*2.4. DMR-Related Genes and Bicolored Flowers on Individual Trees*

DMRs can be used to identify methylation differences between individuals or developmental stages and their involvement in gene transcriptional regulation [50,51]. In the present study, 13,468 DMRs—10,121 hypermethylated and 3347 hypomethylated—were predicted between WT and RT types using DSS software. These DMRs, whose lengths were normally distributed (Figure S8a), were methylated at levels of approximately 38.5% (CG), 31.5% (CHG), and 8.4% (CHH) (Figure S8b). DMR chromosomal distributions and levels of significance are displayed in Figure 4a and Figure S8c. In each gene functional region (except for other regions in CG and CHG contexts), the number of DMRs between WT and RT exhibiting hypermethylation was higher than those with hypomethylation. In the CHH context, many more hypermethylated DMRs were detected than hypomethylated ones, and the TSS and TES regions contained few DMRs (Figure 4b). Heat maps of methylation levels of CG, CHG, and CHH DMRs revealed that variation was present in the methylation of WT and RT samples (Figure 4c). As shown in Figure 4d, these DMRs were overlapped with 4376 gene bodies and 4622 gene promoters. A total of 80 genes containing mCG, mCHG, and mCHH sites in their transcribed region were detected; similarly, 57 gene promoters were predicted containing all three types of methylated sites. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 7 of 27 DMR chromosomal distributions and levels of significance are displayed in Figure 4a and Figure S8c. In each gene functional region (except for other regions in CG and CHG contexts), the number of DMRs between WT and RT exhibiting hypermethylation was higher than those with hypomethylation. In the CHH context, many more hypermethylated DMRs were detected than hypomethylated ones, and the TSS and TES regions contained few DMRs (Figure 4b). Heat maps of methylation levels of CG, CHG, and CHH DMRs revealed that variation was present in the methylation of WT and RT samples (Figure 4c). As shown in Figure 4d, these DMRs were overlapped with 4376 gene bodies and 4622 gene promoters. A total of 80 genes containing mCG, mCHG, and mCHH sites in their transcribed region were detected; similarly, 57 gene promoters were predicted containing all three types of methylated sites.

**Figure 4.** Distribution, methylation level, and predicted genes of differentially methylated regions (DMRs) in white petal tissues (WT) and red petal tissues (RT) samples. (**a**) Circos plots of CG, CHG, and CHH DMRs and transposable element (TE) and gene densities on each chromosome of *Prunus mume*. Track order (outside to inside) is as follows: scatter plot of hypermethylation (Hyper); TE density (TE); gene density (Gene); and scatter plot of hypermethylation (Hypo). Red, blue, and purple dots indicate CG, CHG, and CHH DMRs, respectively. (**b**) Distribution of CG, CHG, and CHH DMRs within gene functional regions. TSS, transcriptional start site; TES, transcriptional end site. (**c**) Heat maps of methylation levels of CG, CHG, and CHH DMRs. (**d**) Venn diagrams of predicted genes linked with CG, CHG, and CHH DMRs. "Genebody" and "promoter" indicate predicted genes anchored within gene body and promoter regions, respectively. Following DMR detection, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and **Figure 4.** Distribution, methylation level, and predicted genes of differentially methylated regions (DMRs) in white petal tissues (WT) and red petal tissues (RT) samples. (**a**) Circos plots of CG, CHG, and CHH DMRs and transposable element (TE) and gene densities on each chromosome of *Prunus mume*. Track order (outside to inside) is as follows: scatter plot of hypermethylation (Hyper); TE density (TE); gene density (Gene); and scatter plot of hypermethylation (Hypo). Red, blue, and purple dots indicate CG, CHG, and CHH DMRs, respectively. (**b**) Distribution of CG, CHG, and CHH DMRs within gene functional regions. TSS, transcriptional start site; TES, transcriptional end site. (**c**) Heat maps of methylation levels of CG, CHG, and CHH DMRs. (**d**) Venn diagrams of predicted genes linked with CG, CHG, and CHH DMRs. "Genebody" and "promoter" indicate predicted genes anchored within gene body and promoter regions, respectively.

Genomes (KEGG) annotations were performed to explore the functions of DMR-related genes. These analyses uncovered the enrichment of 896 CG–DMR-anchored genes (543 hypermethylated and 361 hypomethylated), 704 CHG–DMR-anchored genes (432 hypermethylated and 288 hypomethylated),

Following DMR detection, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed to explore the functions of DMR-related genes. These analyses uncovered the enrichment of 896 CG–DMR-anchored genes (543 hypermethylated and 361 hypomethylated), 704 CHG–DMR-anchored genes (432 hypermethylated and 288 hypomethylated), and 3777 CHH–DMR-anchored genes (2531 hypermethylated and 292 hypomethylated). In addition, 696, 654, and 3292 genes with promoters overlapping with CG, CHG, and CHH DMRs, respectively, were found to be enriched. The identified genes have important molecular functions in various biological processes, especially phenylalanine metabolism and the biosynthesis of phenylpropanoids, carotenoids, flavonoids, and plant hormones. Since these are critical processes in flower color formation, their over-representation among DMR-anchored genes suggests that variation in methylation levels of DMRs affected the color of WT and RT samples (Figures S9–S12).
