Identification of regulatory genes involved in anthocyanin accumulation in radish leaves using bulk segregant analysis

Anthocyanins, a type of flavonoid, are crucial metabolites in most vacuolar plants, significantly contributing to plant coloration and defense responses. Owing to their potent anti-cancer and anti-inflammatory properties, anthocyanins have gained popularity, with extensive application prospects in the health industries. Although the mechanism underlying anthocyanin accumulation in radish fleshy roots has been explored, the processes governing its accumulation in radish leaves remain largely unexplored. In the present study, we aimed to investigate the genetic regulatory mechanisms underlying anthocyanin accumulation, which is responsible for leaf color in radish. We developed self-crossing, hybrid F1 generations, back-crosses, and F2 generation populations using M17 and RA9 pure-line cultivars with purple and green leaves, respectively. Our findings revealed that the color of radish leaves is determined by nuclear genes, with purple being the semi-dominant trait. In the F2 generation, two populations with extreme traits were selected to construct gene sequencing mixing pools for bulked segregant analysis sequencing. Moreover, combined delta (SNP-index) and delta (Indel-index) analyses identified a 200 kb candidate region on chromosome 7, which contains 39 annotated genes. Gene annotation query and quantitative reverse transcription polymerase chain reaction analysis demonstrated that 8 genes were highly expressed in M17, while 13 genes were highly expressed in RA9. Among these, MYB114-like, which was specifically expressed in M17, was the only gene that clearly demonstrated a role in anthocyanin synthesis. This result suggests that MYB114-like is likely the primary gene regulating radish leaf color in this candidate region. Overall, our study provides an additional reference for enhancing the regulatory network of anthocyanin synthesis in radish.