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

Lianxue Fan,Liwang Liu,Jiali Ying,Mingjia Tang,Yan Wang,Liang Xu,Junhui Dong,Cui Li,Xiaoli Zhang

doi:10.1038/s41438-020-00385-y

Abstract

Radish (Raphanus sativus L.) taproot contains high concentrations of flavonoids, including anthocyanins (ATCs), in red-skinned genotypes. However, little information on the genetic regulation of ATC biosynthesis in radish is available. A genome-wide association study of radish red skin color was conducted using whole-genome sequencing data derived from 179 radish genotypes. The R2R3-MYB transcription factor production of anthocyanin pigment 2 (PAP2) gene was found in the region associated with a leading SNP located on chromosome 2. The amino acid sequence encoded by the RsPAP2 gene was different from those of the other published RsMYB genes responsible for the red skin color of radish. The overexpression of the RsPAP2 gene resulted in ATC accumulation in Arabidopsis and radish, which was accompanied by the upregulation of several ATC-related structural genes. RsPAP2 was found to bind the RsUFGT and RsTT8 promoters, as shown by a dual-luciferase reporter system and a yeast one-hybrid assay. The promoter activities of the RsANS, RsCHI, RsPAL, and RsUFGT genes could be strongly activated by coinfiltration with RsPAP2 and RsTT8. These findings showed the effectiveness of GWAS in identifying candidate genes in radish and demonstrated that RsPAP2 could (either directly or together with its cofactor RsTT8) regulate the transcript levels of ATC-related genes to promote ATC biosynthesis, facilitating the genetic enhancement of ATC contents and other related traits in radish.

Highlights

Radish (Raphanus sativus L.) taproot is a valuable source of phytochemicals such as flavonoids
The RsPAP2 gene exhibited high expression levels in tissues with abundant ATC accumulation, such as the root skin, leaf veins, stem, sepals, petals, and anthers of the red-skinned genotypes (Fig. 2e). These results indicated that the RsPAP2 gene is an important regulator of the red skin color of radish taproots
The results showed that AtC4H, AtCHS, AtANS, and AtTT8 were significantly upregulated in both the roots and leaves of the RsPAP2-OEs compared with the wild-type Arabidopsis plants (WTs), whereas AtDFR was downregulated

Summary

Introduction

Radish (Raphanus sativus L.) taproot is a valuable source of phytochemicals such as flavonoids. Anthocyanins (ATCs) are a subgroup of flavonoids that exhibit strong antioxidant activity that is beneficial for human health[1]. Previous studies have reported that the major components of ATC pigments in red radish epidermal tissue are acylated pelargonidin glycosides with a combination of p-coumaric, ferulic, or malonic acids[2,3]. Pelargonidin exhibits an orange-red hue, and acylation is expected to shift the hue to red and improve stability[2], leading to the formation of red skin in radish. ATCs are synthesized via the flavonoid branch of the general phenylpropanoid pathway[4]. The initial steps of ATC biosynthesis pathway have been isolated and well characterized in plant species such as Arabidopsis, apple, eggplant, and grape[1,7,8,9,10]

Methods

Results

Conclusion