Location: Cereal Crops Improvement Research
Project Number: 3060-21000-046-026-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Jul 1, 2023
End Date: Jun 30, 2025
Objective:
1) to provide resistant germplasm and populations for breeders;
2) to localize Rptm2 with high resolution;
3) to develop user-friendly markers for high throughput genotyping to assist breeding selection;
4) to validate the Rptm1 candidate gene.
Approach:
1). Developing segregating population using Pinnacle (S) and PI67381 (R)
Pinnacle is widely used by breeding program of NDSU, but it is susceptible many Ptm isolates. Genetic analysis revealed that SFNB resistance to the ND isolate FGO is controlled by the major QTL Rptm1 and Rptm2 in PI67381 (Tamang et al., 2019). The F2 population and recombinant inbred lines (RILs) have been developed from the cross between Pinnacle and PI67381. A total of 200 F2s will be genotyped with flanking markers for the two loci, and the individuals segregating only at the Rptm2 locus will be selected to develop near-isogenic lines (NILs). Meanwhile, 180 RILs will be used to identify heterozygous residuals at the Rptm2 locus for construction of NIL.
2). Fine mapping of Rptm2
At least 3000 F3 plants will be used for genetic mapping. Inoculum isolate preparation and inoculation will follow the methods established by Nepane et al. (2015). Briefly, two-week-old seedlings growing in a greenhouse will be spray-inoculated, followed by incubation for 24 hours in a mist chamber. The inoculated plants will be then transferred to a growth chamber. Ninety-four RILs will be used first for SNP genotyping with the barley 50k iSelect SNP Array. To assisted breeding selection, the linked SNPs will be converted to PCR-based markers, such as STARP (semi-thermal asymmetric reverse PCR) and KASP (competitive allele-specific PCR) markers.
3). Disease phenotyping and data analysis
Disease reactions will be evaluated seven days after inoculation using a 1 to 5 scale as previously described (Neupane et al. 2015) by which 1 is highly resistant and 5 is highly susceptible. The average disease severities from three independent replicates of the phenotyping assays will be used for mapping of SFNB resistance/susceptibility. A permutation test with 1000 iterations will be performed to find a LOD threshold for QTL analysis at a significance level of a = 0.05 and 0.01. Linkage analysis and QTL mapping will be conducted using the software program Joinmap.
4) Functional validation of the Sptm1 candidate gene and development of diagnostic markers
The transgenic plants will be assessed if spot form net blotch resistance is increase by gene transfer. After the candidate gene is validated, Sptm1 alleles will be sequenced with the barley CAP (Coordinated Agricultural Project) population to identify the SNP haplotype associated with resistance/susceptibility to SFNB. Diagnostic markers will be developed based on the discovered SNPs.
