Construction of a High-Density Genetic Map Based on Specific-Locus Amplified Fragment Sequencing and Identification of Loci Controlling Anthocyanin Pigmentation in Yunnan Red Radish

Radish (Raphanus sativus L.) belongs to the family Brassicaceae. The Yunnan red radish variety contains relatively large amounts of anthocyanins, making them important raw materials for producing edible red pigment. However, the genetic mechanism underlying this pigmentation has not been fully characterized. Here, the radish inbred line YAAS-WR1 (white root skin and white root flesh) was crossed with the inbred line YAAS-RR1 (red root skin and red root flesh) to produce F-1, F-2, BC1P1, and BC1P2 populations. Genetic analyses revealed that the pigmented/non-pigmented and purple/red traits were controlled by two genetic loci. The F-2 population and the specific-locus amplified fragment sequencing (SLAF-seq) technique were used to construct a high-density genetic map (1230.16 cM), which contained 4032 markers distributed in nine linkage groups, with a mean distance between markers of 0.31 cM. Additionally, two quantitative trait loci (QAC1 and QAC2) considerably affecting radish pigmentation were detected. A bioinformatics analysis of the QAC1 region identified 58 predicted protein-coding genes. Of these, RsF3 ' H, which is related to anthocyanin biosynthesis, was revealed as a likely candidate gene responsible for the purple/red trait. The results were further verified by analyzing gene structure and expression. Regarding QAC2, RsMYB1.3 was determined to be a likely candidate gene important for the pigmented/non-pigmented trait, with a 4-bp insertion in the first exon that introduced a premature termination codon in the YAAS-WR1 sequence. Assays demonstrated that RsMYB1.3 interacted with RsTT8 and activated RsTT8 and RsUFGT expression. These findings may help clarify the complex regulatory mechanism underlying radish anthocyanin synthesis. Furthermore, this study's results may be relevant for the molecular breeding of radish to improve the anthocyanin content and appearance of the taproots.