Non-vernalization requirement for flowering in Brassica rapa conferred by a dominant allele of FLOWERING LOCUS T.

KEY MESSAGE: We identified and characterized a dominant FT allele for flowering without vernalization in Brassica rapa, while demonstrating its potential for deployment in breeding to accelerate flowering in various Brassicaceae crops. Controlling the timing of flowering is key to improving yield and quality of several agricultural crops including the Brassicas. Many Brassicaceae crops possess a conserved flowering mechanism in which FLOWERING LOCUS C (FLC) represses the transcription of flowering activators such as FLOWERING LOCUS T (FT) during vernalization. Here, we employed genetic analysis based on next-generation sequencing to identify a dominant FT allele, BraA.FT.2-C, for flowering in the absence of vernalization in the Brassica rapa cultivar 'CHOY SUM EX CHINA 3'. BraA.FT.2-C harbors two large insertions upstream of its coding region and is expressed without vernalization, despite FLC expression. We show that BraA.FT.2-C offers an opportunity to introduce flowering without vernalization requirement into winter-type brassica crops, including B. napus, which have many functional FLC paralogs. Furthermore, we demonstrated the feasibility of using B. rapa harboring BraA.FT.2-C as rootstock for grafting to induce flowering in radish (Raphanus sativus), which requires vernalization for flowering. We believe that the ability of BraA.FT.2-C to overcome repression by FLC can have significant applications in brassica crops breeding to increase yields by accelerating or delaying flowering.

Sat-BSA: an NGS-based method using local de novo assembly of long reads for rapid identification of genomic structural variations associated with agronomic traits.

Advances in next generation sequencing (NGS)-based methodologies have accelerated the identifications of simple genetic variants such as point mutations and small insertions/deletions (InDels). Structural variants (SVs) including large InDels and rearrangements provide vital sources of genetic diversity for plant breeding. However, their analysis remains a challenge due to their complex nature. Consequently, novel NGS-based approaches are needed to rapidly and accurately identify SVs. Here, we present an NGS-based bulked-segregant analysis (BSA) technique called Sat-BSA (SVs associated with traits) for identifying SVs controlling traits of interest in crops. Sat-BSA targets allele frequencies at all SNP positions to first identify candidate genomic regions associated with a trait, which is then reconstructed by long reads-based local de novo assembly. Finally, the association between SVs, RNA-seq-based gene expression patterns and trait is evaluated for multiple cultivars to narrow down the candidate genes. We applied Sat-BSA to segregating F2 progeny obtained from crosses between turnip cultivars with different tuber colors and successfully isolated two genes harboring SVs that are responsible for tuber phenotypes. The current study demonstrates the utility of Sat-BSA for the identification of SVs associated with traits of interest in species with large and heterozygous genomes.

Next-generation sequencing-based bulked segregant analysis for QTL mapping in the heterozygous species Brassica rapa.

KEY MESSAGE: 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.