4.3.4. Evaluation of Methylation Level and Distribution

Methylation level was calculated as ML(mC) = reads(mC)/(reads(mC) + reads(C)), where mC is methylcytosine, C is non-methylated cytosine, and ML(mC) is the methylcytosine level. As recommended in a previous study [91], the parameter ML(mC) was corrected to ML(corrected) according to the following formula: ML(corrected) = (ML(mC) − r)/(1 − *r*), where *r* represents the bisulfite non-conversion rate. Methylcytosine sequence contexts—mCG, mCHG, and mCHH (where H represents A, T, or C)—were analyzed. Methylation level densities and methylcytosine distributions in each chromosome and gene functional region (promoter, exon, intron, and 2-kb upstream and downstream regions) were also analyzed [43,92,93]. Differences in global methylation levels and methylcytosine distributions in gene structural regions (including 2-kb upstream and downstream) were compared between samples [72]. To focus on petal color variation, WT1, WT2, and WT3 samples were merged together as three biological replicates of WT; similarly, RT1, RT2, and RT3 served as the three RT biological replicates.
