Development and Validation of KASP Assays for Functional Genes in Wheat

Kompetitive Allele Specific PCR (KASP) technology provides an excellent opportunity for low-density high-throughput genotyping at low cost for functional markers, the most valuable markers for crop breeding programs. We developed and validated KASP assays for genes that underpin economically important traits in bread wheat including adaptability, grain yield, quality, and biotic and abiotic stress resistances. In total, 140 KASP assays either developed in this study or obtained from public databases were validated for reliability in application. The validation of KASP assays were conducted by i) comparing the assays with available gel-based PCR markers on 23 diverse wheat accessions, ii) validation of the derived allelic information using phenotypes of an association mapping panel comprised of 300 diverse cultivars from China and 13 other countries, and iii) additional testing, where possible, of the assays in four segregating populations. All KASP assays being reported were significantly associated with the relevant phenotypes in the association panel and bi-parental populations, thus revealing potential application in wheat breeding programs. The results revealed 45 times superiority of the KASP assays in speed and greater cost-effectiveness than gel-based PCR markers. KASP has recently emerged as single-plex high-throughput genotyping technology; however, this is the first successful report on deploying a large number of functional genes for high-throughput screens in a major crop. Such assays greatly accelerate the characterization of crossing parents and advanced lines for marker-assisted selection, and can complement the non-flexible, high-density, chip-based SNP assays. Our results offer a robust and reliable molecular marker toolkit and represent a significant step towards maximizing genetic gains in wheat breeding programs.