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.

中文翻译：

---

LDOX基因的DNA甲基化导致桃花色变异

桃是全球重要的水果和观赏植物。花卉的杂色往往会引起消费者的注意，杂色植物具有很高的观赏价值。为了确定DNA甲基化与表型之间的关系，我们通过亚硫酸氢盐测序获得了桃花品种的第一个单核苷酸分辨率DNA甲基化。在这项研究中，确定了类似的甲基化率，杂色花蕾 (VF) 为 12.90%，红色花蕾 (RF) 为 11.96%。甲基-CG (mCG) 是两个样品中的主要背景。我们在所有染色体中鉴定了 503 个差异甲基化区域 (DMR)。这些 DMR 集中在 96 个基因和 156 个启动子上。与转录和蛋白质组分析相关，106 个不同表达的基因和 52 个不同的蛋白质具有不同程度的甲基化。沉默基因表现出比表达基因更高的甲基化水平。根据亚硫酸氢盐测序 PCR (BSP) 结果，VF 中无色花青素双加氧酶 (LDOX) 启动子的甲基化状态在花期 2 (FS2) 时高于 RF。此外，进一步的实验表明，RF 中的 LDOX 基因表达和酶活性高于 VF。DNA甲基化趋势由基因表达和花色表型组成。BSP 序列上的几个顺式作用调控元件与植物激素、转录因子和光反应有关，它们可能影响基因表达。高水平的LDOX基因表达促进了彩色花青素的合成，从而导致花瓣上出现红斑。一起，该研究通过亚硫酸氢盐测序 (BS) 确定了每个位点的甲基化背景和水平。这些结果有助于揭示桃杂色花瓣形成的机制。