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Development and validation of KASP assays for genes underpinning key economic traits in bread wheat

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Abstract

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We developed and validated a robust marker toolkit for high-throughput and cost-effective screening of a large number of functional genes in wheat.

Abstract

Functional markers (FMs) are the most valuable markers for crop breeding programs, and high-throughput genotyping for FMs could provide an excellent opportunity to effectively practice marker-assisted selection while breeding cultivars. Here we developed and validated kompetitive allele-specific PCR (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, 70 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 (a) comparing the assays with available gel-based PCR markers on 23 diverse wheat accessions, (b) validation of the derived allelic information using phenotypes of a panel comprised of 300 diverse cultivars from China and 13 other countries, and (c) 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 cultivars 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 than gel-based PCR markers. KASP has recently emerged as single-plex high-throughput genotyping technology; this is the first report on high-throughput screening of a large number of functional genes in a major crop. Such assays could greatly accelerate the characterization of crossing parents and advanced lines for marker-assisted selection and can complement the inflexible, high-density SNP arrays. Our results offer a robust and reliable molecular marker toolkit that can contribute towards maximizing genetic gains in wheat breeding programs.

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Acknowledgments

The authors acknowledge Profs. Robert McIntosh (University of Sydney), Yunbi Xu (CIMMYT-CAAS, China), Guihua Bai (K-State, USA) and Yuanfeng Hao (CIMMYT) for critical reviews of the manuscript. They are thankful to Drs. Yanwen Xiong and Jian Zhang (Huazhi Rice Bio-Tech Co. Ltd, Changsha, China) for providing technical support in SNPline® application. They are also thankful to Profs. Jizeng Jia, Xueyong Zhang (ICS-CAAS), Daowen Wang (IGDB-CAS) for providing the sequences of genes for marker development. This study was supported by the National Natural Science Foundation of China (31461143021), Beijing Municipal Science and Technology Project (D151100004415003), International Science and Technology Cooperation Program of China (2013DFG30530, 2014DFG31690), and the China Agricultural Research System (CARS-3-1-3).

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Communicated by D. E. Mather.

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Rasheed, A., Wen, W., Gao, F. et al. Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theor Appl Genet 129, 1843–1860 (2016). https://doi.org/10.1007/s00122-016-2743-x

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