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

Location: Hard Winter Wheat Genetics Research

2023 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 pathogens: stripe rust (YR), leaf rust (LR), stem rust (SR), Fusarium head blight (FHB), virus, and Hessian fly (HF), as well as abiotic tolerance (heat and drought) and nutritional quality. In the five years of this project, ARS researchers at Manhattan, Kansas, annually screened about 6,000 breeding lines for field resistance to YR in Rossville, Kansas. Trials included entries from regional nurseries, mapping populations, elite breeding lines from 14 public and private breeding programs and ARS germplasm in development. Also annually, about 1,300 public and private breeding lines were screened for SR resistance in Manhattan, Kansas, 2,000 ARS breeding lines for LR resistance near Castroville, Texas and 1,300 lines in Kansas. Over 5,000 lines were screened for HF resistance. Over 1,200 ARS lines evaluated for wheat streak mosaic virus resistance near Dighton, Kansas. A new broad-spectrum SR resistance gene was discovered and designated as Sr64. It was associated with the Wsm1 wheat streak mosaic virus resistance gene containing a chromosome introgression from a wheatgrass species on chromosome 4D. We demonstrated that this introgression did not have negative effects on yield or quality. Quantitative trait loci (QTL) associated with YR field resistance were identified in the hard winter wheats ‘Overley’ and ‘Overland.’ An FHB resistance QTL (QFhb.hwwg-2DS) was identified on chromosome arm 2DS, and one major FHB resistance QTL was identified on chromosome 2DL of a Chinese cultivar, Ji5265. Native FHB resistance QTL were identified in the cultivars ‘Lyman’ and ‘Everest.’ Objective 2: Incorporate genetic traits into high-yielding winter wheat germplasm and distribute to the breeding community. Work continued toward stacking slow rusting resistance genes from cultivars ‘Kingbird’ and ‘Roelfs F2007’. Lines with homozygous markers for the combination of resistance genes Sr2/Yr30, Sr12, Lr34/Yr18/Sr57, Lr46/Yr29/Sr58, Lr68, Lr77 and Lr78 are ready for yield testing and disease resistance evaluation. Sr2 is the most important slow-rusting SR resistance gene but often is associated with an undesirable trait, pseudo-black chaff (PBC). We developed four mapping populations to identify modifiers of PBC. Breeding lines with the combination of YR resistance genes Yr5 and Yr15 had excellent YR resistance and yield performance in field trials. Lines were tested in statewide yield trials in Oklahoma, Kansas, and Nebraska in cooperation with public breeding programs. Breeding lines with YR resistance genes Yr40, Yr47, Yr51, Yr57, Yr63 and LR resistance genes Lr42 and Lr19 were evaluated in yield testing and distributed for regional breeder evaluation. Pacific Northwest wheat cultivar ‘SY Touchstone’ has high resistance to YR. A new mapping population was developed to analyze this resistance, and one year of field phenotyping data was collected. A new multi-parent mapping population of 798 recombinant inbred lines combining the YR-resistant cultivars ‘Joe’ and ‘Gallagher’ with three susceptible parents was created, and one year of field YR phenotyping was obtained. Recombinant hard winter wheat lines with Sr2 in coupling with Fhb1, FHB resistance gene, were developed, evaluated in field FHB and YR nurseries, and seed distributed for regional breeding program evaluation. The Sr2-Fhb1 cassette lines were used as a donor into the slow-rusting pyramid and crossed into two adapted hard winter wheat backgrounds. Wheat germplasm with pyramids of three major Ug99 race complex effective SR genes, Sr22, Sr26 and Sr35, was developed in three commercial wheat backgrounds and is in advanced yield testing in Oklahoma, Nebraska, and Kansas. The pyramid was expanded by backcrossing resistance genes Sr50 and SrTA10187 into Sr22+Sr26+Sr35 lines. Research continued to develop gene pyramids for FHB resistance. Fhb1, two resistance loci on chromosome 5A, and the Ji5625 QTL on chromosome 2DL were combined in the ‘Everest’ and Overland backgrounds. Resistant lines with 2-3 QTLs (Fhb1/Fhb5/2DL QTL) were sent to regional hard winter wheat breeding programs. We worked to improve the utility of the new Fhb7 FHB resistance gene, transferred to wheat as a wheatgrass species alien translocation. Unfortunately, Fhb7 is associated with an unacceptable high yellow flour pigment gene and the linkage is very difficult to uncouple. Two white flour, FHB-resistant lines were isolated from a mutant population of Fhb7 obtained from an ARS scientist in Fargo, North Dakota. A new project was initiated to transfer Fhb1+Fhb7+2DL QTL to 15 hard winter wheat lines from 7 breeding programs. We evaluated winter wheat germplasm developed from crosses with winter synthetic hexaploid wheats with resistance to wheat streak mosaic virus. Field evaluation under severe virus infection demonstrated limited resistance in these lines. The complex of viruses present in the western Kansas field trials was characterized using sequencing and identified wheat streak mosaic virus, Triticum mosaic virus and a wide array of other viruses. High-temperature HF- resistant breeding lines derived from crosses to durum landraces were evaluated in yield trials and distributed for evaluation to regional breeding programs. High-temperature resistance was confirmed to both the Great Plains biotype and a new Texas HF biotype identified in 2022. This resistance presently is being mapped. We developed a unique germplasm resource, 1,700 chromosome introgression hard winter wheat lines from 27 crosses with diverse wild emmer (WE, Triticum dicoccoides) selections. Wild emmer is the ancestral species for two of the genomes of modern bread wheat. This germplasm is being evaluated to identify new genetic sources of resistance to Ug99 stem rust, YR, wheat stem sawfly (WSSF), drought and heat stress, and to identify sources of improved grain protein concentration and quality. In cooperation with Colorado State University (CSU), three cycles of field selection under severe WSSF pressure were applied to breeding germplasm from WE introgressions. Lines with very limited stem cutting and acceptable agronomic characteristics were harvested from Akron, Colorado in 2023. Objective 3: Develop efficient marker technologies for genetic traits. New trait-specific markers were developed for 1AL.1RS, 1BL.1RS, Rht8, Bdv2, Cmc4, FHB-2DL QTL, Fhb1, Fhb7, Gb5, H13, H34, H35, Lr21, Lr42, Lr68, PHS1, Sr2, Sr35, Yr5, Yr15, Yr17 and Yr34. Annually, 1500-2200 samples were tested for trait-specific markers generating 200,000-300,000 datapoints and 2000-4000 samples for genome-wide markers, generating 20 to 50 million datapoints. Samples were regional breeder’s experimental lines, including cooperative nurseries. A genotyping-by-multiplexed-sequencing assay was developed containing 120 trait-specific markers. Current bioinformatic pipelines do not provide reliable genotypic calls. These 120 markers were added to three commercial multiplexed marker platforms. Work continued to optimize the multiplexed restriction amplicon sequencing (MRAseq) protocol. New primers were developed for library construction. Genomic selection was tested for 1,500 breeding lines from the Kansas State University - Hays program. The MRASeq markers produced the same selection efficiency as genotyping by sequencing (GBS) markers. MRASeq thus can be used for genomic selection without patent limitation and at lower cost than GBS. We advanced the exome capture method as a tool for genome-wide genotyping. This method collects sequence information for coding regions of the genome. Exome capture sequence data was collected for over 1,500 wheat lines at varying sequence depths and identified > 1 million variants across the wheat genome. The key advantage of the method is that different numbers of lines can be combined per sequencing run to produce deep sequencing of critical lines and shallow sequencing for early-generation lines that is cost-effective and repeatable. A Hard Winter Wheat Practical Haplotype Graph was developed from deep-sequencing data. This tool is now used to impute high density sequence variants from lower density data. Objective 4: Characterize molecular foundations of critical plant-microbe and plant-insect interactions. Work continued to sequence genes for putative effector proteins from HF, barley midge and oat midge. Candidate effector genes encoding secreted proteins and specifically expressed in salivary glands were identified. Transgenic wheat lines with HF effectors were generated, and wheat proteins that may interact with HF effectors were identified by immunoprecipitation. Work continued to backcross six LR-resistant mutant lines into adapted winter wheat germplasm. Three derivative lines demonstrated reduced LR symptoms in a field trial in Castroville, Texas. The causal resistance mutation in two mutant lines was mapped and a candidate gene was identified for one line. Transgenic and RNAi lines are being developed. Mapping populations with four other mutant lines were advanced to F6. A telomere-to-telomere genome was assembled of the LR pathogen Puccinia triticina race BBBD . The new genome assembly has the expected two nuclei of 125 Mbp and 18 chromosomes each. This reference genome was used in genome-wide association analysis of a random mating P. triticina population in which six effector genes were mapped. Six genomes of P. triticina races were sequenced and assembled. These races represent new lineages found in the North American LR field populations.

Accomplishments
1. Skim exome capture genotyping method developed. Plant breeders increasingly use DNA sequence information to improve the efficiency of their breeding efforts. The very large (16Gb) and complex (over 85% repetitive material) genome of wheat limits the value of tools commonly used in less complex staple crops like rice and maize. Therefore, ARS scientists at Manhattan, Kansas, developed a new method called skim exome capture that combines the strengths of existing sequencing and genotyping technologies by enabling targeted sequencing of the genic regions of wheat. This results in a highly informative snapshot of the wheat genome at a reduced cost per sample compared to traditional re-sequencing and capture approaches. This new approach is repeatable and can be scaled to the desired price point to generate meaningful genotypic data for use in downstream breeding applications, such as genome-wide association studies or genomic selection, to efficiently produce improved wheat varieties.

2. New broad-spectrum stem rust resistance gene discovered. Stem rust has re-emerged as one of the most important diseases of wheat. The pathogen continues to evolve new, aggressive races that threaten wheat global production, and wheat breeders urgently need new genes for development of resistant cultivars for farmers. ARS researchers in Manhattan, Kansas, identified a new broad-spectrum resistance gene that confers resistance to all known races of stem rust fungus from wheat, including Ug99. The stem rust resistance gene was given the designation Sr64. This stem rust resistance gene is tightly linked to the resistance gene Wsm1, which is effective against Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV). The two genes are on the same translocation from a wheat wild relative, and therefore they will be inherited together in breeding. New markers were developed so that breeders can easily select for the combined resistance to WSMV, TriMV, and stem rust.

3. Discovery of role of brassinosteroid signaling on wheat height reduction. Modern wheat cultivars usually carry either Rht-B1b or Rht-D1b semi-dwarfing genes to reduce plant height and thus reduce lodging. However, the two genes also reduce nitrogen-use efficiency and produce smaller grains. ARS researchers in Manhattan Kansas, collaborated with scientists from thein China Agricultural University to discover a new mechanism to reduce wheat height using a natural mutation that deleted the genes Rht-B1b and ZnF-B. Mutant plants were shorter and had a more compact plant architecture with higher grain yield in field trials. Deletion of ZnF-B induced the semi-dwarf trait by attenuating brassinosteroid perception, and deletion of Rht-B1 improved yield. The findings provide a creative strategy to design high-yield semi-dwarf wheat varieties by manipulating the brassinosteriod signaling pathway to increase wheat production.

4. A complete genome of the wheat leaf rust pathogen, Puccinia triticina, has been assembled. Leaf rust is the most common worldwide wheat pathogen of wheat, causing substantial yield loss for farmers who plant susceptible cultivars. Plant breeders have used genetic resistance to combat leaf rust. However, resistance often is short-lived as the pathogen adapts and produces new, virulent strains. How these new strains develop is unclear. Understanding the genome of the leaf rust pathogen will help scientists to identify proteins involved in infection. ARS researchers at Manhattan, Kansas, through an international collaboration with scientists at Agriculture and Agri-Food Canada, a completed the assembly of the leaf rust genome was accomplished. P. triticina consists of two nuclei, each with 18 chromosomes made of 125 million base pairs of DNA. This new leaf rust genome will be a useful tool for scientists working to understand how the leaf rust pathogen interacts with the wheat plant and may lead to improved strategies for breeding resistant cultivars for farmers.

5. New highly virulent strains of wheat stem rust may result from recombination of ancestral lineages. Stem rust is among the most significant diseases that threaten global wheat production. For decades, plant breeders have successfully used disease resistance genes to combat the fungus. However, new virulent strains recently have emerged in eastern Africa and southern Europe that overcome most of the resistance genes currently used in wheat cultivars around the world. The origin of these new strains has been unclear. Researchers from ARS in Manhattan, Kansas, and St. Paul, Minnesota, and scientists at Kansas State University, identified 12 major clades or subclades of the stem rust fungus. Members of 7 of the 12 groups appear to have arisen by sexual or asexual recombination between distinct ancestral populations. These population admixture events appear to play a role in the origin of new highly virulent races. This new understanding how these virulent races evolved may lead to better strategies for breeding to ensure durable resistance in cultivars for farmers.

Review Publications
Garst, N., Belamkar, V., Easterly, A., Stoll, H., Ibrahim, A., Guttieri, M.J., Baenziger, P. 2023. Evaluation of pollination traits important for hybrid wheat development in Great Plains germplasm. Crop Science. https://doi.org/10.1002/csc2.20926.
Guo, Y., Betzen, B., Salcedo, A., He, F., Bowden, R.L., Fellers, J.P., Jordan, K., Akhunova, A., Rouse, M.N., Szabo, L., Akhunov, E. 2022. Population genomics of Puccinia graminis f.sp. tritici highlights the role of admixture in the origin of virulent wheat rust races. Nature Communications. https://doi.org/10.1038/s41467-022-34050-w.
Guttieri, M.J., Bowden, R.L., Zhang, G., Haley, S., Frels, K., Hein, G., Jordan, K. 2022. Agronomic and quality impact of a shortened translocation for Wheat Streak Mosaic Virus resistance. Crop Science. https://doi.org/10.1002/csc2.20876.
Ruolin, B., Liu, N., Xu, Y., Su, Z., Cai, L., Bernardo, A.E., St Amand, P.C., Fritz, A., Zhang, G., Rupp, J., Akhunov, E., Jordan, K., Bai, G. 2023. Quantitative trait loci for rolled leaf trait in a wheat EMS mutant from Jagger. Journal of Theoretical and Applied Genetics. https://doi.org/10.1007/s00122-023-04284-3.
Mustahsan, W., Guttieri, M.J., Bowden, R.L., Garland Campbell, K.A., Jordan, K., Bai, G., Zhang, G. 2023. Mapping the quantitative field resistance to stripe rust in a hard winter wheat population ‘Overley’ × ‘Overland’. Crop Science. https://doi.org/10.1002/csc2.20977.
Xu, X., Mornhinweg, D.W., Bai, G., Li, G., Bian, R., Bernardo, A.E., Armstrong, J.S. 2022. Characterization of Rsg3, a novel greenbug resistance gene from the Chinese barley landrace PI 565657. The Plant Genome. 16(1). Article e20287. https://doi.org/10.1002/tpg2.20287.
Xu, X., Li, G., Bai, G., Carver, B.F., Bian, R., Bernardo, A.E., Armstrong, J.S. 2023. Genomic location of Gb1, a unique gene conferring wheat resistance to greenbug biotype F. The Crop Journal. https://doi.org/10.1016/j.cj.2023.02.002.
Chen, H., Su, Z., Tian, B., Hao, G., Trick, H., Bai, G. 2022. TaHRC suppresses the calcium-mediated immune response and triggers wheat Fusarium head blight susceptibility. Plant Physiology. 190(3):1566-1569. https://doi.org/10.1093/plphys/kiac352.
Zhao, L., Su, P., Hou, B., Wu, H., Fan, Y., Li, W., Zhao, J., Ge, W., Xu, S., Wu, S., Ma, X., Li, A., Bai, G., Wang, H., Kong, L. 2022. The black necrotic lesion enhanced Fusarium graminearum resistance in wheat. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2022.926621.
Li, X., Li, D., Xuan, Y., He, Z., Zhao, L., Hao, Y., Ge, W., Xu, S., Hou, B., Wang, B., Guo, J., Liu, W., Li, M., Han, Y., Bo, C., Bao, Y., Qi, Z., Xu, S.S., Bai, G., Wang, H., Kong, L. 2023. Elimination of the yellow pigment gene PSY-E2 tightly linked to the Fusarium head blight resistance gene Fhb7 from Thinopyrum ponticum. The Crop Journal. 11(3):957-962. https://doi.org/10.1016/j.cj.2022.12.005.
Tene, M., Adhikari, E., Cobo, N., Jordan, K.W., Matny, O., del Blanco, I.A., Roter, J., Ezrati, S., Govta, L., Manisterski, J., Yehuda, P.B., Chen, X., Steffenson, B., Akhunov, E., Sela, H. 2022. GWAS for stripe rust resistance in wild emmer wheat (Triticum dicoccoides) population: Obstacles and solutions. Crops. 2(1):42-61. https://doi.org/10.3390/crops2010005.
He, F., Wang, W., Rutter, W.B., Jordan, K., Ren, J., Akhunova, A., Szabo, L.J., Rouse, M.N., Akhunov, E. 2022. Genomic variants affecting homoeologous gene expression dosage contribute to agronomic trait variation in allopolyploid wheat. Nature Genetics. 13(1):1-15. https://doi.org/10.1038/s41467-022-28453-y.