Research Project: Genetic Improvement of Biotic and Abiotic Stress Tolerance and Nutritional Quality in Hard Winter Wheat

Location: Hard Winter Wheat Genetics Research

2020 Annual Report

Objectives
OBJECTIVE 1: Strategically characterize wheat genetic resources for priority traits including resistance to damaging fungal pathogens (stripe rust, leaf rust, stem rust, Fusarium head scab), resistance to viruses, resistance to Hessian fly, tolerance to heat and drought stress, and nutritional quality. Subobjective 1A: Characterize wheat genetic resources for resistance to stripe rust, leaf rust, and stem rust. Subobjective 1B: Characterize wheat genetic resources for resistance to Fusarium head blight. Subobjective 1C: Characterize wheat genetic resources for resistance to Wheat streak mosaic. Subobjective 1D: Characterize wheat genetic resources for resistance to Hessian Fly. Subobjective 1E: Characterize wheat genetic resources for improved nutritional quality traits. OBJECTIVE 2: Efficiently and effectively incorporate genetic traits into high yielding winter wheat germplasm, and distribute germplasm to the breeding community. Subobjective 2A: Incorporate resistance to stripe rust, leaf rust, and stem rust. Subobjective 2B: Incorporate resistance to Fusarium head blight. Subobjective 2C: Incorporate resistance to Wheat streak mosaic. Subobjective 2D: Incorporate resistance to Hessian Fly. Subobjective 2E: Incorporate tolerance to heat and drought stress. Subobjective 2F: Incorporated improved nutritional quality traits. OBJECTIVE 3: Develop efficient molecular marker technologies for genetic traits and transfer these technologies to the breeding community. Subobjective 3A: Develop new trait-specific SNP markers for important genes. Subobjective 3B: Develop new genome-wide multiplexed amplicon sequencing assay and imputation protocols for important genes. Subobjective 3C: Transfer genotyping data and information to the breeding community. OBJECTIVE 4: Characterize molecular foundations of critical plant-microbe and plant-insect interactions toward development of effective and durable host plant resistance. Subobjective 4A: Characterize molecular foundations of virulence and resistance for Hessian fly. Subobjective 4B: Characterize molecular foundations of virulence and resistance for leaf rust.

Approach
Production of hard winter wheat is limited by recurring intractable problems including diseases, insects, heat, and drought stress. Both nutrient deficiencies and antinutrient excesses affect nutritional quality of wheat products. Our first objective is to identify germplasm with improved resistance to leaf rust, yellow rust, stem rust, Hessian fly, Fusarium head blight, and viruses; improved tolerance to heat and drought stress; as well as increased iron and zinc and lower phytate and cadmium concentrations. The second objective is to transfer these traits into adapted backgrounds and release new germplasm for use as parents in cultivar development. Innovative male-sterile approaches will be used to efficiently construct disease resistance gene pyramids in adapted backgrounds and to isolate coupling-phase recombinants. We will launch a novel full-season wheat recurrent selection project to extend floral initiation and grain filling while maintaining test weight and yield under heat stress. The third objective is to develop more efficient wheat breeding techniques using high-throughput genotyping technologies and large-scale data mining techniques. New allele-specific PCR assays, multiplexed amplicon sequencing assays, and genomic databases will be developed and used to characterize breeding material for the presence of genes of interest. Phenotype and genotype data will be distributed to the breeding community through USDA-supported databases. The fourth objective is to characterize the molecular basis for interaction between wheat plants and leaf rust or Hessian fly. Greater understanding of avirulence effectors in the Hessian fly and the leaf rust pathogen may lead to better strategies for durable resistance.

Progress Report
Objective 1 is to strategically characterize wheat genetic resources for priority traits including resistance to damaging fungal pathogens (stripe rust, leaf rust, stem rust, Fusarium head scab), resistance to viruses, resistance to Hessian fly, tolerance to heat and drought stress, and nutritional quality. To this end, approximately 6,500 wheat breeding lines were screened for field resistance to stripe rust at Rossville, Kansas. The test included entries from the regional nurseries, gene mapping populations, and advanced lines from 12 public and private breeding programs. Approximately 2,100 lines were screened for resistance to stem rust at Manhattan, Kansas. Approximately 8,400 lines were screened for resistance to leaf rust at Manhattan and Hutchinson, Kansas. Wheat lines with introgressions from wheat’s wild relatives, Ambylopyrum muticum and Triticum urartu, have been screened for leaf rust, stripe rust, stem rust, virus, and Hessian Fly resistance. Hard winter wheat breeding populations have been developed to use these new sources of resistance. Stripe rust mapping populations were all advanced one or two generations, and four populations were evaluated at Rossville. The field evaluation of near-isolines for stripe rust resistance gene Yr36 was completed in replicated trials at four test sites and yield evaluation was completed at two test sites. The ‘Overland’/’Everest’ population was tested in two greenhouse cycles by spray inoculation to assess resistance to initial infection by Fusarium head scab. Selections were made for resistance to Wheat streak mosaic virus from 91 breeding populations grown in the field in Hays, Kansas. We screened over 5,000 wheat lines from wheat breeders and geneticists across the region for resistance to Hessian fly. We screened more than 5,000 plants to genetically map the Hessian fly resistance gene H34 from Clark. Winter wheat breeding lines derived from crosses to high-zinc germplasm were grown in yield trials at two locations to provide grain for analysis of zinc concentration. Objective 2 is to efficiently and effectively incorporate genetic traits into high yielding winter wheat germplasm and distribute germplasm to the breeding community. Toward this objective, a second cycle of field evaluation and selection was completed on the first available families for introgression of new stripe rust resistance genes, including Yr40, Yr47, Yr51, Yr57, Yr63, and Yr79. The first cycle of field evaluation and selection was completed for families with linked combinations of adult plant stripe rust resistance genes and mapping populations were advanced one breeding cycle for the Pacific Northwest sources of adult plant resistance. Potential recombinants that couple the durable stem rust resistance gene, Sr2, and the major Fusarium head blight (FHB) resistance gene, Fhb1, were evaluated in the field and demonstrated both Fusarium head blight resistance and excellent stem rust resistance. Progeny of crosses to isolate coupling phase recombinants of the stem rust resistance gene SrTA10171 and the multi-rust resistance locus Lr34/Yr18/Sr57 were selected under strong stem rust pressure in the field and await marker evaluation. In addition, crosses were made to incorporate a coupled Sr2 and Fhb1 from spring wheat germplasm. Pyramids of five or more stem rust resistance genes were evaluated in disease nurseries at five locations, and agronomic performance was tested in multi-location replicated yield trials. Sister lines varying for each of the three new major stem rust resistance genes were selected in the greenhouse in preparation for seed increase. Introgression germplasm of stem rust pyramids was increased in Arizona in preparation for fall 2020 yield trials. In cooperation with the USDA-ARS in Fargo, North Dakota, segregating progeny of backcrosses of the winter wheats ‘Ruth’ and ‘Smith’s Gold’ to the spring wheat source of the FHB resistance were screened for new genetic markers for the FHB resistance and with markers for the winter-habit gene, which is in close proximity. Plants were identified which have both the necessary winter-habit characteristic and markers for the FHB resistance. These recombinant plants will be used as donor parents in additional backcrosses. Fhb1 and resistance genes on chromosomes 5A and 2DL were pyramided in Everest and Overland backgrounds. Twenty selected lines from each pyramiding project were evaluated in the Kansas State University Plant Pathology field FHB nursery and showed high FHB resistance in the two hard winter wheat backgrounds. Families from crosses to 13 durum landraces with high-temperature Hessian fly resistance were screened with Hessian flies, and 39 families with 100% resistant progeny were identified. Seed was produced from these families for further characterization. In addition, selections were made from 220 field-grown rows derived from resistant selections. Two generations of advancement were conducted on backcrosses of bread wheat to ancestral wild emmer wheat. The heat tolerance dominant male sterile population was grown in the field near Hutchinson, Kansas, and Cycle 2 of field selection was completed with the harvest of 656 individual male-sterile plants from the population. Winter-hardy inbred families of four crosses of hard winter wheat to high-zinc spring lines were selected in the field near Rossville, Kansas and breeding selections were made from 948 rows breeding populations with the low phytate trait. A combined total of 418 new crosses were made in the greenhouse to support the ongoing development of improved hard winter wheat germplasm. Agronomic performance of inbred lines was evaluated in 3,908 yield trial plots. Objective 3 is to develop efficient molecular marker technologies for genetic traits and transfer these technologies to the breeding community. We developed and validated more than 50 new high-throughput markers for important traits in wheat. The first version of the haplotype database was built with the latest version of Practical Haplotype Graph (PHG) software. It is running on the ARS SciNet high performance computer cluster. Currently the hard winter wheat database is made of 65 cultivars used in wheat breeding programs across the Great Plains region of the United States. The objective for this database is to be able to use low cost, low density genetic data to predict high density genetic data, which will reduce the cost of genetic characterization of wheat germplasm. The first version of the haplotype database improved the prediction accuracy to 95-96%, which represents a 3% increase over the current standard imputation software, Beagle. A new protocol called genotyping by multiplexing amplicon sequencing (GBMAS) is under development to characterize wheat lines for a large number known target traits with a single assay. Data are now available for 100 genetic targets using this new protocol. However, genetic markers have not yet been definitively identified because the data processing pipeline is under development. Haplotypes around agriculturally important traits have been constructed, and winter wheat germplasm lines are sorted into haplotype groups. We are currently designing markers to efficiently screen breeding lines for haplotype classification for carriers of traits of interest for quick identification for lines to retain in breeding programs. Database construction for haplotypes of these traits is underway. More than 15,000 wheat samples from 15 research and breeding programs were analyzed for molecular markers this year. More than 350 million genome-wide and 210,000 sequence-specific data points were generated for wheat geneticists and breeders. The two hard winter wheat regional wheat performance and germplasm observation nurseries were characterized with more than 120 sequence-specific markers linked to important traits. The genotypic data were used in conjunction with phenotypic data by wheat researchers for characterizing and selecting breeding lines with desired combinations of agronomic and pest resistance traits. Objective 4 is to characterize the molecular foundations of critical plant-microbe and plant-insect interactions toward development of effective and durable host plant resistance. We continued working on family-1 effectors from Hessian fly and their distribution in infested wheat plant tissues and the dynamics in Hessian fly tissues at different developmental stages. We continued work on identification of mutant host genes that affect infection by leaf rust. Mutant resistant lines were backcrossed to cultivar Thatcher and progeny were sequenced for expression analysis and mutant detection. Genomic DNA was sent for exome capture analysis of seven of the mutants. To aid the identification of fungal effectors, the genome of the leaf rust fungus has been further assembled using new computer programs. The genome is complex because it is contained in two nuclei and there are regions that are highly similar and difficult to separate. Using new sequencing technologies, the full phased diploid genome of the reference strain of the leaf rust fungus is nearing completion.

Accomplishments
1. Dominant male sterility opens new strategies in wheat breeding. Wheat is a self-pollinated crop, so the production of a large number of hybrid seeds by cross-pollination is technically demanding and labor-intensive. Genetic dominant male sterility (DMS) can be used to facilitate cross-pollination but has not been commonly used by breeders due to inability to sort sterile from fertile plants in a timely manner. An ARS researcher at Manhattan, Kansas, has identified a DNA marker for DMS that is breeder-friendly and efficient. This marker enables wheat breeders to identify sterile plants at the seedling stage and then use breeding strategies like those used in corn such as mass selection, half-sib selection, and recurrent selection. It will also enable large-scale marker-assisted backcrossing and gene pyramiding. This will speed the development of elite new wheat cultivars with stacks of desirable traits. 301 1 A 2018

2. Sulfur fertilization improves wheat yield and grain quality. Wheat growers are challenged to produce high yielding crops with desirable grain protein quality, and the milling industry is challenged to produce flour with favorable strength and tolerance to mixing. ARS researchers in Manhattan, Kansas, demonstrated that addressing sulfur deficiency can improve both yield and quality of the wheat crop, to the benefit of both producers and flour millers. Furthermore, asparagine, which is an essential amino acid precursor of protein in wheat grain, can accumulate in grain produced under low sulfur conditions. In the preparation of some foods, asparagine is converted to acrylamide, which is a health concern. Asparagine concentration was reduced 8-fold by sulfur application to wheat growing in low sulfur soil. This research demonstrates the need for producers to monitor sulfur conditions in their soils and address deficiency where it exists. 301 1 A 2018

3. Resistance to three wheat rusts identified in wheat wild relative Ambylopyrum muticum. Cereal rust diseases are a constant problem in wheat production. New resistant varieties are released each year, but within a few years, rust pathogens usually overcome the resistance. For that reason, plant breeders are in a constant search for new resistance genes. Ambylopyrum muticum is a wild relative of wheat and previous hybridization work has integrated segments of the genome into 28 wheat lines. ARS researchers in Manhattan, Kansas found multiple lines with resistance to leaf rust, stem rust, or stripe rust. These resistant lines can now be used by breeders to improve the rust resistance of new wheat cultivars. 301 1 A 2018

4. Markers developed for four Hessian fly resistance genes in wheat. Hessian fly is an important destructive insect pest of wheat that causes stunting of seedlings and lodging of mature tillers. Genetic resistance is the best control method for Hessian fly. Traditional selection techniques for resistance use live colonies of Hessian fly biotypes and are laborious, slow, and expensive. Marker-assisted selection is much easier, faster, and cheaper but requires development of DNA markers for each resistance gene. ARS researchers in Manhattan, Kansas, developed new breeder-friendly DNA markers for Hessian fly resistance genes h4, H7, H35, and H36. These markers will help select future wheat cultivars with enhanced resistance to the Hessian fly. 301 1 A 2018

5. Hessian fly acquired fructan metabolism by horizontal gene transfer. Hessian fly is an important insect pest of wheat that causes stunting and lodging. ARS researchers in Manhattan, Kansas, found that Hessian fly has at least ten genes for enzymes that digest fructan, which is a storage polymer of fructose in plants. Most animals do not have enzymes that digest fructans. These ten genes were most similar to those from bacteria. The results indicate that an ancient horizontal gene transfer from bacteria to Hessian fly was followed by gene duplication and possibly functional specialization of the ten copies. Deeper understanding of the carbohydrate metabolism of Hessian fly could lead to innovative methods of control. 301 3 A 2018

Review Publications
Guttieri, M.J., Bowden, R.L., Reinhart, K., Marshall, D.S., Jin, Y., Seabourn, B.W. 2020. Registration of hard white winter wheat germplasms KS14U6380R5, KS16U6380R10, and KS16U6380R11 with adult plant resistance to stem rust. Journal of Plant Registrations. 1-7. https://doi.org/10.1002/plr2.20004.
Wilson, T., Guttieri, M.J., Nelson, N., Fritz, A., Tilley, M. 2020. Nitrogen and sulfur effects on hard winter wheat quality and asparagine concentration. Journal of Cereal Science. 93:102969. https://doi.org/10.1016/j.jcs.2020.102969.
Singh, N., Steeves, R., Chen, M., El Bouhssini, M., Pumphrey, M., Poland, J. 2020. Registration of hessian fly resistant germplasm KS18WGRC65 carrying H26 in hard red winter wheat ‘overley’ background. Journal of Molecular Biology. 14:206–209. https://doi.org/10.1002/plr2.20003.
Zhang, G., Martin, T.J., Frtiz, A.K., Regan, R., Bai, G., Chen, M., Bowden, R.L., Jin, Y., Chen, X., Kolmer, J.A., Chen, Y.R., Seabourn, B.W. 2020. Registration of ‘KS Venada’ hard white winter wheat. Journal of Plant Registrations. 14:153–158. https://doi.org/10.1002/plr2.20026.
Niu, F., Xu, Y., Liu, X., Zhao, L., Bernardo, A.E., Yaoguang, L., Guixia, L., Chen, M., Cao, L., Hu, Z., Xu, X., Bai, G. 2020. The Hessian fly recessive resistance gene H4 mapped to chromosome 1A of the wheat cultivar ‘Java’ using genotyping-by-sequencing. Journal of Theoretical and Applied Genetics. https://doi.org/10.1007/s00122-020-03642-9.
Rudd, J.C., Devkota, R.N., Ibrahim, A.M., Baker, J.A., Baker, S., Sutton, R., Simoneauz, B., Opena, G., Hathcoat, D., Awika, J.M., Nelson, L.R., Liu, S., Xue, Q., Bean, B., Neely, C.B., Duncan, R.W., Seabourn, B.W., Bowden, R.L., Jin, Y., Chen, M., Graybosch, R.A. 2019. ‘TAM 204’ wheat, adapted to grazing, grain, and graze-out production systems in the southern High Plains. Journal of Plant Registrations. https://doi.org/10.3198/jpr2018.12.0080crc.
Guttieri, M.J. 2020. Ms3 dominant genetic male sterility for wheat improvement with molecular breeding. Crop Science. https://doi.org/10.1002/csc2.20091.
Varella, A.C., Weaver, D.K., Blake, N.K., Hofland, M.L., Heo, H., Cook, J.P., Lamb, P.F., Jordan, K.W., Akhunov, E., Chao, S., Talbert, L.E. 2019. Analysis of recombinant inbred line populations derived from wheat landraces to identify new genes for wheat stem sawfly resistance. Theoretical and Applied Genetics. 132(8):2195-2207. https://doi.org/10.1007/s00122-019-03347-8.
Dewitt, N., Guedira, M., Lauer, E., Sarinelli, M., Tyagi, P., Fu, D., Hao, Q., Murphy, J. P., Marshall, D.S., Akhunova, A., Jordan, K., Akhunov, E., Brown Guedira, G.L. 2019. Sequence based mapping identifies a candidate transcription repressor underlying awn suppression at the B1 locus in wheat. New Phytologist. 225:326–339. https://doi.org/10.1111/nph.16152.
Fellers, J.P., Matthews, A., Fritz, A., Rouse, M.N., Grewal, S., Hubbart-Edwards, S., King, I., King, J. 2020. Resistance to wheat rusts identified in wheat/Amblyopyrum muticum chromosome introgressions. Crop Science. https://doi.org/10.1002/csc2.20120.
Kolmer, J.A., Bernardo, A.N., Bai, G., Hayden, M.J., Anderson, J.A. 2020. Thatcher wheat line RL6149 carries Lr64 and a second leaf rust resistance gene on chromosome 1DS. Theoretical and Applied Genetics. 132:2809–2814. https://doi.org/10.1007/s00122-019-03389-y.
Zhu, Z., Hao, Y., Mergoum, M., Bai, G., Humphreys, G., Cloutier, S., Xia, X., He, Z. 2019. Breeding wheat for resistance to Fusarium head blight in the Global North: China, USA and Canada. The Crop Journal. https://doi.org/10.1016/j.cj.2019.06.003.
Ahmed, W., Xia, Y., Li, R., Bai, G., Siddique, K.M., Guo, P. 2019. Non-coding RNAs: Functional roles in regulation of stress response in Brassica crop. Genomics. 140:96-104. https://doi.org/10.1016/j.ygeno.2019.08.011.
Mourad, A., Sallam, A., Belamkar, V., Wegulo, S., Bai, G., Mahdy, E., El-Wafa, A., Jin, Y., Baenziger, S.P. 2019. Molecular marker dissection of stem rust resistance in Nebraska bread wheat germplasm. Scientific Reports. 9:11694. https://doi.org/10.1038/s41598-019-47986-9.
Chen, H., Ino, M., Shimono, M., Ganpatrao, S., Kobayashi, K., Yaeno, T., Yamaoka, N., Bai, G., Nishiguchi, M. 2019. A single amino acid substitution in the intervening region of 129K protein of cucumber green mottle mosaic virus resulted in attenuated symptoms. Phytopathology. https://doi.org/10.1094/PHYTO-12-18-0478-FI.
Wu, Y., Wu, Y., Gao, T., Mu, Q., Wang, J., Li, X., Tian, B., Wang, M.L., Bai, G., Ramasamy, P., Trick, H.N., Bean, S.R., Ismail, D.M., Turinsta, M.R., Morris, G., Tesfaye, T.T., Yu, J., Li, X. 2019. Allelochemicals targeted to balance competing selection forces in African agroecosystems. Nature Plants. https://doi.org/10.1038/s41477-019-0563-0.
Wang, H., Sun, S., Ge, W., Zhao, L., Hou, B., Wang, K., Lyu, Z., Chen, L., Xu, S., Guo, J., Xu, S.S., Bai, G. 2020. Horizontal gene transfer of Fhb7 from fungus underlies Fusarium head blight resistance in wheat. Science. https://doi.org/10.1126/science.aba5435.
Liu, S., Bai, G., Lin, M., Luo, M., Zhang, D., Jin, F., Tian, B., Trick, H., Yan, L. 2020. Identification of candidate chromosome region of Sbwm1 for Soil-borne wheat mosaic virus resistance in wheat. Scientific Reports. https://doi.org/10.1038/s41598-020-64993-3.
Zhao, L., Abdelsala, N., Xu, Y., Chen, M., Feng, Y., Kong, L., Bai, G. 2020. Identification of two novel Hessian fly resistance genes H35 and H36 in a hard winter wheat line SD06165. Journal of Theoretical and Applied Genetics. https://doi.org/10.1007/s00122-020-03602-3.
Pradhan, Sumit, Babar, M., Bai, G., Khan, J., Shahi, D., Avci, M., Guo, J., McBreen, J., Asseng, S., Gezan, S., Baik, B.V., Blount, A., Harrison, S. 2019. Genetic dissection of heat-responsive physiological traits to improve adaptation and increase yield potential in soft winter wheat. BMC Genomics. 66:941–950. https://doi.org/10.1007/s10722-019-00742-4.