RNA-Seq bulked segregant analysis enables the identification of high-resolution genetic markers for breeding in hexaploid wheat

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The identification of genetic markers linked to genes of agronomic importance is amajor aim of crop research and breeding programmes. Here, we identify markers for Yr15, a major disease resistance gene for wheat yellow rust, using a segregating F2 population. After phenotyping, we implemented RNA sequencing (RNA-Seq) of bulked pools to identify single nucleotide polymorphisms (SNP) associated with Yr15. Over27,000 genes with SNPs were identified between the parents, and then classified basedon the results from the sequenced bulks. We calculated the bulk frequency ratio (BFR)of SNPs between resistant and susceptible bulks, selecting those showing six-foldenrichment/depletion in the corresponding bulks (BFR > 6). Using additional filteringcriteria we reduced the number of genes with a putative SNP to 175. The 35 SNPs withthe highest BFR values were converted into genome-specific KASP assays using an automated bioinformatics pipeline (PolyMarker) which circumvents the limitations associated with the polyploid wheat genome. Twenty-eight assays were polymorphic of which twenty two (63%) mapped in the same linkage group as Yr15. Using thesemarkers, we mapped Yr15 to a 0.77 cM interval. The three most closely linked SNPswere tested across varieties and breeding lines representing UK elite germplasm. Twoflanking markers were diagnostic in over 99% of lines tested, thus providing a reliablehaplotype for marker-assisted selection in these breeding programmes. Our results demonstrate that the proposed methodology can be applied in polyploid F2 populations to generate high-resolution genetic maps across target intervals.