Next-generation sequencing-based bulked segregant analysis for QTL mapping in the heterozygous species Brassica rapa
An improved protocol of QTL-seq, an NGS-based method for bulked segregant analysis we previously developed in rice, allowed successful mapping of QTLs of interest in the highly heterozygous genome of B. rapa, demonstrating the power of this elegant method for genetic analyses in heterozygous species of economic importance.
Recent advances in next-generation sequencing (NGS) and the various NGS-based methods developed for rapidly identifying candidate genes of interest have accelerated genetic analysis mainly in the model plants rice and Arabidopsis. Brassica rapa includes several economically important crops such as Chinese cabbage, turnip and various leafy vegetables. The application of NGS-based approaches for the analysis of B. rapa has been limited mainly due to its highly heterozygous genome and poor quality of the reference genome sequence currently available for this species. In this study, we have improved QTL-seq, a method for NGS-based bulked segregant analysis we previously developed in rice, extending its applicability for accelerating the genetic analysis and molecular breeding of B. rapa. Addition of new filters to the original QTL-seq pipeline allowed removal of spurious single-nucleotide polymorphisms caused by alignment/sequencing errors and variability between parents, significantly improving accuracy of the analysis. As proof of principle, we successfully applied the new approach to identify candidate genomic regions controlling flowering and trichome formation using segregating F2 progeny obtained from crosses made between cultivars of B. rapa showing contrasting phenotypes for these traits. We strongly believe that the improved QTL-seq method reported here will extend the applicability of NGS-based genetic analysis not only to B. rapa but also to other plant species of economic importance with heterozygous genomes.
This study was funded by the JSPS KAKENHI Grant Number 15H05611 and the Mitani Foundation for Research and Development 2018. The computations were partially performed on the National Institute of Genetics (NIG) supercomputer at Research Organization of Information and Systems (RIOS) located in Mishima (Shizuoka), Japan. Local landraces/cultivars of Brassica rapa used in this study were obtained from the National Agriculture and Food Research Organization (NARO) Genebank and Matsushita seed.
- Diederichsen A (2001) Brassica rapa group. In: Hanelt P (ed) Mansfeld’s encyclopedia of agricultural and horticultural crops (except ornamentals). Springer, Berlin, pp 1446–1453Google Scholar
- Sato K, Nishio T, Kimura R, Kusaba M, Suzuki T, Hatakeyama K, Ockendon DJ, Satta Y (2002) Coevolution of the S-locus genes SRK, SLG and SP11/SCR in Brassica oleracea and B. rapa. Genetics 162:931–940Google Scholar