Enabling High-Resolution Mapping of the Ug99 Resistance Gene SrWLR via RNAseq

The emergence and spread of the highly virulent Puccinia graminis f. sp. tritici Ug99 lineage in eastern Africa has stimulated research directed toward the discovery and mapping of Ug99 resistance genes in wheat. A potentially new source of Ug99 (TTKSK) resistance was identified in the spring wheat landrace PI626573-2. The resistance was found to be conferred by a single gene (SrWLR) through Chi-square analysis and was mapped to a 1.6 cM region on the long arm of chromosome 2B delimited by GWM47 and the cosegregating iSelect SNP markers IWA6121, IWA6122, IWA7620, IWA8295, and IWA8362. To facilitate positional cloning efforts a mapping population consisting of 6,382 recombinant gametes was created through crosses with the susceptible parent LMPG-6. Sixty-seven critical recombinants were identified via PCR using GWM47 and high-resolution melt curve (HRM) markers created from the iSelect assay. Synteny analysis of the region between two of the genic iSelect markers was performed using the Wheat Zapper program. The Wheat Zapper program identified 493 wheat ESTs syntenic with ESTs in rice, sorghum, and Brachypodium. Markers were developed from the ESTs and the region was reduced to 0.44 cM. To further identify SNPs in the region RNAseq was performed on the resistant and susceptible parents using the Ion Torrent PGM. The PI626573-2 and LMPG-6 libraries produced 6.3 and 4.6 million reads with an average read lengths of 125 and 108 base pairs, respectively. HRM Markers were developed from this data to further reduce the region and assist with future anchoring of a physical map.