RNA-Seq Bulked Segregant Analysis Enables the Identification of High-Resolution Genetic Markers for Breeding in Hexaploid Wheat

Next Generation Sequencing (NGS) is providing new methodologies to improve and complement traditional genetic approaches. These strategies, collectively termed NGS-enabled genetics, consist of identifying variation in bulks of plants that have been grouped based on a specific phenotype of interest. We used NGS-enabled genetics in hexaploid wheat to identify single nucleotide polymorphisms (SNP) linked to the yellow (stripe) rust resistance locus Yr15. We used RNA-Seq on resistant and susceptible F₂ bulks to identify polymorphic SNPs. Variants enriched in the resistant bulk were expected to be linked to Yr15 in coupling and KASPar assays were designed using PolyMarker. The validated SNPs were closely linked to Yr15 (<1 cM) and were diagnostic of Yr15 in >99% of the European breeding lines surveyed (N=117), making them suitable for marker assisted selection (MAS). The automated primer design pipeline was released as PolyMarker (http://polymarker.tgac.ac.uk), a web-service to facilitate the design of genome-specific SNP assays, a previously time consuming process. The pipeline uses the recently published IWGSC Chromosome-arm Survey Sequence (CSS) to find the different homoeologues and identify genome-specific variations that are incorporated into the primer design. This increases the chances of SNP assays being genome-specific, a feature which is especially important for MAS. PolyMarker and additional bioinformatics tools developed in the project are available to the community (https://github.com/TGAC/bioruby-polyploid-tools).