DNA methylation of LDOX gene contributes to the floral colour variegation in peach

Abstract Peach is an important fruit and ornamental plant around the globe. Variegation in flowers often captures consumers’ attention, and variegated plants are of high ornamental value. To determine the relationship between DNA methylation and phenotype, we obtained the first single-nucleotide resolution DNA methylation of variegation cultivars in peach through bisulfite sequencing. In this study, a similar methylation rate of 12.90% in variegated flower buds (VF) and 11.96% in red flower buds (RF) was determined. The methyl-CG (mCG) was the main context in both samples. We identified 503 differentially methylated regions (DMRs) in all chromosomes. These DMRs were focused on 96 genes and 156 promoters. Associated with the transcriptional and proteome analysis, 106 differently expressed genes and 52 different proteins had varying degrees of methylation. Silent genes exhibited higher methylation levels than expressed genes. The methylation state of the leucoanthocyanidin dioxygenase (LDOX) promoter in VF was higher than RF at flower stages 2 (FS2) based on bisulfite sequencing PCR (BSP) results. Moreover, further experiments showed LDOX gene expression and enzyme activity in RF was higher than VF. The DNA methylation trend consisted of the gene expression and flower colour phenotype. Several cis-acting regulatory elements on BSP sequences were involved in phytohormones, transcription factors, and light responsiveness, which could affect gene expression. The higher level of LDOX gene expression promoted synthesis of colourful anthocyanidins, which caused red spots on the petal. Together, this study identified the context and level of methylation at each site with bisulfite sequencing (BS). These results are helpful in uncovering the mechanism of variegated flower petal formation in peach.