Combined QTL-Seq and Traditional Linkage Analysis to Identify Candidate Genes for Purple Skin of Radish Fleshy Taproots

Abstract

Taproot skin color is a crucial visual and nutritional quality trait of radish, and purple skin is most attractive to consumers. However, the genetic mechanism underlying this character is unknown. Herein, F2 segregating populations were constructed to investigate radish genomic regions with purple skin genes. Segregation analysis suggested that pigment presence was controlled by one dominant gene, Rsps. A bulk segregant approach coupled to whole-genome sequencing (QTL-seq) and classical linkage mapping narrowed the Rsps location to a 238.51-kb region containing 18 genes. A gene in this region, designated RsMYB1.1 (an Arabidopsis PAP1 homolog), was a likely candidate gene because semiquantitative RT-PCR and quantitative real-time PCR revealed RsMYB1.1 expression in only purple-skinned genotypes, sequence variation was found between white- and purple-skinned radishes, and an InDel marker in this gene correctly predicted taproot skin color. Furthermore, four RsMYB1.1 homologs (RsMYB1.1-1.4) were found in “XYB36-2” radish. RsMYB1.1 and the previously mapped and cloned RsMYB1.4 (contributing to red skin) were located on different chromosomes and in different subclades of a phylogenetic tree; thus, they are different genes. These findings provide insight into the complex anthocyanin biosynthesis regulation in radish and information for molecular breeding to improve the anthocyanin content and appearance of radish taproots.

A genome-wide association study uncovers a critical role of the RsPAP2 gene in red-skinned Raphanus sativus L.

“…The RsMyb1/RsMYB90 gene is a crucial regulator of radish root skin color 23,26 . Liu et al reported that the RsMYB1.1 gene was located within the mapping region for the purple skin of radish root 24 . These findings suggested that MYB TFs are the crucial determinants of ATC accumulation in red-skinned radish and that MYB genes from different plant materials might control skin color inheritance via different mechanisms.…”

“…A previous study indicated that the genetic mechanisms determining radish root skin color differ among different genotypes 24 . Although several ATC-related RsMYB genes have been identified by QTL-seq, traditional linkage analysis and genome resequencing using hybrid populations 23 , 24 , 26 , the available information is far from complete to fully explain the red skin color variations among diverse radish genotypes. In the present study, to enrich the understanding of ATC biosynthesis regulation, the GWAS approach was first applied to address potential genes responsible for red skin color among various red-skinned radish genotypes.…”

“…In addition, a few members of the bHLH TF family, such as AtTT8 (bHLH42) in Arabidopsis and StbHLH1 in potato, also result in enhanced activity at promoters containing a cis-regulatory element that is conserved in several ATC biosynthetic genes 19,20 . Most of the structural genes known to be involved in ATC biosynthesis have been reported in radish 21 ; however, only a few TFs have been clearly identified as being related to the transcriptional regulation of the ATC biosynthesis pathway in radish, such as RsMYB1, RsMYB1a, RsMYB90, and RsTT8 [22][23][24][25][26][27] .…”

Targeted amplicon sequencing + next-generation sequencing–based bulked segregant analysis identified genetic loci associated with preharvest sprouting tolerance in common buckwheat (Fagopyrum esculentum)

“…The development of genome databases makes it easy to do QTL analysis by BSA (QTL-seq) in major crops and model plant species, such as rice, barley, and Arabidopsis [18][19][20]. By WGS in buckwheat, we developed the BGDB [11], but its small scaffold size so far prevents its use in QTL-seq analysis.…”

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“…However, the genetic basis for these two interesting behavioral traits is still unclear. Benefit from the highthroughput sequencing-based bulked segregant analysis (BSA), the trait-related loci can be identified rapidly using individuals with extreme phenotypes in a mapping population (Takagi et al, 2013;Zegeye et al, 2018;Liu et al, 2019). The feasible construction of mapping population between the wild silkworm and the domestic silkworm also enables the identification of domestic behavior-related loci in silkworm.…”