Effect of structural variation in the promoter region of RsMYB1.1 on the skin color of radish taproot.

Accumulation of anthocyanins in the taproot of radish is an agronomic trait beneficial for human health. Several genetic loci are related to a red skin or flesh color of radish, however, the functional divergence of candidate genes between non-red and red radishes has not been investigated. Here, we report that a novel genetic locus on the R2 chromosome, where RsMYB1.1 is located, is associated with the red color of the skin of radish taproot. A genome-wide association study (GWAS) of 66 non-red-skinned (nR) and 34 red-skinned (R) radish accessions identified three nonsynonymous single nucleotide polymorphisms (SNPs) in the third exon of RsMYB1.1. Although the genotypes of SNP loci differed between the nR and R radishes, no functional difference in the RsMYB1.1 proteins of nR and R radishes in their physical interaction with RsTT8 was detected by yeast-two hybrid assay or in anthocyanin accumulation in tobacco and radish leaves coexpressing RsMYB1.1 and RsTT8. By contrast, insertion- or deletion-based GWAS revealed that one large AT-rich low-complexity sequence of 1.3-2 kb was inserted in the promoter region of RsMYB1.1 in the nR radishes (RsMYB1.1nR), whereas the R radishes had no such insertion; this represents a presence/absence variation (PAV). This insertion sequence (RsIS) was radish specific and distributed among the nine chromosomes of Raphanus genomes. Despite the extremely low transcription level of RsMYB1.1nR in the nR radishes, the inactive RsMYB1.1nR promoter could be functionally restored by deletion of the RsIS. The results of a transient expression assay using radish root sections suggested that the RsIS negatively regulates the expression of RsMYB1.1nR, resulting in the downregulation of anthocyanin biosynthesis genes, including RsCHS, RsDFR, and RsANS, in the nR radishes. This work provides the first evidence of the involvement of PAV in an agronomic trait of radish.

An improved Raphanus sativus cv. WK10039 genome localizes centromeres, uncovers variation of DNA methylation and resolves arrangement of the ancestral Brassica genome blocks in radish chromosomes.

KEY MESSAGE: This study presents an improved genome of Raphanus sativus cv. WK10039 uncovering centromeres and differentially methylated regions of radish chromosomes. Comprehensive genome comparison of radish and diploid Brassica species of U's triangle reveals that R. sativus arose from the Brassica B genome lineage and is a sibling species of B. nigra. Radish (Raphanus sativus L.) is a key root vegetable crop closely related to the Brassica crop species of the family Brassicaceae. We reported a draft genome of R. sativus cv. WK10039 (Rs1.0), which had 54.6 Mb gaps. To study the radish genome and explore previously unknown regions, we generated an improved genome assembly (Rs2.0) by long-read sequencing and high-resolution genome-wide mapping of chromatin interactions. Rs2.0 was 434.9 Mb in size with 0.27 Mb gaps, and the N50 scaffold length was 37.3 Mb (40-fold larger assembly compared to Rs1.0). Approximately 38% of Rs2.0 was comprised of repetitive sequences, and 52,768 protein-coding genes and 4845 non-protein-coding genes were predicted and annotated. The improved contiguity and coverage of Rs2.0, along with the detection of highly methylated regions, enabled localization of centromeres where R. sativus-specific centromere-associated repeats, full-length OTA and CRM LTR-Gypsy retrotransposons, hAT-Ac, CMC-EnSpm and Helitron DNA transposons, and sequences highly homologous to B. nigra centromere-specific CENH3-associated CL sequences were enriched. Whole-genome bisulfite sequencing combined with mRNA sequencing identified differential epigenetic marks in the radish genome related to tissue development. Synteny comparison and genomic distance analysis of radish and three diploid Brassica species of U's triangle suggested that the radish genome arose from the Brassica B genome lineage through unique rearrangement of the triplicated ancestral Brassica genome after splitting of the Brassica A/C and B genomes.

Identification of candidate domestication regions in the radish genome based on high-depth resequencing analysis of 17 genotypes.

KEY MESSAGE: This study provides high-quality variation data of diverse radish genotypes. Genome-wide SNP comparison along with RNA-seq analysis identified candidate genes related to domestication that have potential as trait-related markers for genetics and breeding of radish. Radish (Raphanus sativus L.) is an annual root vegetable crop that also encompasses diverse wild species. Radish has a long history of domestication, but the origins and selective sweep of cultivated radishes remain controversial. Here, we present comprehensive whole-genome resequencing analysis of radish to explore genomic variation between the radish genotypes and to identify genetic bottlenecks due to domestication in Asian cultivars. High-depth resequencing and multi-sample genotyping analysis of ten cultivated and seven wild accessions obtained 4.0 million high-quality homozygous single-nucleotide polymorphisms (SNPs)/insertions or deletions. Variation analysis revealed that Asian cultivated radish types are closely related to wild Asian accessions, but are distinct from European/American cultivated radishes, supporting the notion that Asian cultivars were domesticated from wild Asian genotypes. SNP comparison between Asian genotypes identified 153 candidate domestication regions (CDRs) containing 512 genes. Network analysis of the genes in CDRs functioning in plant signaling pathways and biochemical processes identified group of genes related to root architecture, cell wall, sugar metabolism, and glucosinolate biosynthesis. Expression profiling of the genes during root development suggested that domestication-related selective advantages included a main taproot with few branched lateral roots, reduced cell wall rigidity and favorable taste. Overall, this study provides evolutionary insights into domestication-related genetic selection in radish as well as identification of gene candidates with the potential to act as trait-related markers for background selection of elite lines in molecular breeding.