*3.2. Methylcytosine Modification Affected the Expression of Anthocyanin-Related Genes*

Cytosine methylation is a common form of DNA modification that is closely interwoven with the process of gene transcription. Although important for the epigenetic regulation of endogenous genes, the extent to which this type of DNA modification regulates genomes remains elusive [59,60]. DNA methylation occurs at higher levels in heterochromatin than euchromatin, and performs specific functions across different species [43,48,61,62]. In this study, we mapped the first-reported methylomes of *P. mume* flower petals using the single base resolution technique. We also compared methylomes and transcriptomes to elucidate the relationship of methylcytosine and gene expression. We discovered that 11.29–14.83% of cytosine sites within genomes were methylated, and the patterns of methylation (mCG, mCHG and mCHH) were similar to those reported in *Arabidopsis* [60,63], rice [64,65], maize [66], soybean [44,67], tomato [43], apple [48], and cotton [45,47]; specifically, the highest and lowest levels of methylation were at the CG and CHH sites, respectively, with significant differences observed within both WT and RT tissues. We also found that the mCG/CG ratios were highest in exon and intron regions; in contrast, mCHG/CHG and mCHH/CHH ratios were lower in the exon and intron regions than in the promoter and TE regions.

Gene transcription is influenced or regulated by DNA methylation [59,68], with methylcytosine levels negatively correlated with gene expression in general [69]. In our study, we observed a similar pattern. We also found that methylcytosine levels of RT samples were lower than those of WT samples, which suggests that anthocyanidin-related genes are suppressed by the high levels of methylcytosine in WT genomes. However, a contradictory result was seen in gene body regions, where methylcytosine levels of hypomethylated CG-DMR-related genes were positively correlated with gene expression. This latter observation is consistent with the finding that genes methylated in transcribed regions are highly expressed and constitutively active in *A. thaliana* [60], and that CG methylation is often linked to increased gene expression [68,70,71].

Next, we searched for DMRs and investigated the expression of DMR-related genes (especially DEGs). We focused on DMRs in functional regions, as gene body methylation is conserved across species among constitutively expressed genes [60,64,71], and because methylation within promoters is highly tissue-specific in nature and strongly associated with transcriptional repression in plants [60,69,72]. We detected 13,468 DMRs associated with 4376 gene bodies (285 DEGs) and 4622 gene promoters (282 DEGs). These associated DEGs were enriched in KEGG terms such as flavonoid biosynthesis, the biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, plant hormone signal transduction, and transcription factor activity. In previous studies, gene methylation has been observed to influence floral pigmentation. For instance, red pigmentation is observed in the flowers of the transgenic petunia line 17-R upon hypomethylation of the 35S promoter driving the *A1* gene [26], while mosaic red anthocyanin in lip crests, sepals, and petals of yellow flowers of *Oncidium* "Gower Ramsey" may be attributed to activation of the *OgCHS* gene resulting from the demethylation of the five-upstream promoter region [27]. As a third example, methylation levels of *MYB* genes are associated with the formation of red-skinned pears and apples [37,38]. Thus, we suggest that the methylation or demethylation of genes participating in the anthocyanin regulation pathway is responsible for flower color chimerism in *P. mume* "Danban Tiaozhi".
