Research Project: Improvement of Biotic Stress Resistance in Durum and Hard Red Spring Wheat Using Genetics and Genomics

Location: Cereal Crops Research

2020 Annual Report

Objectives
OBJECTIVE 1: Identify novel sources of disease and pest resistance in durum wheat and goatgrass to enhance crop resilience. OBJECTIVE 2: Map and characterize novel genes governing resistance/susceptibility to tan spot, Septoria nodorum blotch, stem rust, and Hessian fly in wheat and goatgrass to develop the knowledge and tools for their deployment in the development of wheat varieties with improved resistance. OBJECTIVE 3: Characterize genetic mechanisms associated with wheat-pathogen interactions to increase our understanding and knowledge of the biological mechanisms associated with resistance and susceptibility. OBJECTIVE 4: Utilize and develop genetic resources and molecular tools for the improvement of wheat and provide genotyping services to expedite the development of improved wheat, barley and oat varieties. OBJECTIVE 5: Genetically improve barley by the application of molecular genetics and genomics to increase resistance to head and foliar diseases such as Fusarium head blight, net blotch and spot blotch.

Approach
Durum, hard red spring wheat (HRSW) and barley varieties with improved resistance to diseases and pests are needed to meet the demands of the world’s growing population. This challenge must be met through the discovery, characterization, and deployment of genes for resistance to biotic stresses. In this project, we will identify new sources of resistance to Septoria nodorum blotch, tan spot, and stem rust in durum, to Hessian fly in goatgrass, and to Fusarium head blight and spot-form net blotch in barley. Molecular mapping and genetic analyses will be used to identify and characterize genes and quantitative trait loci governing resistance to tan spot, Septoria nodorum blotch, stem rust, Fusarium head blight and spot-form net blotch. This work will yield knowledge of the genetic mechanisms controlling these traits, the development of markers for marker-assisted selection, and genetic stocks and germplasm useful for gene deployment. Additional work on the molecular characterization of the genes and genetic pathways associated with wheat/barley-pathogen interactions will be conducted as part of this project and will yield basic knowledge useful for devising novel strategies for developing disease and pest resistant varieties. Finally, genetic resources and tools for the development of improved wheat, durum and barley cultivars will be generated, including stocks for the genetic analyses of Septoria nodorum blotch susceptibility genes and Hessian fly resistance genes, adapted germplasm with low cadmium and resistance to sawfly, Fusarium head blight, and stem rust, and a reference sequence-based genetic map for durum wheat. In addition, genotyping services will be provided to regional wheat, durum, barley, and oat breeders to expedite the development of improved varieties.

Progress Report
To determine the prevalence of necrotrophic effector (NE) sensitivity genes in durum wheat, we have screened the Durum Wheat Reference Collection (DWRC) with SnTox1, SnTox2, SnTox3, and SnToxA in two replications and with SnTox5 in one replication, and we have begun to analyze the data. This work will provide information on the prevalence of five NE sensitivity genes in durum wheat. Furthermore, analysis of this data together with inoculation data will provide knowledge regarding the importance of the four NE sensitivity genes in conferring disease susceptibility in durum. This work is aligned with Subobjective 1C. Toward the identification and characterization of the necrotrophic effector sensitivity gene Snn5 in wheat, the appropriate high-resolution mapping populations have been constructed, and they have been genotyped and phenotyped. Molecular markers have been developed that define a genomic region containing few candidate genes, one of which has homology to a known necrotrophic effector sensitivity gene. The candidate genes are being analyzed in ethyl methane sulfonate (EMS)-induced knockout mutants and characterized for expression. This work is aligned with Subobjective 2C. To functionally characterize the barley ortholog of wheat FHB1, namely HvHRC, in susceptibility to Fusarium head blight, we have designed vectors of clustered regularly interspaced short palindromic repeats (CRISPR) for gene knockout and constructs for complementation test. A platform for Agrobacterium-mediated barley transformation has been established. Immature embryos have been isolated as target tissue for transformation. At least 300 embryos were inoculated with the Agrobacterium strain AGL1 containing the designed vectors. The induced calli are being selected with appropriate antibiotics. This work pertains to Subobjective 5A. Using the Pyrenophora teres f. maculata (Ptm) isolate 13IM8.3 and recombinant inbred line (RIL) population derived from CI9214 x CI 9776, we conducted three replicates of pathogen inoculation to identify resistance or susceptibility genes against net form net blotch in barley. A total of 146 lines were genotyped with the Barley 50k iSelect single nucleotide polymorphism (SNP) array. Four loci with significant effect were detected through quantitative trait locus (QTL) analysis. Molecular markers are being developed to assist breeding selection and to confirm genetic locations for the identified QTL. This work pertains to Subobjective 5B. Towards genotyping and phenotyping of a Global Durum Wheat Panel (GDP), which was previously named Durum Wheat Reference Collection (DWRC), we have imported, quarantined, and increased the 960 accessions. These accessions have been genotyped and analyzed for genetic diversity, population structure and genetic relationships. A subset of 513 lines consisting of modern durum cultivars and landraces have been evaluated for resistance to two isolates (Sn4 and Sn2000) of fungal pathogen causing Septoria nodorum blotch. The data are currently being used for genome-wide association analysis, which will lead to the identification of novel genes for Septoria nodorum blotch resistance and/or susceptibility. Towards developing near-isogenic lines (NILs) with different Hessian fly resistance genes (Subobjective 4B), the synthetic wheat lines SW8 (PI 639730) and W7984, which carry H26/H26B and H32, respectively, have been backcrossed to Newton through five backcrosses. New molecular markers for each of the genes have been developed and will be used for marker-assisted selections of the NILs carrying the three genes, respectively, in the subsequent generations. To continue providing SNP genotyping options for breeders and geneticists, we have collected representative samples from our barley and wheat breeding community to test a new multi-species assay that evaluates specific genomic positions that allow prediction of the surrounding haplotype. If useful in the North Central region, it will provide a lower-cost option to facilitate genomic selection in breeding programs. We also have just begun the generation of a novel graph-based representation of lines to facilitate high-accuracy prediction of genome-side SNPs from data already acquired. This will allow us to improve genotyping resolution of previously assayed lines. Finally, we have also begun development of a targeted long-read genotyping platform to aid in fine-mapping QTL to discover causative genes. This will also be utilized to genotype complex QTL where SNPs cannot be assayed.

Accomplishments
1. A wheat gene with broad resistance to a fungal pathogen. Tan spot is a serious fungal disease of wheat. Interactions between wheat plants and the tan spot fungus are usually specific and rely on specific effector proteins in the fungus that, when recognized by specific genes in the plant, cause cell death and disease. ARS researchers in Fargo, North Dakota, conducted genetic analyses of an ancestor of wheat, known as wild emmer, and evaluated the plant response to multiple strains of the tan spot fungus that are known to produce a variety of effector proteins. The researchers identified an accession of wild emmer wheat that was resistant to all strains of the tan spot pathogen. Detailed molecular genetic analyses indicated that the wild emmer plant carried a single gene that conferred the broad-spectrum resistance to all strains of the fungus. Furthermore, the researchers transferred the gene to elite wheat lines to render them resistant to tan spot as well. The identification and deployment of the tan spot resistance gene by wheat and durum breeders will lead to higher yielding wheat varieties that require less fungicide applications.

2. Functional characterization of a major wheat domestication gene. Wheat domestication occurred about 10,000 years ago in the Fertile Crescent of the Middle East and involved specific genetic mutations that made wheat more amenable to harvesting and processing by early farmers. One of these mutations occurred in the wheat Q gene and rendered the seed free-threshing, essentially allowing wheat to be harvested on a massive scale and leading to the rise of modern civilization. Genetic characterization of the Q gene is important to provide understanding of the genetic networks and processes controlled by the Q gene that may be refined to optimize wheat production. ARS researchers in Fargo, North Dakota, conducted a wide range of genetic, physiologic, and molecular experiments to identify genetic pathways and processes controlled by the Q gene. The results revealed that Q is a master regulator controlling numerous traits including plant architecture, cell wall thickness, photosynthesis, pollen fertility, and ultimately, seed production and yield. These discoveries provide insights into the significance of the genetic mutation that gave rise to the Q gene, and they provide information necessary for the development of more productive and resilient wheat varieties by wheat breeders that will have increased tolerance to harsh environments and reduced yield losses under a changing global climate.

3. Characterization of a gene family involved in regulation of tobacco lateral branching. The lateral shoot resulting from outgrowth of axillary buds in tobacco, commonly called suckers, seriously compromises leaf yield and quality. Genetic control of sucker growth in tobacco is an environmentally sustainable means to eliminate the secondary branches. Identification and functional characterization of genes involved in regulation of tobacco lateral branching may provide perspectives for genetic manipulation of shoot architecture. ARS researchers in Fargo, North Dakota, addressed the function of an important gene family (BRC1) in outgrowth of axillary buds. Using clustered regularly interspaced short palindromic repeats (CRISPR), gene transformation and RNA interference (RNAi) technologies, the researchers tested five BRC1 genes in tobacco. The results showed that the BRC1 genes have opposing regulatory roles in lateral branching. Some family members are suppressors, but others are activators for outgrowth of axillary buds. This study provides clues to understanding how lateral growth could be manipulated in crop plants.

4. Genetic mapping of a gene controlling pod coiling direction in Medicago truncatula. Handedness in plants introduced by helical growth of organs is frequently observed, and it has fascinated plant scientists for decades. However, the genetic mechanism underlying plant handedness is unknown. Pod coiling is commonly found in the legume genus Medicago, including the model plant M. truncatula. The coiling directions vary within and among the species of Medicago, and coiling can be either clockwise or counterclockwise, providing a model for genetic study of handedness in plants. ARS researchers in Fargo, North Dakota, determined the genomic position of the SPC gene controlling pod coiling direction in Medicago truncatula. The SPC gene was delimited to a 250 kb-region on chromosome 7, and two promising candidate genes were selected for functional validation. The result of this fundamental research provides further insights for plant biologists into the mechanisms underlying plant development and architecture, which may influence how plants respond to different environments.

5. Genetic diversity of Global Durum Wheat Panel (GDP). A collection of unique lines that represent global genetic diversity is an excellent resource for genetic studies and breeding for crop improvement. However, a global core collection of durum wheat has not previously been established. ARS researchers in Fargo, North Dakota, participated in an international initiative in assembling and characterizing a global durum wheat panel (GDP) of 1,011 lines obtained from various gene banks and breeding programs globally. The GDP consists of a wide representation of modern and historic durum wheat cultivars along with a selection of wild relatives and durum wheat progenitors to maximize diversity. The GDP accessions have been genotyped and analyzed for genetic diversity, population structure and genetic relationships. The GDP accessions and their genetic and genomic data from this study will facilitate international collaboration for the identification and utilization of beneficial genes to improve the productivity and quality of durum wheat.

6. Cloning and introgression of a gene for resistance to Fusarium head blight. Fusarium head blight (FHB), a disease caused by fungal pathogens that produce food toxins, currently devastates wheat production worldwide, yet few resistance resources have been discovered in wheat germplasm. ARS researchers in Fargo, North Dakota, and Manhattan, Kansas, participated in an international team to clone the Fhb7 functional gene for FHB resistance based on assembling the genome of a wild wheatgrass species. The results revealed that Fhb7 confers broad resistance to the fungal pathogens causing FHB via detoxification of food toxins. The wheatgrass gained Fhb7 via gene transfer from a fungal species that colonizes temperate grasses. When transferred into wheat, Fhb7 confers FHB resistance in diverse wheat backgrounds without yield penalty, providing a solution for FHB resistance breeding in wheat. Moreover, the assembled wheatgrass genome and molecular and biochemical characterization of the cloned Fhb7 provide new knowledge and tools to assist scientists across a wide range of communities, including those working on plant-pathogen interactions and genetic modification of plants. The wheat lines carrying Fhb7 are useful germplasm for developing new FHB-resistant varieties for wheat growers.

7. Identification of genes controlling fungal disease resistance in small grains. Wheat, durum, and barley are susceptible to many fungal pathogens that cause foliar disease, which decreases yield and end-use quality. Economic losses due to these diseases can be most easily prevented by cultivating lines that natively resist the pathogens. In three studies published this year, ARS researchers in Fargo, North Dakota, investigated the genetic determinants of fungal disease (tan spot, powdery mildew, spot blotch) important in our growing region by purposefully inoculating test populations with the pathogen and using advanced molecular analysis to identify specific gene regions that influence disease. Another study formally summarized all the published data of one disease (tan spot) in wheat and used statistical analysis to identify the genes durably found to influence disease year after year and hone in on their genomic location. These studies have identified several new genes, validated previously discovered ones, and have established precise molecular tools to rapidly deploy these genes in breeding programs. Using molecular breeding techniques, elite varieties that contain these identified genes can now be generated that natively combat foliar disease caused by these pathogens. These varieties will not require timely fungicide application, which decreases the risk of yield loss and increases profits to the producer.

Review Publications
Ding, N., Qin, Q., Wu, X., Miller, R., Zaitlin, D., Li, D., Yang, S. 2020. Antagonistic regulation of auxiliary bud outgrowth by the BRANCHED genes in tobacco. Plant Molecular Biology. https://doi.org/10.1007/s11103-020-00983-3.
Faris, J.D., Overlander, M., Kariyawasam, G.K., Carter, A., Xu, S.S., Liu, Z. 2019. Identification of a major dominant gene for race-nonspecific tan spot resistance in wild emmer wheat. Theoretical and Applied Genetics. 133:829-841. https://doi.org/10.1007/s00122-019-03509-8.
Adeleke, E., Millas, R., McNeal, W., Faris, J.D., Taheri, A. 2020. Variation analysis of root system development in wheat seedlings using a root phenotyping system. Agronomy. 10(2):206. https://doi.org/10.3390/agronomy10020206.
Zhang, Z., Li, A., Song, G., Geng, S., Gill, B.S., Faris, J.D., Mao, L. 2019. Comprehensive analysis of Q gene near isogenic lines reveals key molecular pathways for wheat domestication and improvement. Plant Journal. 102:299-310. https://doi.org/10.1111/tpj.14624.
Liu, Y., Salsman, E., Fiedler, J.D., Hegstad, J.B., Liu, Z., Faris, J.D., Xu, S.S., Li, X. 2019. QTL mapping of resistance to tan spot induced by race 2 of Pyrenophora tritici-repentis in tetraploid wheat. Theoretical and Applied Genetics. 133:433-442. https://doi.org/10.1007/s00122-019-03474-2.
Galagedara, N., Liu, Y., Fiedler, J.D., Shi, G., Chao, S., Xu, S.S., Faris, J.D., Li, X., Liu, Z. 2020. Genome-wide association mapping of tan spot resistance in a worldwide collection of durum wheat. Theoretical and Applied Genetics. 133:2227–2237. https://doi.org/10.1007/s00122-020-03593-1.
Liu, Y., Salsman, E., Wang, R., Galagedara, N., Zhang, Q., Fiedler, J.D., Liu, Z., Xu, S.S., Faris, J.D., Li, X. 2020. Meta-QTL analysis of tan spot resistance in wheat. Theoretical and Applied Genetics. 133:2363-2375. https://doi.org/10.1007/s00122-020-03604-1.
Leng, Y., Zhao, M., Fiedler, J.D., Dreiseitl, A., Chao, S., Li, X., Zhong, S. 2020. Molecular mapping of loci conferring susceptibility to spot blotch and resistance to powdery mildew in barley using the sequencing-based genotyping approach. Phytopathology. 110(2):440-446. https://doi.org/10.1094/PHYTO-08-19-0292-R.
Zhang, M., Zhang, W., Zhu, X., Sun, Q., Chao, S., Yan, C., Xu, S.S., Fiedler, J.D., Cai, X. 2020. Partitioning and physical mapping of wheat chromosome 3B and its homoeologue 3E in Thinopyrum elongatum by inducing homoeologous recombination. Theoretical and Applied Genetics. 133:1277-1289. https://doi.org/10.1007/s00122-020-03547-7.
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.
Yu, X., Qin, Q., Wu, X., Li, D., Yang, S. 2020. Genetic localization of the SPC gene controlling pod coiling direction in Medicago truncatula. Genes and Genomics. 42:735-742. https://doi.org/10.1007/s13258-020-00947-3.