Figure 4
Analysis of single-base resolution methylome.
(a–b), Targeted and whole-genome bisulfite sequencing (TBS, WGBS) reveals methylation changes in specific cytosine residues (a) and the overall Cnr promoter region (b) in the SlCMT-silenced Cnr fruit. Bar-chart shows the methylation levels in the Cnr gene locus in epimutant fruit at breaker stage (Cnr), SlCMT3-silenced Cnr fruit at breaker stage (VIGS) and in wild-type fruit at immature (IM), mature green (MG), breaker (Br), ripening stages (Ripen) and LeMADS-RIN ChIP-Seq (RIN binding). The location of the two differentially methylated regions (DMR1 and DMR2) and the epi-allele in the promoter region of Cnr are shown. (c–d), Genome-wide hypomethylation caused by SlCMT3 silencing. Kernel density plots of the loss of CG (c), CHG (d) and CHH (e) methylation in the SlCMT3-silenced Cnr fruit at breaker stage. Methylation differences (methylation level of Cnr minus SlCMT3 silenced Cnr fruit at breaker stage) of the whole-genome (bin = 1000 bp), annotated gene regions, repeats and the LeMADS-RIN bindings sites are shown and regions with zero methylation are discarded29. (f), SlCMT3 silencing causes global demethylation in Cnr fruit. Boxplot showing the delta-methylation levels of Cnr and SlCMT3-silenced fruits at the breaker stage. For calculation of the global methylation delta, genome is divided into 200-bp bins and the methylation levels of each bin are calculated. Gene and the repeat are defined according to the ITAG v2.5 annotation. RIN binding sites are called as previously described29.
