A comprehensive and precise set of intervarietal substitution lines to identify candidate genes and quantitative trait loci in oilseed rape (Brassica napus L.).

KEY MESSAGE: A set of intervarietal substitution lines were developed in rapeseed by recurrent backcrossing and marker-assisted selection and employed for mapping both qualitative and quantitative traits. Intervarietal substitution lines (ISLs) may be assembled into advanced secondary mapping populations that have remarkable potential for resolving trait loci and mapping candidate genes. To facilitate the identification of important genes in oilseed rape (canola, Brassica napus), we developed 89 ISLs using an elite cultivar 'Zhongyou 821' (ZY821) as the recipient and a re-synthesized line 'No.2127' as the donor. In the whole process of ISLs development, the target chromosome segments were selected based on the genotypes of 300 microsatellite markers evenly distributed across the genome. Eighty-nine ISLs fixed at BC5F4 were genotyped by sequencing using double digestion to survey the lengths of target substitution segments from the donor parent and the background segments from the recurrent parent. The total length of the substituted chromosome segments was 3030.27 Mb, representing 3.56 × of the Darmor-bzh reference genome sequence (version 4.1). Gene mapping was conducted for two qualitative traits, flower colour and seed-coat colour, and nine quantitative traits including yield- and quality-related traits, with 19 QTLs identified for the latter. Overlapping substitution segments were identified for flower colour and seed-coat colour loci, as well as for QTLs consistently detected in 2 or 3 years. These results demonstrate the value of these ISLs for locus resolution and subsequent cloning, targeted mutation or editing of genes controlling important traits in oilseed rape.

Development of a core set of single-locus SSR markers for allotetraploid rapeseed (Brassica napus L.).

Brassica napus (AACC) is a recent allotetraploid species evolved through hybridization between two diploids, B. rapa (AA) and B. oleracea (CC). Due to extensive genome duplication and homoeology within and between the A and C genomes of B. napus, most SSR markers display multiple fragments or loci, which limit their application in genetics and breeding studies of this economically important crop. In this study, we collected 3,890 SSR markers from previous studies and also developed 5,968 SSR markers from genomic sequences of B. rapa, B. oleracea and B. napus. Of these, 2,701 markers that produced single amplicons were putative single-locus markers in the B. napus genome. Finally, a set of 230 high-quality single-locus SSR markers were established and assigned to the 19 linkage groups of B. napus using a segregating population with 154 DH individuals. A subset of 78 selected single-locus SSR markers was proved to be highly stable and could successfully discriminate each of the 45 inbred lines and hybrids. In addition, most of the 230 SSR markers showed the single-locus nature in at least one of the Brassica species of the U's triangle besides B. napus. These results indicated that this set of single-locus SSR markers has a wide range of coverage with excellent stability and would be useful for gene tagging, sequence scaffold assignment, comparative mapping, diversity analysis, variety identification and association mapping in Brassica species.