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

Location: Hard Winter Wheat Genetics Research

2021 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: Strategically characterize wheat genetic resources for priority traits including resistance to damaging fungal pathogens (stripe rust, leaf rust, stem rust, Fusarium head blight), resistance to viruses, resistance to Hessian fly, tolerance to heat and drought stress, and nutritional quality. We screened approximately 5,200 wheat breeding lines for field resistance to stripe rust at Rossville, Kansas. The test included entries from the regional nurseries, gene mapping populations, and elite breeding lines from 14 public and private breeding programs. Approximately 2,200 breeding lines were screened for stem rust resistance at Manhattan, Kansas. Approximately 1,000 breeding lines were screened for leaf rust resistance at Castroville, Texas; Manhattan, Kansas; and Hutchinson, Kansas (Goal 1A.1). Hessian fly resistance of over 5,400 wheat lines from breeders and geneticists was evaluated (Goal 1D.1). ARS scientists collaborated with scientists at Kansas State University to evaluate remote sensing technology for assessing Hessian fly infestation in a field trial with caged flies applied to resistant and susceptible cultivars at varying densities and cage sizes. ARS germplasm under development was characterized for resistance to stripe rust, leaf rust, stem rust, Fusarium head scab, and viruses. We screened nearly 1,200 ARS breeding lines for resistance to wheat streak mosaic virus (Goal 1C.1). Over 500 ARS breeding lines in yield testing were evaluated for resistance to Fusarium head scab, stem rust, stripe rust (Rossville, Kansas and Hutchinson, Kansas), and leaf rust (Castroville, Texas; Hutchinson, Kansas; and Manhattan, Kansas). ARS breeding germplasm from early generation segregating populations was selected in disease nurseries at these locations. In total, over 15,000 rows were evaluated for breeding selection in ARS germplasm. Three biparental mapping populations and 76 near-isogenic lines were grown in a field trial at a high-Cadmium site in Nebraska (Goal 1E.1). A mutant population derived from the cultivar ‘Jagger’ was screened to find knock-out mutants for leaf rust resistance gene Lr37 and stem rust resistance gene Sr38 in a cooperative effort to clone those genes. Germplasm development continued on hard winter wheat lines with resistance genes Sr50, SrTA10171, and SrTA10187. Phenotypic and genotypic selections continued on stacking slow rusting resistance genes from cultivars ‘Kingbird’ and ‘Roelfs F2007’. Four mapping populations derived from cultivars with stripe rust resistance were screened for the second year and were also genotyped. We advanced four mapping populations designed to identify modifiers of the pseudo-black chaff trait associated with the stem rust resistance gene Sr2 to the F3 generation. Sr2 also was associated with necrotic blotch symptoms known as bacterial leaf blight in a mapping population. Bacteria were not consistently associated with symptoms, so it appears that the disease was mis-characterized and is, in fact, a pleiotropic effect of Sr2. Data were collected to map the trait in two additional populations. Those populations also were scored for necrotic spots associated with chloride deficiency. A gene associated with stay-green trait under heat stress was fine-mapped to a narrow region (1.5 cM) on chromosome 3BS at the Sr2 locus. Sr2 was associated with lower chlorophyll retention under heat stress. The two contrasting alleles of the gene were crossed to cultivars ‘Ruth’ and ‘Denali’ to test performance. Objective 2: Incorporate genetic traits into high-yielding winter wheat germplasm and distribute to the breeding community. Breeding germplasm selected for the combination of stripe rust resistance genes Yr5 and Yr15 continued to demonstrate exceptional resistance in field experiments. Multi-location yield trials of 147 of these selections was completed, and some selections were among the highest yielding germplasm tested. Breeding germplasm with stripe rust resistance genes Yr40, Yr47, Yr51, Yr57, Yr63, and leaf rust resistance genes Lr42 and Lr19 were selected under high stripe rust and leaf rust disease pressure and advanced to yield testing (Goal 2A.1). Bread wheat introgression lines with chromosome fragments from wheat wild relatives Amblyopyrum muticum, T. urartu, and T. timopheevii were screened for leaf, stem, and stripe rust resistance. These lines also were planted in Rossville and Prosper, North Dakota for field evaluation for disease traits. Introgression lines are being backcrossed into two adapted hard winter wheat backgrounds (Goal 2A.1). A total of 327 crosses were made for this project in fiscal year (FY) 21, and lines are being advanced to the next backcross (BC2F1). Three recombinant winter wheat lines with Sr2 in coupling with the Fusarium head blight (FHB) resistance gene Fhb1 were evaluated for FHB resistance and stem rust resistance in field nurseries, and yield was evaluated in preliminary yield trials (Goal 2A.2). Development of wheat germplasm with pyramids of three major stem rust genes, Sr22, Sr26, and Sr36 continued (Goal 2A.3). Sixty-one breeding lines with combinations of these genes in three commercial wheat genetic backgrounds were included in yield tests in multiple Kansas environments. A cooperative research and development agreement (CRADA) partner also tested 35 of these lines in three Oklahoma environments, and six of these 35 lines were advanced to the next stage of yield testing by the CRADA partner. Pyramids of Fusarium head blight resistance traits were evaluated in field and greenhouse trials, and promising lines were identified for subsequent yield testing (Goal 2B.1). Fhb1 and two resistance loci on chromosome 5A from GP-80, and Fhb1 and a resistance on chromosome 2DL from Ji5625 were combined in Everest and Overland backgrounds. Twenty selected breeding lines (BC2F5) from each pyramiding project were evaluated in a field nursery and showed high Fusarium head blight resistance. We evaluated winter wheat germplasm derived from crosses with winter synthetic hexaploid wheats that confer resistance to wheat streak mosaic virus. Field evaluation under severe virus infection demonstrated limited resistance in breeding germplasm (Goal 2C.1). Breeding germplasm derived from crosses to durum landraces with high-temperature Hessian fly resistance was evaluated in yield tests was conducted at Ashland and Hutchinson, Kansas (Goal 2D.1). Seed increase of 498 F5-derived introgression lines of T. dicoccoides in hard winter wheat backgrounds was conducted in Yuma, Arizona to provide seed for subsequent experiments (Goal 2E.1). Grain for end-use quality evaluation was produced from 283 F3-derived T. dicoccoides introgression lines grown in a field experiment at Hutchinson, Kansas. High-Zinc germplasm was tested in replicated yield trials at 6 locations, and mineral analyses are pending (Goal 2F.1). Low phytate breeding lines were evaluated in preliminary yield trials. We produced a total of 5,157 yield trial plots in support of the breeding and research efforts. Objective 3: Develop efficient marker technologies for genetic traits. We validated the imputation accuracy of a new bioinformatic technique, the practical haplotype graph (PHG) database (Goal 3B.1). A PHG database with 345 wheat genotypes from all ARS regional genotyping labs has been constructed and contains >1.7 million variants for imputation. This database is available to other genotyping labs on SciNet. The genotyping by multiplexing amplicon sequencing (GBMAS) marker technology was evaluated on 192 wheat genotypes for 114 single nucleotide polymorphism (SNP) targets. This assay genotyped 63 SNP targets with average depth of coverage of 73 reads, however depths were highly variable. Sequence data was analyzed for 10 populations genotyped using multiplex restriction amplicon sequencing (MRAseq), and numerous raw variants were identified for each population. Population-specific filtering parameters drastically reduced marker coverage: on average, 600-1000 high quality markers per population were identified for mapping or background selection. We have begun to test the concept of skim exome capture combined with haplotype-based imputation as a method for inexpensive genome-wide genotyping, which could efficiently deliver high-density genomic information on wheat breeding lines. We analyzed more than 7,000 wheat samples from 15 research and breeding programs for molecular markers (Goal 3C.1). Over 100 million genome-wide and 200,000 sequence-specific data points were generated for regional wheat researchers. The two regional wheat performance and germplasm observation nurseries were characterized with 90 sequence-specific markers linked to important traits. Objective 4: Characterize molecular foundations of critical plant-microbe and plant-insect interactions. We sequenced genes for putative effector proteins from Hessian fly, barley midge, and oat midge (Goal 4A.1). Transgenic wheat lines with selected Hessian fly effectors have been generated and are under propagation and characterization (4A.2). Seven mutants of ‘Thatcher’ wheat and the wild-type Thatcher were sequenced using exome capture (Goal 4B.1). Bulk RNA sequencing analysis was completed on mutant lines 1995 and 2048. Mapping populations with mutant lines 1995, 2948, and 2348 were advanced to the F4 for genetic mapping. A near-complete genome of the leaf rust pathogen Puccinia triticina race BBBD was assembled (4B.2). The new genome assembly is much more complete and is a much better representation of each chromosome pair in this fungus. Using a draft assembly, virulence mutants for leaf rust resistance genes Lr2A, Lr2C, and Lr52 were aligned to the genome. Seven P. triticina genes were identified as possible candidate Lr2 effector/avirulence gene in the fungus. For Lr52, 24 P. triticina genes were identified as candidates for virulence. More mutants are being isolated to narrow the candidate gene list.

Accomplishments
1. A new haplotype-based genotyping method in wheat. Wheat breeding programs increasingly depend on genotyping of DNA markers to help select the best lines with the best traits for the wheat industry. Unfortunately, current genotyping methods target individual markers that may suffer from high costs, intellectual property constraints, and high false positive or negative results. ARS researchers at Manhattan, Kansas, worked with other ARS and university colleagues to apply a new genotyping method called PHG (practical haplotype graph) to wheat. Haplotypes are combinations of nearby markers along the chromosome and collectively have much better trait predictive power than individual markers. The heart of the PHG is a database of high-coverage haplotype sequences from 345 wheat cultivars and experimental lines. The PHG allows breeders to leverage this haplotype database to impute genotypes of breeding lines using only low-cost, low-coverage skim sequencing information as input. In computer trials, the PHG was significantly more accurate than traditional imputation tools. The PHG is flexible and expandable and has the potential to be an efficient, inexpensive, and powerful platform for genotyping in wheat.

2. Identification of durable leaf rust resistance in cultivar ‘Roelfs F2007’. Leaf rust is one of the most important diseases of wheat worldwide. Unfortunately, most resistance to leaf rust is race-specific, and the pathogen population can rapidly adapt and overcome it. Roelfs F2007, a Mexican spring wheat cultivar, was reported to carry a high level of durable race-nonspecific adult plant resistance to leaf rust. To map the resistance, we crossed Roelfs F2007 with the adapted hard white winter wheat, ‘Lakin’, which is moderately susceptible. Researchers at Manhattan, Kansas, identified resistance genes on chromosome arms 3BS, 5DS, and 7BL of Roelfs F2007, which correspond to resistance genes Lr74, Lr78, and Lr68, respectively. In addition, a novel resistance gene on chromosome arm 7BS was discovered in Lakin. Together, the four genes conferred a high level of adult plant resistance to leaf rust that can be used to enhance durable resistance to leaf rust in adapted cultivars.

3. Chromosome level assembly of the Puccinia triticina genome. The leaf rust pathogen is genetically complex, with two nuclei and 18 chromosomes. This complexity creates challenges for identifying the fungal genes for virulence on wheat. The initial draft assembly of the leaf rust fungal genome only represented half of the genome, and it was very fragmented, and it lacked many of the key fungal genes. Researchers at Manhattan, Kansas, used new sequencing technology and computer programs enabled a better knitting together of DNA sequences to produce a near complete genome. The complete assembly will help scientists find important genes needed for infection, and to find mutations that cause leaf rust to overcome wheat disease resistance genes.

4. Efficient markers for Fhb7. Fhb7, a gene with a major effect on Fusarium head blight resistance, was recently cloned as a gene encoding a glutathione S-transferase (GST), which originated from chromosome 7E of Thinopyrum ponticum and confers broad resistance to Fusarium species. DNA markers are not available for high-throughput detection of the gene in wheat breeding programs. Therefore, researchers at Manhattan, Kansas, developed Kompetitive Allele Specific PCR (KASP) markers using a key sequence variation in the GST gene and single nucleotide polymorphisms (SNPs) tightly linked to Fhb7. Four markers were validated in both biparental and natural populations. These markers are diagnostic and will facilitate the deployment of Fhb7 in wheat breeding programs to improve FHB resistance.

5. Genetic tools for wheat streak mosaic virus. Wheat streak mosaic virus is an endemic and potentially devastating disease of wheat. Only three genes for resistance are known. Wsm1 is a potentially useful resistance gene that provides resistance to both wheat streak mosaic virus and Triticum mosaic virus, but its use has been limited to one commercial cultivar, ‘Mace,’ because of a significant detrimental effect of the translocation from Thinopyrum intermedium that provided the resistance. A shortened version of the translocation has become available for breeding. Researchers at Manhattan, Kansas, demonstrated that nearly identical sister lines, with and without the virus resistance from this short translocation, have similar grain yield. To support breeding efforts with this useful and shorter translocation, researchers at Manhattan, Kansas, developed codominant Kompetitive Allele Specific PCR (KASP) markers that are diagnostic for the translocation and will facilitate the use of Wsm1 in wheat breeding programs to improve wheat mosaic virus resistance.

Review Publications
Zhang, G., Martin, T.J., Fritz, A.K., Li, Y., Bai, G., Bowden, R.L., Chen, M., Chen, X., Kolmer, J.A., Seabourn, B.W., Chen, Y., Marshall, D.S. 2020. Registration of ‘KS Dallas’ hard red winter wheat. Journal of Plant Registrations. https://doi.org/10.1002/plr2.20108.
Zhang, G., Fritz, A.K., Haley, S., Li, Y., Bai, G., Bowden, R.L., Chen, M., Jin, Y., Chen, X., Kolmer, J.A., Seabourn, B.W., Chen, Y., Marshall, D.S. 2020. Registration of ‘KS Western Star’ hard red winter wheat. Journal of Plant Registrations. https://doi.org/10.1002/plr2.20104.
Nyine, M., Adhikari, E., Clinesmith, M., Jordan, K., Alan, F., Akhunov, E. 2020. Genomic patterns of introgression in interspecific populations created by crossing wheat with its wild relative. G3, Genes/Genomes/Genetics. 10:3651-3661. https://doi.org/10.1534/g3.120.401479.
Liu, S., Wang, D., Lin, M., Sehgal, S., Dong, L., Bai, G. 2020. Artificial selection in breeding extensively enriched a functional allelic variation in TaPHS1 for pre-harvest sprouting susceptibility in wheat. Journal of Theoretical and Applied Genetics. 134:339–350. https://doi.org/10.1007/s00122-020-03700-2.
Zhang, G., Martin, T., Fritz, A., Bowden, R.L., Bai, G., Chen, M., Kolmer, J.A., Seabourn, B.W., Chen, Y., Marshall, D.S. 2021. Registration of ‘KS Silverado’ hard white winter wheat. Journal of Plant Registrations. https://doi.org/10.1002/plr2.20106.
Zhao, L., Liu, S., Abdelsalam, N., Carvar, B., Bai, G. 2021. Characterization of wheat curl mite resistance gene Cmc4 in OK05312. Journal of Theoretical and Applied Genetics. 134:993–1005. https://doi.org/10.1007/s00122-020-03737-3.
Zhang, T., Hua, C., Li, L., Sun, Z., Yuam, M., Bai, G., Humphreys, G., Li, T. 2020. Integration of meta-QTL discovery with omics: Towards a molecular breeding platform for improving wheat resistance to Fusarium head blight. The Crop Journal. https://doi.org/10.1016/j.cj.2020.10.006.
Baenziger, S.P., Graybosch, R.L., Rose, D., Xu, L., Guttieri, M.J., Regassa, T., Klein, R., Kruger, G., Santra, D., Hergert, G., Wegulo, S., Jin, Y., Kolmer, J.A., Hein, G., Bradshaw, J., Chen, M., Bai, G., Bowden, R.L. 2020. Registration of ‘NE10589’ (Husker genetics brand ‘Ruth’) hard red winter wheat. Journal of Plant Registrations. https://doi.org/10.1002/plr2.20068.
Xu, X., Li, G., Bai, G., Bernardo, A., Carver, B.F., St Amand, P., Armstrong, S. 2020. Development of KASP markers for wheat greenbug resistance gene Gb5. Crop Science. 61(1):490–499. https://doi.org/10.1002/csc2.20339.
Xu, X., Li, G., Bai, G., Bernardo, A.E., Carver, B.F., St Amand, P.C., Bian, R. 2021. Characterization of an incomplete leaf rust resistance gene and development of KASP markers for the leaf rust resistance gene Lr47 in wheat. Phytopathology. https://doi.org/10.1094/PHYTO-07-20-0308-R.
Li, J., Zhao, L., Cheng, X., Bai, G., Li, M., Wu, J., Yang, Q., Chen, X., Yang, Z., Zhao, J. 2020. Molecular cytogenetic characterization of a novel wheat–Psathyrostachys huashanica Keng T3DS-5NsL•5NsS and T5DL-3DS•3DL dual translocation line with powdery mildew resistance. Biomed Central (BMC) Plant Biology. 20:163. https://doi.org/10.1186/s12870-020-02366-8.
Guo, J., Pradhan, S., Rshahi, D., Khan, J., Mcbreen, J., Bai, G., Murphy, P., Babar, M. 2020. Increased prediction accuracy using combined genomic information and physiological traits in a soft wheat panel evaluated in multi-environments. Scientific Reports. 10. Article 7023. https://doi.org/10.1038/s41598-020-63919-3.
Gong, X., He, X., Zhang, Y., Li, L., Xun, Z., Bai, G., Singh, P.K., Li, T. 2020. Development of an evaluation system for Fusarium resistance in wheat grains and its application in assessment of the corresponding effects of Fhb1. Plant Disease. https://doi.org/10.1094/PDIS-12-19-2584-RE.
Zhang, P., Guo, C., Liu, Z., Bernardo, A.E., Ma, H., Jiang, P., Song, G., Bai, G. 2020. Quantitative trait loci for Fusarium head blight resistance in wheat cultivars Yangmai 158 and Zhengmai 9023. The Crop Journal. https://doi.org/10.1016/j.cj.2020.05.007.
Liu, G., Liu, X., Xu, Y., Bernardo, A.E., Chen, M., Li, Y., Fuan, N., Zhao, L., Bai, G. 2020. Reassigning Hessian fly resistance genes H7 and H8 to chromosomes 6A and 2B of the wheat cultivar ‘Seneca’ using genotyping-by-sequencing. Crop Science. 60(3):1488-1498. https://doi.org/10.1002/csc2.20148.
Pang, Y., Liu, C., Wang, D., St Amand, P.C., Bernardo, A.E., Li, W., He, F., Li, L., Wang, L., Yuan, X., Bai, G., et al. 2020. High resolution genome-wide association study identified genomic regions and candidate genes for important agronomic traits in wheat. Molecular Plant. https://doi.org/10.1016/j.molp.2020.07.008.
Brucker, P., Berg, J., Lamb, P., Kephart, K., Eberly, J., Miller, J., Chen, C., Torrion, J., Pradhan, G., Ramsfield, R., Nash, D., Holen, D., Cook, J., Gale, S.W., Jin, Y., Kolmer, J.A., Chen, X., Bai, G. 2020. Registration of ‘Bobcat’ hard red winter wheat. Journal of Plant Registrations. 2020:1-6. https://doi.org/10.1002/plr2.20057.
Jordan, K., He, F., Fernandez De Soto, M., Akhunova, A., Akhunov, E. 2020. Differential chromatin accessibility landscape reveals structural and functional features of the allopolyploid wheat chromosomes. Genome Biology. 21. Article 176. https://doi.org/10.1186/s13059-020-02093-1.
Zhu, L., Yuan, J., O'Neal, J., Brown, D., Chen, M. 2020. Analyzing molecular basis of heat-induced loss-of-wheat resistance to hessian fly (Diptera: Cecidomyiidae) Infestation using RNA-sequencing. Journal of Economic Entomology. 113(3):1504–1512. https://doi.org/10.1093/jee/toaa058.
Andreas, P., Kisiala, A., Emery, R.N., De Clerck-Floate, R., Tooker, J.F., Price, P.W., Miller, D.G., Chen, M., Connor, E.F. 2020. Cytokinins are abundant and widespread among insect species. Plants. 9(2):208. https://doi.org/10.3390/plants9020208.
Jordan, K., Bradbury, P., Miller, Z., Nyine, M., He, F., Guttieri, M.J., Brown Guedira, G.L., Buckler Iv, E.S., Jannink, J., Akhunov, E., Ward, B.P., Bai, G., Bowden, R.L., Fiedler, J.D., Faris, J.D. 2021. Development of the Wheat Practical Haplotype Graph Database as a Resource for Genotyping Data Storage and Genotype Imputation. G3 Genes/Genomes/Genetics. https://doi.org/10.1101/2021.06.10.447944.
Tian, W., Wilson, T.L., Chen, G., Guttieri, M.J., Nelson, N., Fritz, A., Smith, G., Li, Y. 2021. Effects of environment, nitrogen, and sulfur on total phenolic content and phenolic acid composition of winter wheat grain. Journal of Agricultural and Food Chemistry. https://doi.org/10.1002/cche.10432.