martinez, S.A., Shorinola, O., Conselman, S., See, D.R., Skinner, D.Z., Uauy, C., Steber, C.M. 2020. Exome sequencing of bulked segregants identified a novel TaMKK3-A allele linked to the wheat ERA8 ABA-hypersensitive germination phenotype. Theoretical and Applied Genetics. 133:719-736. https://doi.org/10.1007/s00122-019-03503-0.
Ghimire, B., Hulbert, S., Garland Campbell, K.A., Steber, C.M., Sanguinet, K. 2020. Characterization of root traits for improvement of spring wheat in the Pacific Northwest. Agronomy Journal. 112(1):228-240. https://doi.org/10.1002/agj2.20040.