Genome-wide analysis of DNA methylation in photoperiod- and thermo-sensitive male sterile rice Peiai 64S

Abstract

Background: Epigenetic modifications play important roles in the regulation of plant development. DNA methylation is an important epigenetic modification that dynamically regulates gene expression during developmental processes. However, little studies have been reported about the methylation profiles of photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice during the fertility transition.

Results: In this study, using methylated DNA immunoprecipitation sequencing (MeDIP-seq), the global DNA methylation patterns were compared in the rice PTGMS line PA64S under two different environments (different temperatures and day lengths). The profiling of the DNA methylation under two different phenotypes (sterility and fertility) revealed that hypermethylation was observed in PA64S (sterility), and 1258 differentially methylated regions (DMRs) were found between PA64S (sterility) and PA64S (fertility). Twenty differentially methylated genes of them were further validated through bisulfite sequencing, and four of these genes were analyzed by qRT-PCR. Especially, a differentially methylated gene (LOC_Os08g38210), which encoded transcription factor BIM2, is a component of brassinosteroid signaling in rice. The hypermethylated BIM2 gene may suppress some downstream genes in brassinosteroid signaling pathway, and thus affect the male fertility in PA64S.

Conclusions: The results presented here indicated that hypermethylation was observed in PA64S (sterility). Gene Ontology (GO) analysis and KEGG analysis revealed that flavone and flavonol biosynthrsis, circadian rhythm, photosynthesis and oxidative phosphorylation pathways were involved in sterility-fertility transition of PA64S.

Figure 1

Cytological observation of pollen morphology. (a) and (c), the anther phenotypes of PA64S under two environmental conditions. (b) and (d) 1% I₂-KI staining of the pollen grains of PA64S under two environmental conditions.

Figure 2

Distribution of reads around CpG Islands and gene body. (a) Distribution of reads around CpG Islands; (b) Distribution of reads around gene body. The upstream and downstream 2 kb regions were split into 20 equal regions. In the gene body, each gene was split into 40 equal regions. For each region, the normalized number of reads was calculated. The “Y” axis is the average of the normalized depth for each region.

Figure 3

Different distributions of PA64S(S) and PA64S (F). (a) Peak distribution in different components of the PA64S(S) and PA64S (F) genome. (b) Distribution of methylated peaks of different repeat types in PA64S (S) and PA64S (F). (c) Distribution of CpG (O/E) in PA64S(S) and PA64S (F).

Figure 4

Boxplots showing the 5mC content (read count, y axis) in 1 kb tiled windows for PA64S(S) and PA64S (F). (a) Total reads of PA64S(S) and PA64S (F); (b) Reads for each elements of PA64S(S) and PA64S (F). The asterisks indicate significant differences between PA64S(S) and PA64S (F), as determined by Student’s t test (**P < 0.01, ***P < 0.001).

Figure 5

DNA methylation patterns of four selected DMR-associated genes validated by bisulfite sequencing. (a) LOC_Os10g10560 (invertase/pectin methylesterase inhibitor, IPMI); (b) LOC_Os05g12210 (chalcone synthase); (c) LOC_Os02g17780(ent-kaurene synthase, chloroplast precursor); and (d) LOC_Os12g08770 (photosystem I reaction center subunit N). The red, green and blue columns in the histograms refer to the collective methylation levels (in percentage) of CG, CHG, and CHH, respectively.

Figure 6

DNA methylation patterns and gene expression of the four selected DMR-associated genes. (a) Bisulfite sequencing and qRT-PCR analysis of LOC_Os03g27770 (heme oxygenase 2); (b) Bisulfite sequencing and qRT-PCR analysis of LOC_Os03g51200 (H2A); (c) Bisulfite sequencing and qRT-PCR analysis of LOC_Os06g16270 (heat shock factor binding protein 2); and (d) Bisulfite sequencing and qRT-PCR analysis of LOC_Os08g38210 (transcription factor BIM2). The red, green and blue columns in the histograms refer to the collective methylation levels (in percentage) of CG, CHG, and CHH, respectively.

Figure 7

The genomic organization of the rice OsBIM2 (LOC_Os08g38210) loci is compared to those of the BIM1 , BIM2 and BIM3 genes in Arabidopsis. Note: This genomic organization is modified on the basis of Xing et al., 2013 [30].

Figure 8

Expression analysis of genes related to BR signaling pathway.

Figure 9

Gene categories and distribution of differential methylated genes between PA64S(S) and PA64S (F).