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

Location: Cereal Crops Research

2023 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
This is the final report for the project 3060-21000-038-000D. Research will continue under a new research project once it is reviewed and approved. Throughout the life of the project, significant progress was made in multiple areas. The following summary of progress over the life of the project relates to the achievement of the objectives of the project. The septoria nodorum blotch (SNB) pathogen produces several proteins known as effectors. ARS researchers in Fargo, North Dakota, used several strains of the SNB fungus that varied in the number of effectors that they produced. With some strains, the amount of disease a strain could cause was a cumulative effect of all its effectors, but with others only one or two effectors were responsible for disease even though all were recognized by the plant. This study showed that nonrecognition of all fungal effector proteins by the plant is key to achieving resistance, and it provides knowledge that may lead to novel approaches to control disease through manipulation of fungal effector proteins. Sub-objectives 3A and 3B. Wheat domestication involved specific genetic mutations that made wheat more amenable to harvesting and process by early farmers. ARS researchers in Fargo, North Dakota, compared the genetic components of threshability in a primitive wheat known as ‘emmer’ and modern domesticated durum wheat and found that three genes were involved in governing the free-threshing trait, and all three had undergone mutations during the evolution of emmer to domesticated durum wheat. Researchers may use this information to better understand wheat domestication and acquisition of beneficial genes from wheat relatives for improvement of modern varieties. Objective 4. The wheat pathogen Fusarium graminearum, which causes the devastating wheat disease Fusarium head blight (FHB) and seedling rot, is one of the fungal pathogens known to produce KP4 killer toxin (KP4). ARS researchers in Fargo, North Dakota, found that the FHB fungus produces four KP4 proteins all involved in causing seedling rot. This work provides insights regarding the significance of KP4 proteins in causing seedling rot in wheat. Sub-objective 3D. Fusarium head blight (FHB), commonly known as wheat scab, is a devastating disease of durum wheat. Through genetic analysis and gene mapping, ARS researchers in Fargo, North Dakota, and their collaborators at North Dakota State University identified three FHB resistance genes in a durum line and confirmed the successful transfer of a major gene from bread wheat into durum wheat. The durum line carrying FHB resistance genes from bread wheat is useful germplasm for developing new durum varieties with FHB resistance. Objective 4. ARS researchers in Fargo, North Dakota, developed 200 new synthetic wheat lines. Most of the synthetic wheat lines were more resistant to FHB than their AB-genome wheat parents, indicating that the D genome may play a major role in reducing disease infection. Objective 1. Durum wheat is a widely grown cereal crop mainly for making pasta products. ARS researchers in Fargo, North Dakota, participated in an international effort to sequence the durum genome. The team generated a genome sequence of the modern durum wheat cultivar ‘Svevo’ and made an in-depth comparison between modern durum and its ancestor wild emmer. Sub-objectives 4D and 4E. Disease resistance (R) genes from wild relatives of wheat are a valuable resource for breeding crops. ARS researchers in Fargo, North Dakota, participated in an international team in developing a new method (AgRenSeq) for rapid discovery and cloning of R genes. This method is a major advance in discovering and isolating R genes for engineering resistance against a wide range of pathogen races in crops. Objective 3. Tan spot is a serious fungal disease of wheat. ARS researchers in Fargo, North Dakota, conducted genetic analyses of numerous populations and genetic stocks of wheat and related species and identified several genes governing either resistance or susceptibility to the disease. Several DNA markers were developed for each of the genes for selection and monitoring of desirable alleles in varieties and germplasm. Sub-objectives 1B, 2A, and 2B. The wheat Q gene is a major domestication gene. 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 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. Objective 4. ARS researchers in Fargo, North Dakota, participated in an international initiative to characterize a global durum wheat panel (GDP). 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 was 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. Objective 1. ARS researchers in Fargo, North Dakota, 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. When transferred into wheat, Fhb7 confers FHB resistance in diverse wheat backgrounds without yield penalty, providing a solution for FHB resistance breeding in wheat. Objective 3. Spot blotch and powdery mildew are two major fungal diseases of barley that attack the leaves and decrease yield and quality of the grain. ARS researchers in Fargo, North Dakota, identified genes that govern resistance to spot blotch and powdery mildew in barley. These results provide novel information about the disease mechanism of spot blotch and validate previous studies in powdery mildew research. Objective 5. Septoria nodorum blotch (SNB) is a severe fungal disease of wheat worldwide. ARS researchers in Fargo, North Dakota, isolated a new gene in wheat, Snn3-D1, that makes wheat susceptible to SNB. The researchers found that a portion of the Snn3-D1 gene involved in the recognition of the SNB pathogen in wheat plants has similarity to a component of a nematode gene involved in sperm movement. This is the first research to show common features between a nematode sperm-movement gene and a pathogen resistance gene in plants. This knowledge will allow researchers to devise appropriate strategies for the development of disease resistant crops. Objective 3. Septoria tritici blotch (STB) is a severe fungal disease of wheat worldwide. ARS researchers in Fargo, North Dakota, along with collaborators in France and The Netherlands, discovered a gene from wheat that provides resistance to STB. The gene, Stb16, belongs to a class of genes that have not previously been associated with disease resistance in plants. This gene is important because it makes wheat resistant to many strains of the STB fungus and very useful for plant breeders as they develop new STB resistant varieties. Objective 3. Stem rust is one of the most damaging diseases in durum and bread wheat and a threat to wheat production worldwide. ARS researchers in Fargo, North Dakota, identified several new forms of stem rust resistance genes in durum and wheat lines. These new alleles can be used by breeders to develop new wheat varieties with robust stem rust resistance. Sub-objectives 1A and 2D. Plant chloroplasts are miniature factories of photosynthesis and critical for crop biomass and grain yield. However, chloroplast development is a complicated process, and the underlying chloroplast formation pathways have not been fully revealed. ARS researchers in Fargo, North Dakota, genetically characterized a mutated strain of barley known as grandpa1 (gpa1) that is defective in chloroplast formation. This research provides knowledge for barley geneticists to potentially manipulate Gpa1 to improve photosynthesis for higher yields. Objective 5. Durum wheat is used to make pasta and other semolina-based products. ARS researchers in Fargo, North Dakota, conducted genetic analysis of yield-related traits in durum and a relative of durum known as emmer wheat. Greenhouse and field-based studies revealed the positions of genes within the genome of emmer wheat that improved the yields of durum wheat under multiple environments. Durum breeders will benefit from this study because it provides them with knowledge of emmer genes that can be used to improve durum wheat through conventional hybridization means. Objective 4. A disease known as spot form net blotch (SFNB) is one of the most destructive leaf diseases of barley in the U.S. and worldwide. ARS researchers in Fargo, North Dakota, conducted genetic analysis of barley resistance to SFNB. The researchers identified a total of ten genes that contributed different levels of SFNB resistance, with one of the genes contributing high levels of resistance. DNA markers associated with the genes were developed and will be useful to expedite the transfer of the resistance genes into new barley varieties. Therefore, the knowledge and tools developed in this research will help to mitigate losses in barley yields due to SFNB, which will lead to increased income for barley producers. Objective 5.

Accomplishments
1. Tan spot resistance genes in durum wheat. Tan spot is a devastating disease of durum wheat and causes significant losses in yield and revenue in most durum-growing regions of the world. ARS researchers in Fargo, North Dakota, conducted genetic analysis of a panel of durum varieties from all over the world to identify genes governing tan spot resistance. Six genes were identified among the durum lines that governed resistance to tan spot disease. In addition, the researchers identified molecular markers associated with the genes that can be used to track their inclusion in breeding programs. Durum breeders and geneticists will benefit from this research in that they will now be able to efficiently characterize durum lines for presence/absence of the genes and retain lines with desirable gene combinations for the development of new, high-performing durum varieties, which, as a result, will suffer less yield losses due to tan spot.

2. Genes for disease resistance in winter wheat. Septoria nodorum blotch and tan spot are two devastating foliar diseases of winter wheat globally and can cause significant yield losses that result in decreased profits for farmers. ARS researchers in Fargo, North Dakota, conducted genetic analysis of winter wheat lines from around the world to identify genes governing resistance to septoria nodorum blotch and tan spot. The researchers identified two genes for tan spot and eight genes for septoria nodorum blotch among the winter wheat lines that governed resistance. Additionally, the researchers developed DNA markers that can be used by breeders to track the genes in their wheat lines. Winter wheat breeders and geneticists will benefit from this research in that they have new resistance genes to select for in their programs, along with markers to use to increase their efficiency in developing new, high-performing winter wheat varieties that are resistant to septoria nodorum blotch and tan spot. Winter wheat producers will see better yields thanks to the development of new disease-resistant varieties.

3. A gene governing virulence of a wheat fungal pathogen. Some strains of the fungus that cause the disease known as spot blotch in wheat carry a gene known as ToxA that aids the fungus in causing disease on plants that carry a gene known as Tsn1. ARS researchers in Fargo, North Dakota, in collaboration with colleagues at North Dakota State University collected over 300 strains of the spot blotch fungus, and they evaluated the strains for the presence of the ToxA gene to determine its prevalence among the strains. The ToxA gene was present in over a third of the strains. Strains that harbored the ToxA gene produced more disease on wheat plants that had the Tsn1 gene than strains that lacked the ToxA gene, indicating that the ToxA gene increased virulence of the spot blotch pathogen. The results of this research show that wheat breeders should strive to eliminate the Tsn1 gene from their breeding lines to gain increased resistance to spot blotch disease.

4. Identification of genes for enhanced yield in durum wheat. Crop yields need to increase to feed the growing global population, including durum wheat, which is used to make pasta and other semolina-based products. ARS researchers in Fargo, North Dakota, conducted genetic analysis of yield-related traits in durum and a relative of durum known as emmer wheat, which may provide a source of genetic variation to improve durum yield. Field-based studies identified 25 genomic regions associated with yield in two populations that were present under multiple environments. Many of these genomic regions had the gene contributing to increased yield coming from cultivated emmer. Durum breeders will benefit from this study because it provides knowledge of both durum and cultivated emmer genes that can be used to improve durum wheat. Higher yielding durum varieties will result in increased profits for producers and more food for pasta and semolina consumers.

5. Crown rust resistance in oats. Crown rust (CR) is an important disease of oat that has the potential to cause significant yield losses worldwide. ARS researchers in Fargo, North Dakota, in collaboration with ARS colleagues in St. Paul, Minnesota, conduced genetic analysis of a large multi-parent population that was evaluated for CR at the adult growth stage. The researchers identified six genes that provide resistance and concluded that they would provide durable protection if used together. The researchers developed high-throughput molecular markers to track these genes and have released the source germplasm to the public. With this knowledge and the new tools in hand, improved varieties can be rapidly developed to combat this destructive disease with a robust resistance mechanism.

6. Leaf rust resistance in cultivated emmer wheat. Leaf rust is a serious threat to global wheat production with the ability to cause wide-spread epidemics. To combat this disease efficiently, new genetic sources of resistance need to be identified and evaluated. ARS researchers in Fargo, North Dakota, and Albany, California, in collaboration with colleagues at North Dakota State University evaluated a diverse global collection of emmer wheat, which was commonly cultivated before durum wheat was domesticated. Upon genetic analysis of this population after screening for leaf rust reactions, the researchers identified many resistant lines and over 90 different gene regions, highlighting the importance of undomesticated germplasm pools as sources of new traits. Several of the identified gene regions have already been deployed in commercial wheat varieties, but the majority have not been previously identified and provide a rich deposit for future characterization.

7. Genetic control of morphological, growth, and disease traits in diverse oat populations. Grown as a source of food, feed, and cosmetics, cultivated oat is a major cereal crop with growing market potential. Historically, geneticists have focused on evaluating populations for grain yield, agronomic traits, and disease resistance, but little attention has been given to other traits, such as morphological characteristics of the panicle, that structure on which oat grains develop. ARS researchers in Fargo, North Dakota, conducted image analysis of diverse oat lines, which revealed novel and reproducible models of panicle architecture and development and their relationships with plant growth, physiology, and disease resistance. Genetic mapping in these populations identified regions of the oat genome associated with many important traits and functionally relevant candidate genes. The results will facilitate novel approaches to breeding and selection in oat and offer a unique perspective on the genetic control of panicle architecture in cereal crops.

8. A gene controlling barley architecture for higher yield. An erect leaf in cereal crops is a highly desirable trait that enhances light capture capacity and photosynthetic efficiency under dense planting. ARS researchers in Fargo, North Dakota, identified a gene regulating leaf angle in barley and found that this gene encoded a protein that regulates other genes. Mutant plants that don’t have this gene have a disruption in the formation of certain structures at the base of barley leaves, which results in a more erect leaf trait. More importantly, the researchers found that the mutant shows similar yield and agronomic traits to non-mutant plants. Therefore, this research provides genetic material for breeders to improve barley yield using a suitable architecture for dense planting, and it provides a new gene target for barley geneticists to optimize light-use efficiency under field conditions.

9. Spot form net blotch susceptibility gene in barley. A fungal disease known as spot form net blotch (SFNB) occurs worldwide and can cause significant yield losses in barley. ARS researchers in Fargo, North Dakota, in collaboration with colleagues at North Dakota State University and Washington State University conducted genetic analysis to precisely determine the genomic location of a specific barley gene that, when present, renders the barley plant susceptible to the disease. This gene, referred to as Sptm1, was located to a small segment of a specific barley chromosome. Further analysis of the segment indicated that it harbors a gene known as a protein kinase, which are genes typically associated with disease resistance or susceptibility in plants. The researchers also developed molecular markers that can be used to track the presence of the gene when developing new barley varieties. The results of this research provide a foundation for geneticists to advance our knowledge of how barley plants interact with the pathogen that causes SFNB and provides tools for breeders to develop SFNB-resistant barley varieties.

Review Publications
Poddar, S., Tanaka, J., Running, K.L., Kariyawasam, G., Faris, J.D., Friesen, T.L., Cho, M., Kate, J.H., Staskawicz, B. 2023. Optimization of highly efficient exogenous-DNA-free Cas9-ribonucleoprotein mediated gene editing in disease susceptibility loci in wheat (Triticum aestivum L.). Frontiers in Plant Science. 13. Article 1084700. https://doi.org/10.3389/fpls.2022.1084700.
Peters Haugrud, A., Zhang, Q., Green, A., Xu, S.S., Faris, J.D. 2022. Identification of stable QTL controlling multiple yield components in a durum x cultivated emmer population under field and greenhouse conditions. Genes, Genomes, Genetics. 13(2). https://doi.org/10.1093/g3journal/jkac281.
Kaur, S., Gill, H., Breiland, M., Kolmer, J.A., Gupta, R., Sehgal, S., Gill, U. 2023. Identification of leaf rust resistance loci in a geographically diverse panel of wheat using high-resolution genome-wide association study. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2023.1090163.
Kaur, S., Pennington, T., Conley, E., Green, A., Kolmer, J.A., Anderson, J., Gupta, R., Gill, U. 2022. High resolution melting (HRM) based marker development for wheat leaf rust resistance gene Lr34. Phytopathology. https://doi.org/10.1094/PHYTO-08-22-0313-R.
Liu, J., Wang, T., Qin, Q., Yu, X., Yang, S., Dinkins, R.D., Kuczmog, A., Putnoky, P., Muszynski, A., Griffitts, J., Kereszt, A., Zhu, H. 2022. Paired Medicago receptors mediate broad-spectrum resistance to nodulation by Sinorhizobium meliloti carrying a species-specific gene. Proceedings of the National Academy of Sciences (PNAS). 119(51). https://doi.org/10.1073/pnas.2214703119.
Karmacharya, A., Li, D., Leng, Y., Shi, G., Liu, Z., Yang, S., Du, Y., Dai, W., Zhong, S. 2023. Targeting wheat disease susceptibility genes in wheat through wide hybridization with maize expressing Cas9 and guide RNA. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-01-23-0004-SC.
Hyden, B., Zou, J., Wilkerson, D.G., Carlson, C.H., Robles Riviera, A., Difazio, S., Smart, L. 2023. Structural variation of a sex-linked region confers monoecy and implicates GATA15 as a master regulator of sex in Salix purpurea. New Phytologist. 238(6):2512-2523. https://doi.org/10.1111/nph.18853.
Alhashel, A., Fiedler, J.D., Nandety, R., Skiba, R., Bruggeman, R., Baldwin, T., Friesen, T.L., Yang, S. 2023. Genetic and physical localization of a major susceptibility gene to Pyrenophora teres f. maculata in barley. Theoretical and Applied Genetics. 136. Article e118. https://doi.org/10.1007/s00122-023-04367-1.
Manan, F., Shi, G., Gong, H., Hou, H., Khan, H., Leng, Y., Castell-Miller, C., Ali, S., Faris, J.D., Zhong, S. 2023. Prevalence and importance of the necrotrophic effector gene ToxA in Bipolaris sorokiniana populations collected from spring wheat and barley. Plant Disease. https://doi.org/10.1094/PDIS-08-22-2011-RE.
Peters Haugrud, A., Shi, G., Seneviratne, S., Running, K., Zhang, Z., Singh, G., Szabo-Hever, A., Acharya, K., Friesen, T.L., Liu, Z., Faris, J.D. 2023. Genome-wide association mapping of resistance to the foliar diseases septoria nodorum blotch and tan spot in a global winter wheat collection. Molecular Breeding. 43. Article 54. https://doi.org/10.1007/s11032-023-01400-5.