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ARS Home » Pacific West Area » Pullman, Washington » WHGQ » Research » Research Project #424575

Research Project: Genetic Improvement of Wheat and Barley for Resistance to Biotic and Abiotic Stresses

Location: Wheat Health, Genetics, and Quality Research

2017 Annual Report

Objective 1. Identify and develop wheat germplasm adapted to the Pacific Northwest of the United States with improved tolerance to pre-harvest sprouting, drought stress, cold temperatures, rusts, and soil-borne diseases. 1A. Identify sources of drought, cold, and disease tolerance by phenotyping subsets of the National Small Grains Collection as well as international and regional nurseries. 1B. Reduce production risk by developing germplasm with increased resistance to stripe and stem rust. 1C. Breeding club wheat and hard white winter wheat. Objective 2: Develop more efficient wheat and barley breeding approaches based on high throughput phenotyping and genotyping methods as well as genomic selection models. 2A. Identify and apply SNP markers for basic biology and MAS in wheat and barley. 2B. Develop high-throughput phenotyping methods for measuring freezing and drought tolerance. 2C. Develop statistical models for genotype response to environmental stress that improve the efficiency of selection and breeding. Objective 3: Investigate the mechanisms controlling drought and cold tolerance, pre-harvest sprouting, and rust resistance in wheat. 3A. Identify and combine physiological mechanisms that support yield under water stress in wheat including water-use efficiency, root architecture, and photosynthetic efficiency. 3B. Transcriptome analysis of post cold-acclimation stress response. 3C. Gene Expression profiling and biochemical pathway discovery for stripe rust resistance. 3D. Examine the role of the plant hormones ABA and GA in controlling seed dormancy, germination, and preharvest sprouting tolerance.

Objective 1. We will evaluate a total of 6,356 accessions for resistance to freezing injury, Fusarium crown rot, lesion nematodes, cyst nematodes, and stripe rust. We will conduct these evaluations using facilities at WSU, including controlled environments in the WSU Plant growth facility and at the Spillman Agronomy Farm. We will use the genomic information generated by the T-CAP for the existing core collection to link phenotypes to genotypes. We will also screen germplasm from U.S. regional nurseries. These selections will be genotyped to determine relationships and, on the theory that genetic control of resistance will be different among genetically diverse genotypes, traits from the most diverse will be introgressed into adapted cultivars, and germplasm adapted to various regions of the U.S. carrying unique new sources of resistance and molecular markers that can be used to select for these new resistance loci. Objective 2. Specific areas that are being targeted in SNP development include identification of SNP markers linked to stem and stripe rust resistance genes, climatic resilience and identification of SNP in wheat responsible for regional and market class adaptation. The current small grains single plant core collections are being evaluated for SNP linkages to drought, stripe, leaf and stem rust response. As new, verified markers are identified, they will be made available to the customers of the genotyping laboratory as applicable to the customers’ research and breeding objectives. Our goal is to transition away from single gene selection using SSR markers, genotyping by sequencing and incorporate genome selection utilizing SNPs through SNP-chip platforms. Objective 3. Pathways and mechanisms controlling drought and cold tolerance, pre-harvest sprouting, and rust resistance in wheat will be elucidated. Indirect selection for tolerance to freezing and to drought based on physiological traits associated with drought and freezing tolerance will be carried out as part of the selection process. Plant lines will be selected for higher water use efficiency, deeper roots, and higher photosynthetic efficiency to develop better grain yield and grain-filling under drought stress. Transcriptome analysis will be used to identify pathways and mechanisms responding to freezing stress and stripe rust. Key genes will be identified and their expression monitored under stress conditions, thereby identifying plant lines differing in their abilities to respond to parts of the freezing or infection process. Variation in sensitivity to plant hormones will be investigated as a means to control and improve seedling emergence and preharvest sprouting tolerance. These different abilities and sensitivities will be genetically combined, resulting in improved stress tolerance.

Progress Report
Progress was made on all three objectives and sub-objectives, which fall under National Program 301, Component I, Crop genetic improvement; Component 2, Crop Genetic and Genomic Resources and Information Management, and Component 3, Crop Biological and Molecular Processes. Sub-objective 1A: We collaborated with colleagues to discover and validate new loci associated with winter survival and resistance to the soil borne diseases snow mold and Fusarium crown rot, which severely impact winter survival. We identified the genetic control of resistance to stripe rust (also known as yellow rust) in a new landrace source of resistance and developed molecular markers associated with those genes so that they can easily be combined with existing resistance genes for more durable control. These discoveries represent significant progress on the need for wheat germplasm and cultivars with environmental resilience and cold tolerance to avoid winterkill; disease-resistant root systems to improve water use; and resistance to rust diseases that pose a serious threat to productivity. Sub-objective 1B: Fourteen separate screening trials of over 1,300 genotypes to evaluate resistance to cereal cyst nematode were conducted. We have identified 179 genotypes with lower nematode counts and are retesting these to verify resistance. We evaluated 2019 breeding lines for resistance to multiple races of stripe rust in three environments and reported data to regional breeders. We developed 1328 populations to introgress multiple gene resistance to stripe rust from novel sources into breeding lines adapted to the Pacific Northwest, the Great Plains, and the Mid-Atlantic regions of the U.S. Several of these populations were selected for resistance in field in 2017. The resistant selections will be harvested and returned to breeders so they can begin to work with them. We will advance these to another year of testing in 2018 for release of new sources of resistance in adapted germplasm backgrounds. Sub-ojective 1C: We evaluated 5063 plots and 15,740 head rows in 12 locations in the western U.S. to develop improved club wheat cultivars. Club wheat is only grown in the Pacific Northwest of the U.S. as an export crop. Club wheat represents 12% of the total wheat acreage in that region. The current dominant club wheat, Bruehl, is susceptible to quality reduction due to low falling numbers. Therefore, new club wheat cultivars are needed. We entered nine advanced breeding lines into regional and statewide extension trials. We evaluated early generation breeding lines for resistance to stripe rust and soil borne disease in disease nurseries at three locations. The club wheat breeding line, ARS20060123-31C with excellent stripe rust resistance, tolerance to high aluminum in soils, tolerance to low falling numbers, excellent soft wheat quality and yield potential and moderately early maturity, is being purified and increased for release in 2018. This will provide growers with a club wheat breeding line that is earlier than currently marketed lines giving more options for harvest dates. Good quality grain can be produced without additional fungicide applications. Sub-ojective 2A: Genomic data and disease resistance data to other wheat breeders in the region was provided. A database of sequence variants was developed and used for amplicon sequencing of wheat mapping panels. This technology will allow breeding programs to have a large number of molecular markers that can saturate a genome with known markers linked to important selection targets and allow for genomic selection. Sub-objective 2B: We published our analysis of freezing tolerance in a segregating RIL population and reported that freezing tolerance and snow mold resistance were associated at the Fr2 locus in wheat. Sub-objective 2C: Computer scripts were used to analyze next-generation sequencing data for several bi-parental studies and association mapping trials. The scripts provide pre-processing functions to filter out tags of unacceptable occurrence frequencies and those likely to have sequencing errors. These scripts provide data that can be used to analyze panels for genome wide association studies (GWAS) and to generate genomic selection indices. We completed an evaluation of spatial models for analysis of variety trial data and concluded that correcting for major trends and smaller scale spatial variation improved the predictions in every case over standard randomized complete block and even over alpha-lattice designs. Sub-objective 3A: The evaluation of two large populations for preharvest sprouting, alpha amylase, and falling number using grain grown at two locations and using spike wetting tests conducted at the Washington State University (WSU) plant growth facility were completed. We evaluated hundreds of samples from the statewide extension trials for falling number and provided the data to farmers and the industry in a web-accessed database. The purpose of this research was to generate and provide accurate data and begin to assess effect of the cultivar and the environment on the risk of low falling numbers. These data can be used to aid in variety selection to reduce the economic costs of the problem. Under sub objective 3B we discovered a strong diurnal component to freezing tolerance. In plants grown under a 12h light/12h dark photoperiod at constant 4 degrees Celsius (C), the ability to withstand potentially damaging temperatures (less than -12C) varied by nearly 20% if the plants were exposed to the subfreezing temperature starting at the midpoint of the light phase or the dark phase, compared to the end of either phase. This discovery indicates that the regulation of some of the genes controlling response to freezing stress is closely tied to changes in available light, providing new avenues of research into the control of this critically important trait. Sub-objective 3C: Gene expression profiling and biochemical pathway discovery for stripe rust resistance were conducted. The purpose of this research is to move beyond just identifying genetic markers and to discern the causes and relationships among resistance mechanisms. Sub-objective 3D: We investigated the hormonal control of preharvest sprouting, the germination of mature grain on the mother plant when cool rainy conditions occur before harvest, a major cause of low falling numbers. Sprout tolerance results from seed dormancy, the inability to germinate until grain has been stored dry for a while (after-ripened). The Enhanced Response to abscisic acid (ABA) hormone 8 (ERA8) gene was mapped and molecular marker are being used to cross this locus into adapted winter and spring breeding lines. This research is important because it provides breeders with novel mechanisms to reduce the risk of quality degradation caused by low falling numbers.

1. Identification of new loci and molecular markers for stripe rust resistance in a spring wheat landrace. Stripe rust is the most destructive disease of wheat in the world and even though several genes for resistance exist, the pathogen population rapidly mutates to overcome existing deployed genes. ARS researchers in Pullman, Washington, and collaborators at Washington State University, discovered a new source of resistance in a wheat landrace, PI480035, based on greenhouse and field testing over multiple years and locations. They found a major gene for resistance located on chromosome 1B and a minor gene on chromosome 3B. The identification of these new genes with the tightly linked molecular markers will give breeders additional tools to diversify the deployment of resistance genes in wheat. Because PI480035 also carries resistance to stem rust, it will provide breeders with useful sources of resistance to multiple rust diseases.

2. Widespread losses experienced by western wheat farmers. Recent losses were due to low falling numbers caused by a condition known as Late Maturity Alpha-Amylase (LMA) in response to temperature stress. LMA causes starch degradation resulting in low Hagberg-Perten Falling Numbers and in poor-quality baked goods. ARS researchers in Pullman, Washington, and collaborators in Washington, Idaho, and Montana, discovered that LMA susceptibility is widespread in western wheat varieties. The identification of resistant and susceptible varieties will help farmers choose existing varieties with higher resistance to LMA. This will prevent staggering economic losses such as those experienced in the 2016 crop year.

3. Genome-wide association mapping identified genetic loci linked to two serious problems for Northwest wheat growers. Preharvest sprouting is the germination of grain on the mother plant when rain occurs before harvest. This results in economic losses for farmers when their wheat is discounted for high alpha-amylase as measured by the falling numbers test. ARS researchers in Pullman, Washington, and collaborators at Washington State University, discovered that genetic loci associated with preharvest sprouting based on visible sprouting scores are often different from those associated directly with low falling number. Selection using molecular markers linked to both stable falling numbers and lack of visible sprouting will result in better preharvest sprouting resistance than markers based solely on visible sprouting.

4. Genes that improve tolerance to freezing and snow mold disease are either the same or close to each other in winter wheat. Winter wheat planted in the fall faces several threats to survival during the winter months including cold temperatures and soil borne diseases like snow mold, which survives best with moisture and temperatures just above freezing under snow. ARS researchers in Pullman, Washington, and collaborators at Washington State University, rated a winter wheat population segregating for freezing tolerance and resistance to snow mold and conducted a genetic analysis to determine where the genes for resistance to both traits were located. They discovered the major locus for resistance to both freezing and snow mold tolerance was located on the long arm of chromosome 5A, at a location previously associated with freezing survival, as well as new loci for freezing tolerance and snow mold resistance were located on chromosomes 4B and 6B, respectively. This is the first report of the association between resistance to both freezing tolerance and snow mold. Since both of these problems are difficult to evaluate in the field, these molecular markers can be used to select for snow mold resistance and to improve winter survival in wheat.

5. New PCR assays were developed that could assay over 800 loci in barley in one reaction. Next generation marker systems rely on random sequencing, which results in markers that may or may not be linked to important targets of selection by breeders. ARS researchers at Pullman, Washington, screened over 2,000 primer pairs derived from previously mapped single nucleotide polymorphic (SNP) loci to identify highly informative SNP markers for genomic selection in western germplasm. They supplemented this set with markers derived from known genes that are targets of selection in breeding programs. A targeted amplicon sequencing panel was developed for the regional barley programs with over 500 oligonucleotides that saturate the barley genome that can be utilized in genomic selection. This panel allows next generation sequencing methods to be used in breeding programs with known targets of selection that are important for plant breeding.

6. Differences in gene expression were observed between dormant seeds and seeds that lost dormancy in dry after-ripening storage period. Mechanisms controlling seed dormancy, the major factor determining preharvest sprouting resistance, are not well understood. ARS researchers in Pullman, Washington, analyzed the gene expression, or transcriptome, of dry seeds with high seed dormancy and of seeds that lost seed dormancy through a period of storage under dry conditions, known as after-ripening. This revealed that biological changes occur even in dry seeds. These gene expression differences explain the basis of the changes that occur in seeds during after-ripening.

7. Enhanced freezing tolerance resulting from exposure to freeze-thaw cycles was discovered in winter wheat. Acclimation, or descending temperatures during the fall, and fluctuation in soil temperature of three degrees above and below freezing increase the tolerance of plants to freezing temperatures, but little is known about how long these processes need to occur to result in the maximum tolerance in wheat. ARS researchers in Pullman, Washington, exposed six different wheat varieties to differing lengths of freezing and frost/thaw cycles and rated their survival. The freeze–thaw treatments resulted in increased freezing tolerance only after 6–12 weeks of acclimation, but not before 6 nor after 12 weeks of acclimation. This indicates that a long acclimation period must occur prior to the freeze-thaw cycles but that freezing tolerance does reduce after a saturation point. Two cycles of -3 to 3 degrees Celsius freeze–thaw was consistently more effective than one cycle and variation in the extent and timing of the effectiveness of the freeze–thaw treatments was found among the wheat varieties. This research has discovered genetic variation to prolong freezing tolerance further into the winter months in winter wheat.

8. Resistance to multiple soil-borne pathogens is co-located in the Iranian wheat landrace, IWA8608077 (PI621458). Multiple soil borne pathogens infect wheat and cause yield loss in the western U.S. ARS researchers in Pullman, Washington, and collaborators in Washington and Oregon, evaluated a population derived from a cross between IWA8608077 and an adapted spring wheat cultivar, Louise, for resistance to the diseases Fusarium crown rot, Rhizoctonia root rot, and cyst nematodes and mapped the resistance using molecular markers. The population segregated for resistance to all three soil borne diseases and six regions of the genome were associated with resistance to multiple pathogens. One major gene on chromosome 5A contributed to the resistance of all three diseases. These quantitative trait loci (QTL) are used in wheat breeding programs to incorporate resistance to multiple soil-borne pathogens in wheat cultivars.

9. Correlations between spectral reflectance indices and crop yield are also associated with similar quantitative trait locus (QTL). Selection for grain yield, especially in stress environments, is difficult unless large plot, replicated trials are used. ARS researchers at Pullman, Washington, in collaboration with colleagues at Washington State University, correlated several spectral reflectance indices with crop yield in multiple environments and then conducted genomic analysis to determine the number of loci and their location for both crop yield and individual spectral reflectance indices. The correlations that were observed for the spectral reflectance indices and crop yield were reflected in co-located QTL for both sets of traits in certain environments. All three methods, genomic, spectral reflectance and crop yield assays, are used by plant breeders at different times in the breeding program, depending on seed amounts, labor, and cost considerations to select for productivity specific to an environment.

Review Publications
Erginbas-Orakci, G., Poole, G., Nicol, J., Paulitz, T.C., Dababat, A.A., Garland Campbell, K.A. 2016. Assessment of inoculation methods to identify resistance to Fusarium crown rot in wheat. Journal of Plant Diseases and Protection. 123:19-27.
Evans, M.A., Skinner, D.Z., Koenig, R.T., Hulbert, S.H., Pan, W.L. 2016. Effect of phosphorus, potassium, and chloride nutrition on cold tolerance of winter canola. Journal of Plant Nutrition. 39:1112-1122.
Garland Campbell, K.A., Carter, A.H., Jones, S.S., Chen, X., Demacon, P., Higginbotham, R., Engle, D.A., Guy, S.O., Mundt, C.M., Murray, T.D., Morris, C.F., See, D.R. 2017. Registration of “Pritchett” soft white winter club wheat. Journal of Plant Registrations. 11:152-158.
Sthapit Kandel, J., Krishnan, V., Jiwan, D., Chen, X., Skinner, D.Z., See, D.R. 2017. Mapping genes for resistance to stripe rust in spring wheat landrace PI 480035. PLoS One. 12(5):e0177898.
Kiszonas, A., Boehm, J.D., See, D.R., Morris, C.F. 2017. Identification of SNPs, QTLs, and dominant markers associated with wheat flavor preference using genotyping-by-sequencing. Journal of Cereal Science. 76:140-147.
Kruse, E.B., Carle, S.W., Wen, N., Skinner, D.Z., Murray, T.D., Garland Campbell, K.A., Carter, A.H. 2017. Genomic regions associated with freezing tolerance and snow mold tolerance in winter wheat. G3, Genes/Genomes/Genetics. 7:775-780.
Mahoney, A., Babiker, E., Paulitz, T.C., See, D.R., Okubara, P.A., Hulbert, S. 2016. Characterizing and mapping resistance in synthetic-derived wheat to Rhizoctonia root rot in a green bridge environment. Phytopathology. 106:1170-1176.
Nelson, S.K., Steber, C.M. 2017. Transcriptional mechanisms associated with seed dormancy and dormancy loss in the gibberellin-insensitive sly1-2 mutant of Arabidopsis thaliana. PLoS One. 12:(6)e0179143.
Skinner, D.Z., Bellinger, B.S. 2016. Freezing tolerance of winter wheat as influenced by extended growth at low temperature and exposure to freeze-thaw cycles. Canadian Journal of Plant Science. doi: 10.1139/CJPS-2016-0154.
Lei, Y., Wang, M., Wan, A., Xia, C., See, D.R., Min, Z., Chen, X. 2016. Virulence and molecular characterization of experimental isolates of the stripe rust pathogen (Puccinia striiformis) indicate somatic recombination. Phytopathology. 107:329-344.
Sanad, M., Garland Campbell, K.A., Gill, K. 2016. Developmental program impacts phenological plasticity of spring wheat under drought. Botanical Studies. 57:35. doi: 10.1186/s40529-016-0149-3.
Thompson, A.L., Mahoney, A.K., Smiley, R.W., Paulitz, T.C., Hulbert, S., Garland Campbell, K.A. 2017. Resistance to multiple soil-borne pathogens of the Pacific Northwest is co-located in a wheat recombinant inbred line population. G3, Genes/Genomes/Genetics. 7:1109-1116.