Transposon-induced methylation of the RsMYB1 promoter disturbs anthocyanin accumulation in red-fleshed radish

Abstract

Red-fleshed radish (Raphanus sativus L.) is a unique cultivar whose taproot is rich in anthocyanins beneficial to human health. However, the frequent occurrence of white-fleshed mutants affects the purity of commercially produced radish and the underlying mechanism has puzzled breeders for many years. In this study, we combined quantitative trait location by genome resequencing and transcriptome analyses to identify a candidate gene (RsMYB1) responsible for anthocyanin accumulation in red-fleshed radish. However, no sequence variation was found in the coding and regulatory regions of the RsMYB1 genes of red-fleshed (MTH01) and white-fleshed (JC01) lines, and a 7372 bp CACTA transposon in the RsMYB1 promoter region occurred in both lines. A subsequent analysis suggested that the white-fleshed mutant was the result of altered DNA methylation in the RsMYB1 promoter. This heritable epigenetic change was due to the hypermethylated CACTA transposon, which induced the spreading of DNA methylation to the promoter region of RsMYB1. Thus, RsMYB1 expression was considerably down-regulated, which inhibited anthocyanin biosynthesis in the white-fleshed mutant. An examination of transgenic radish calli and the results of a virus-induced gene silencing experiment confirmed that RsMYB1 is responsible for anthocyanin accumulation. Moreover, the mutant phenotype was partially eliminated by treatment with a demethylating agent. This study explains the molecular mechanism regulating the appearance of white-fleshed mutants of red-fleshed radish.

Keywords: RsMYB1; (Raphanus sativus); CACTA transposon; DNA methylation; radish; taproot flesh color.

Fig. 1.

Phenotypic comparison of MTH01 (left) and JC01 (right). (A) Seeds. (B) Radicles emerging from germinated seeds. The arrow indicates the accumulation of red pigment at the radicle tip. (C) Cotyledons and hypocotyls. (D) 10-day-old seedlings. (E) 3-week-old roots. (F) 7-week-old roots. (G) Flowering branches. (H) Siliques. (I) Total anthocyanin levels in the root flesh and skin of MTH01, JC01, and F₁ hybrid plants. Data presented are the mean ±SD from three individuals. DW, dry weight; F, flesh; S, skin. (This figure is available in colour at JXB online.)

Fig. 2.

Phenotype and inheritance patterns of the white-fleshed mutant. (A) Root flesh and skin colors in the MTH01, JC01, and F₁ and F₂ hybrid plants. (B) Inheritance patterns of the white-fleshed mutant in segregating populations from the MTH01 × JC01 cross. R/G, red flesh and green skin; R/R, red flesh and red skin; W/G, white flesh and green skin. (This figure is available in colour at JXB online.)

Fig. 3.

Identification of RsMYB1 as a candidate gene responsible for anthocyanin accumulation in red-fleshed radish based on QTL-seq and RNA-seq analyses. (A) Δ(SNP-index) graph from the QTL-seq analysis. The x-axis represents the positions on chromosome R07 and the y-axis represents the Δ(SNP-index). A candidate QTL was identified on chromosome R07 (16.25–18.33 Mb interval), with a Δ(SNP-index) greater than 0.7 (P<0.05). (B) Genetic distances of SNP markers and a candidate gene (red1) on chromosome R07 (16.25–18.33 Mb interval) according to linkage analyses involving 646 F₂ individuals. (C) Comparison of the syntenic regions between chromosome R07 (16.25–17.35 Mb interval) from the XYB36-2 genome and six scaffolds from the Aokubi DH genome based on an online BLAST analysis. (D) Differentially expressed genes (indicated by the vertical bars in panel C) in the candidate region and their relative expression levels based on RNA-seq data. The heatmap was prepared with the MeV4.9.0 program: left, MTH01; right, JC01. (This figure is available in colour at JXB online.)

Fig. 4.

Analysis of cytosine methylation of the RsMYB1 promoter region in MTH01 and JC01. (A) Structural representation and localization of the CACTA transposon inserted at the RsMYB1 locus. (B, C) McrBC-sensitive PCR analysis of the RsMYB1-CACTA promoter region (−46 to −303 bp) and the CACTA transposon region (−1009 to −1789 bp) in MTH01 and JC01. + and − indicate whether genomic DNA was treated with McrBC before PCR amplification. The absence of a PCR product in the McrBC-treated samples indicates the DNA was methylated. M, marker DL2000. (D) Analysis of cytosine methylation of the promoter region (BS5; −176 to −1 bp relative to the RsMYB1-CACTA start codon, ATG) by bisulfite sequencing (location information from Supplementary Fig. S2A). The percentage of methylation of each cytosine in the region is indicated by the vertical bars.

Fig. 5.

Treatment with 5-azaC reversed the mutant phenotype of JC01, likely as a result of demethylation. (A) Phenotypes of 5-azaC-treated individuals (T1–T8) and untreated controls at the germination stage. Arrows indicate the accumulation of red pigment at the radicle tip. (B, C) Phenotypes of 5-azaC-treated individuals at the cotyledon expansion and young seedling stages. (D) Relative expression level of RsMYB1-CACTA in JC01, MTH01, and the 5-azaC-treated samples, based on a qRT–PCR analysis. (E) Total anthocyanin levels in the root flesh and skin of JC01, MTH01, and 5-azaC-treated samples, based on an HPLC analysis. DW, dry weight. In (D) and (E), data presented are the mean ±SD from three technical replicates and different letters indicate significant differences (P=0.05). (This figure is available in colour at JXB online.)

Fig. 6.

Functional complementation analysis of RSMYB1-CACTA by VIGS and Agrobacterium transformation. (A) Seedlings injected with pTRV2-RsMYB1-CACTA + pTRV1 or pTRV2 + pTRV1. WT, wild-type control. (B) Relative expression of RsMYB1-CACTA in the petioles collected from seedlings injected with pTRV2-RsMYB1-CACTA + pTRV1 (V1, V2, and V3) or pTRV2 + pTRV1 (mean ±SD, n=3). (C) Anthocyanin accumulation in 35S:GFP and 35S:RsMYB1-CACTA-GFP transgenic JC01 radish calli. (D) Relative RsMYB1-CACTA expression in the transgenic calli (mean ±SD, n=3). (This figure is available in colour at JXB online.)

Fig. 7.

The phenotype of red-fleshed radish is associated with the insertion of the CACTA transposon in the promoter region of RsMYB1. Molecular structures of RsMYB1-CACTA and RsMYB1 alleles with flanking sequences are presented. (A) The insertion sites upstream of RsMYB1-CACTA and RsMYB1 are indicated for a red-fleshed line (MTH011) and a white-fleshed line (Baiyuchun). (B) Images of 14 radish varieties that varied in terms of flesh color, and results of agarose gel electrophoresis for the PCR-based analysis of the RsMYB1-CACTA downstream and upstream junctions and the RsMYB1 promoter region of each variety. Cox1 served as a reference gene. The 1000 bp and 1800 bp fragments corresponding to the RsMYB1-CACTA downstream and upstream junctions are present in the red-fleshed varieties (lanes 1–6), but not in the non-red-fleshed varieties (lanes 7–14). (C) PCR-based analysis of the inserted CACTA transposon in 111 accessions. The data presented in (B) and (C) are provided in Supplementary Table S2. (This figure is available in colour at JXB online.)