*4.5. WGBS and Data Analyses*

Genomic DNA was extracted from 10 day-old seedlings and shipped to the Anoroad Genome Company (Beijing, China) for WGBS. WGBS library preparation and sequencing was performed by the Anoroad Genome Company (Beijing, China).

After downloading, the raw reads were filtered and trimmed to obtain clean reads and the available data were compared with the reference genome of *Oryza\_sativa Japonica* IRGSP-1.0 to obtain the alignment results using Bismark (v0.9.0) [65]. The uniquely mapped reads will be used to call the methylated cytosines (mC) in highly enriched regions. For each cytosine site, the methylation level (%) was calculated by: 100 × (reads-supported methylation)/(total reads depth for the site). For the methylated region, the methylation level (%) was calculated by: 100 × methylation level of all cytosine sites in the region / the total number of cytosine sites in the region. The conversion ratio of Lambda DNA was around 99.5% to 99.6% in all samples. Valid coverage of methylated cytosine is the percentage of methylated C among all C on the reference genome, is associated with methylation level. C sites with read depths less than 5 are eliminated firstly. Then, for each C site, valid coverage is calculated by for all C or C within each pattern (CG, CHG and CHH). Coverage is the count of C sites with more than 5 depth divided by total number of C site by its pattern. Normalized mC number (Figure 1, Table 1) was calculated by: total mC number/valid coverage of methylated cytosine. Relative data are listed in Supplementary Table S7.

The differential analysis of methylation was performed at the region-base level, differentially methylated regions (DMRs). Aberrant DNA methylation was compared with the control group, an increased methylation pattern was defined as hyper-methylation, whereas a decreased methylation pattern was defined as hypo-methylation. DMR detection was performed using a methylKit and eDMR package [66] to estimate the boundary of CpG islands with a mixed bimodal normal distribution assumption of the distance between CpGs.
