Location: Cereal Crops Improvement Research
Project Number: 3060-21000-046-006-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2020
End Date: Aug 31, 2025
Objective:
The objectives of this research project are to:
1. Develop a high-resolution linkage map of the Septoria nodorum blotch susceptibility gene Snn2
2. Develop molecular markers suitable for marker-assisted selection against Snn2
3. Clone the Snn2 gene using map-based cloning, mutagenesis, bioinformatics, and comparative mapping techniques
4. Determine structure, function and diversity of Snn5-B1 gene
5. Map the location of Snn5-B2 and identify candidate genes
6. Use CRISPR/Cas9 to disrupt Snn2 candidate genes for Snn2 validation
Approach:
Septoria nodorum blotch (SNB) is a devastating foliar disease caused by a necrotrophic fungal pathogen and affects all classes of wheat. The wheat Snn2 gene confers susceptibility to strains of the SNB pathogen that produce the protein known as SnTox2. Snn2 is common among modern wheat varieties, and therefore renders them susceptible to SNB. The Snn2 gene was previously mapped to the short arm of wheat chromosome 2D, but markers suitable for marker-assisted selection against Snn2 have not been developed. A saturated genetic linkage map of the Snn2 genomic region has already been developed using a segregating population derived from crossing the Snn2-containing line BG301 with the SnTox2-insensitive line BR34. A large F2 population consisting of at least 5,000 plants derived from the same cross will be used to develop a high-resolution genetic linkage map of the Snn2 locus and identify markers tightly linked to Snn2 that would be suitable for marker-assisted selection. The population will be screen with current flanking markers to identify genetic recombinants between the two markers, and the recombinants will be tested for reaction to SnTox2 to map the Snn2 gene relative to the molecular markers. Codominant markers that cosegregate with Snn2 will be tested on a panel of wheat lines with known SnTox2 reactions to determine the robustness of the markers. Diagnostic markers will be converted to KASP markers for high-throughput screening of lines by breeders and regional genotyping service labs. The wheat line BG301 will be treated with the chemical mutagen ethylmethane sulfonate to develop a population of mutants. The mutant population will be screened for reaction to SnTox2 by infiltrating M2 families with purified cultures. Plants that show insensitivity to the cultures will be considered candidates for Snn2-disrupted mutants. The presence of Snn2 in all the sequenced wheat lines will be determined by infiltrating the lines with the SnTox2 culture. Bioinformatic analysis of the candidate gene region will then be conducted using all sequenced wheat lines to identify candidate genes for Snn2. DNA of the wild type BG301 and of the Snn2-disrupted mutant lines will extracted and the sequences of candidate genes will be compared between the mutants and the wild type to validate or rule out specific genes as Snn2. Once a gene is validated to be Snn2 by mutagenesis, it will undergo a second round of validation using complementation by transformation.
