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ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Cereal Crops Research » Research » Research Project #434261

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

Location: Cereal Crops Research

2019 Annual Report

Objective 1: Identify novel sources of disease and pest resistance in durum wheat and goatgrass to enhance crop resilience. [NP301, C1, PS1B] 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. [NP301, C1, PS1A] 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. [NP301, C3, PS3A] Objective 4: Utilize and develop genetic resources and molecular tools for the improvement of wheat and other small grains will be enhanced by designing, validating, and implementing the adaptation of next-generation sequencing technology to the needs of oat breeders and by providing bioinformatics support to breeders seeking to use new technologies for oat improvement. [NP301, C1, PS1A and PS1B] 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. [NP301, C1, PS1A and PS1B]

Durum and hard red spring wheat (HRSW) 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, and to Hessian fly in goatgrass. 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, and stem rust. 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-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 and durum 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 Snn1 and Snn3 in durum wheat as outlined in objective 3A, we have screened the Durum Wheat Reference Collection (DWRC) with SnTox1 and SnTox3 in two replications, and we have begun to analyze the data. To broaden this objective, we have also screened the same collection of durum lines with SnToxA in two experiments and with SnTox5 in one experiment, and the latter will be performed a second time as well. This work will provide information on the prevalence of four 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. Toward the identification and cloning of the necrotrophic effector sensitivity gene Snn5 in wheat, the appropriate high-resolution mapping populations have been constructed and partially phenotyped. New molecular markers have been developed that delineate the candidate region to a few hundred kilobase pairs of DNA. Several promising candidate genes reside within the region and are currently being analyzed. We have learned that the hexaploid wheat line Chinese Spring carries the Snn5 gene as well. This will greatly aid the cloning of Snn5 because there is a high-quality reference sequence available for Chinese Spring, and we have developed appropriate mapping populations to take advantage of the resource. To identify a stem rust resistance gene in Ae. tauschii accession RL5271, a population of 366 F2 plants from the cross between Ae. tauschii RL5271 and AL8/78 was evaluated for seedling reactions to stem rust race TPMKC at the seedling stage and genotyped with a set of D-genome specific simple sequence repeats (SSR) markers. Using bulked segregant analysis, we mapped the gene to the Sr46 genomic region, which was previously identified on chromosome arm 2DS in Ae. tauschii AUS 18913. Based on the Ae. tauschii AL8/78 genome sequence, we have fine-mapped the Sr gene in RL 5271 within a 0.69 cM interval, which corresponds to a region including Sr46, which encodes a disease resistance protein for stem rust resistance. We are currently conducting sequencing analysis to determine allelic status of the Sr gene in RL 5271. Towards genotyping and phenotyping of a Global Durum Wheat Panel (GDP), which was previously named Durum Wheat Reference Collection, we have imported seeds of the 960 accessions of durum wheat and its closely related subspecies. These accessions have been grown in quarantine and increased. This panel has been extensively genotyped through the wheat iSelect 90K SNP assay, and the genotype call pipeline allowed to retrieve data for more than 45,000 markers. The dataset firstly obtained was refined to nearly 20,000 unique, non-redundant, single Mendelian single nucleotide polymorphisms (SNP) markers that were both genetically and physically mapped. The genotypic information will allow us to characterize the GDP for genetic diversity, population structure and genetic relationships. A subset of 500 lines consisting of modern durum cultivars and landraces have been increased for the 2nd season in greenhouse and they are currently being phenotyped for resistance to tan spot.

1. Genetic interactions between wheat and a fungal pathogen. Septoria nodorum blotch (SNB) is a serious fungal disease of wheat. The SNB pathogen produces several proteins known as effectors that, when recognized by specific genes in the wheat plant, induce a cell death response in the plant, which allows the fungus to grow and cause disease. While much is known about individual plant gene-effector interactions, little is known about how the plant and pathogen respond when there are multiple effectors in the pathogen being recognized by multiple genes in the plant. ARS researchers in Fargo, North Dakota, used several strains of the SNB fungus that varied in the number of effectors that they produced to evaluate their ability to cause disease on wheat plants that carried genes to recognize the effectors. The researchers found that, with some strains, the amount of disease a strain could cause was a cumulative effect of all of its effectors, but with others only one or two effectors were responsible for disease even though all were recognized by the plant. This study shows that plants must be blind to all fungal effector proteins in order to achieve resistance, and it provides knowledge of the wheat-SNB fungus interaction that may lead to novel approaches to control disease through manipulation of fungal effector proteins.

2. Genes governing wheat domestication. 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. The genetic mutations that allowed the wheat seeds to be easily separated from the hulls was particularly beneficial because it greatly decreased the amount of effort required to prepare the wheat for consumption. Understanding the genetic basis of the free-threshing trait would provide insights to the molecular mechanisms associated with grain development. ARS researchers in Fargo, North Dakota, compared the genetic components of threshability in a primitive wheat known as ‘emmer’ and modern domesticated durum wheat. The researchers found that three genes were involved in governing the free-threshing trait, and all three genes had undergone mutations during the evolution of emmer to domesticated durum wheat. These findings provide insights into the timeline and biological mechanisms responsible for the domestication of wheat through mutation of genes governing seed threshability. Researchers may use this information to better understand wheat domestication and acquisition of beneficial genes from wheat relatives for improvement of modern varieties.

3. A fungal virulence protein causes seedling rot in wheat. Numerous plant fungal pathogens produce a protein known as KP4 killer toxin (KP4) that is instrumental in causing disease. 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. However, the role of KP4 in causing FHB in wheat has not been determined. ARS researchers in Fargo, North Dakota, found that the FHB fungus produces four very similar KP4 proteins, and they found that the production of these proteins by the fungus is increased when the fungus infects a wheat plant. Further research indicated that, although the proteins did not play a significant role in the development of FHB, they were involved in causing seedling rot. This work provides insights regarding the significance of KP4 proteins in the fungal pathogen Fusarium graminearum and how it causes seedling rot in wheat.

4. Molecular mapping of genes for Fusarium head blight resistance introgressed into durum wheat. Fusarium head blight (FHB), commonly known as wheat scab, is a devastating disease of durum wheat. To combat the disease, ARS researchers in Fargo, North Dakota, have transferred FHB resistance genes from durum-related species including bread wheat into adapted durum cultivars. However, most of the durum genes for FHB resistance have not been characterized or confirmed. 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 that breeders can use to develop new durum varieties with FHB resistance.

5. Resistance to Fusarium head blight in synthetic wheat. Bread wheat and synthetic wheat both have three closely related genomes (A, B, and D) derived from hybridization between wheat progenitor species carrying A and B genomes and a goatgrass species (Ae. tauschii) with the D genome. Compared to bread wheat, which originated about 8,000 years ago from natural hybridization, synthetic wheat has been artificially created. To introduce useful genes into bread wheat from its AB- and/or D-genome ancestors, ARS researchers in Fargo, North Dakota, developed 200 new synthetic wheat lines. In this study, 149 synthetic wheat lines and their 74 AB-genome wheat parents were evaluated for resistance to Fusarium head blight (FHB). The results showed that the FHB resistance of the synthetic wheat lines varied greatly depending on their Ae. tauschii and AB-genome wheat parents. Most of the synthetic wheat lines were more resistant than their AB-genome wheat parents, indicating that the D genome may play a major role in reducing disease infection. Thirteen synthetic lines consistently showed a high level of FHB resistance across different environments and they may represent a new source of FHB resistance for wheat improvement.

6. Characterization of wheat-Aegilops markgrafii chromosome addition lines. Goatgrass species Aegilops markgrafii is a good source of new genes for resistance to the major diseases of wheat, including stem rust, leaf rust, stripe rust, and powdery mildew. A set of six wheat lines, each carrying a single additional chromosome from Ae. markgrafii designated as B through G, was previously produced. ARS researchers in Fargo, North Dakota, studied these lines to determine which of the Ae. markgrafii chromosomes carry genes for disease resistance, to determine the relationship of the Ae. markgrafii chromosome to the wheat chromosomes, and to discover molecular markers associated with each Ae. markgrafii chromosome. The results showed leaf rust resistance was associated with chromosome B, powdery mildew resistance associated with chromosomes D, E, F, and G, and stem rust resistance associated with chromosomes C and D. Thus, each Ae. markgrafii chromosome conferred resistance to at least one disease. The disease data, molecular markers, and chromosome groupings will facilitate transfer of the Ae. markgrafii genes for resistance to these diseases into wheat.

7. Genome sequencing and analysis in durum wheat. Durum wheat is a widely grown cereal crop mainly for making pasta products. It originated from the taming of wild emmer wheat, a primitive wheat species whose genome was recently sequenced. 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. This comparative analysis revealed that the chromosomal regions exhibiting genetic changes associated with formation of durum wheat and facilitated by human selection were widely present in the durum genome. One such region carries a gene causing high levels of dietary cadmium in grain. This gene is widespread among modern durum cultivars but undetected in wild emmer germplasm collections. The rapid analysis of the structure and function of this gene allowed the effective recovery of the beneficial gene for low cadmium uptake, thus demonstrating the practical utility of the durum genome sequence for wheat improvement.

8. Resistance gene discovery and cloning by sequence capture and association genetics. Disease resistance (R) genes from wild relatives of wheat are a valuable resource for breeding crops. However, current R gene identification and cloning methods require segregating or mutant progenies, which are difficult to generate for many wild relatives due to poor agronomic traits. ARS researchers in Fargo, North Dakota, and St. Paul, Minnesota, participated in an international team to develop a new method (AgRenSeq) for rapid discovery and cloning of R genes using natural populations, genome sequencing, and gene-trait association analysis. In this study, four genes for resistance to wheat stem rust were rapidly discovered and cloned by using natural genome variation in a set of 151 goatgrass (Ae. tauschii) lines. This method is a major advance in discovering and isolating R genes for engineering resistance against a wide range of pathogen races in crops.

Review Publications
Gyawali, Y., Zhang, W., Chao, S., Xu, S.S., Cai, X. 2019. Delimitation of wheat ph1b deletion and development of ph1b-specific DNA markers. Journal of Theoretical and Applied Genetics. 132(1):195–204.
Niu, Z., Chao, S., Cai, X., Whetten, R.B., Breiland, M., Cowger, C., Chen, X., Friebe, B., Gill, B.S., Rasmussen, J.B., Klindworth, D.L., Xu, S.S. 2018. Molecular and cytogenetic characterization of six wheat-Aegilops markgrafii disomic addition lines and their resistance to rusts and powdery mildew. Frontiers in Plant Science.
Szabo-Hever, A., Zhang, Q., Friesen, T.L., Shaobin, Z., Elias, E.M., Cai, X., Jin, Y., Faris, J.D., Chao, S., Xu, S.S. 2018. Genetic diversity and resistance to Fusarium head blight in synthetic hexaploid wheat derived from Aegilops tauschii and diverse Triticum turgidum subspecies. Frontiers in Plant Science.
Lu, S., Faris, J.D. 2019. Fusarium graminearum KP4-like proteins possess root growth-inhibiting activity against wheat and potentially contribute to fungal virulence in seedling rot. Fungal Genetics and Biology. 123:1-13.
Peters Haugrud, A.R., Zhang, Z., Richards, J.K., Friesen, T.L., Faris, J.D. 2019. Genetics of variable disease expression conferred by inverse gene-for-gene interactions in the wheat-Parastagonospora nodorum pathosystem. Plant Physiology. 180(1):420-434.
Maccaferri, M., Harris, N.S., Twardziok, S.O., Gundlach, H., Spannagl, M., Ormanbekova, D., Lux, T., Prade, V., Milner, S., Himmelbach, A., Chao, S., Faris, J.D., Xu, S.S. 2019. Durum wheat genome highlights past domestication signatures and future improvement targets. Nature Genetics. 51(5):885-895.