Genome Wide Methylation Analysis of Organellar DNA in MR219 Indica Rice Cultivar

Purpose: The chloroplast DNA has not been primiarly analyzed in rice plants before. Hence, the objective of this study is to analyze and compare the differential methylation of chloroplast DNA in MR219 indica rice across different tissues and different developmental stages. Methods: We prepared a total of nine sodium bisulfite treated DNA libraries from three developing grain tissues, three leaf tissues at ripening stage and three leaf tissues at vegetative stage and sequenced them in Illumina Miseq platform. We performed quality trimming, alignment followed by methylation calling and differential methylation analysis using Trimmomatic v36, Bismark v16.3 and SeqMonk v40.0 on the sequencing data obtained. Statistical analysis was carried out in SeqMonk software and further validated in SPSS statistical software v22.2. Results: With an optimized data analysis workflow, we mapped around average of 26000 reads to chloroplast genome. Differential CpG and CHG methylation in SeqMonk v40.0 revealed that MR219 chloroplast DNA is differentially methylated in grain and leaf tissues and across vegetative stage and ripening stage in the leaf tissues. Chloroplast DNA from leaf at ripening stage was most methylated, followed by grain tissue and lastly leaf tissue from vegetative stage. Conclusions: Overall, it can be concluded that the organellar DNA in MR219 rice are differentially methylated at different tissues and across different developmental stages. The chloroplast DNA was most methylated in the leaf at ripening stage, followed by grain at ripening stage and leaf at vegetative stage. The functional significance of the differential methylation observed in this study needs to be investigated. MR219 rice developing grain at ripening stage, leaf tissue at ripening stage and leaf tissue at vegetative stage sequencing data were generated from Illumina Miseq in triplicates.

研究目的：此前尚未针对水稻植株的叶绿体DNA开展系统性初步分析。因此本研究的目标为分析并比较MR219籼稻在不同组织及不同发育阶段下的叶绿体DNA差异甲基化情况。
研究方法：本研究共构建9份亚硫酸氢盐处理的DNA文库，样本分别为3份发育中籽粒组织、3份成熟期叶片组织及3份营养生长期叶片组织，随后在Illumina Miseq平台完成测序。针对获取的测序数据，我们分别使用Trimmomatic v36、Bismark v16.3及SeqMonk v40.0完成质量修剪、序列比对、甲基化位点识别及差异甲基化分析。统计分析借助SeqMonk软件完成，并通过SPSS v22.2统计软件进行验证。
研究结果：通过优化后的数据分析流程，我们平均将约26000条读段比对至叶绿体基因组。利用SeqMonk v40.0进行的CpG与CHG位点差异甲基化分析显示，MR219的叶绿体DNA在籽粒与叶片组织间，以及叶片组织的营养生长期与成熟期之间均存在差异甲基化现象。其中，成熟期叶片的叶绿体DNA甲基化水平最高，其次为籽粒组织，营养生长期叶片的甲基化水平最低。
研究结论：综合来看，本研究证实MR219水稻的细胞器DNA在不同组织及不同发育阶段中存在差异甲基化现象。其中，成熟期叶片的叶绿体DNA甲基化水平最高，其次为成熟期籽粒组织，营养生长期叶片的甲基化水平最低。本研究中观测到的差异甲基化现象的生物学意义仍有待进一步探究。本研究中MR219水稻发育中籽粒、成熟期叶片及营养生长期叶片的测序数据均通过Illumina Miseq平台完成，且设置3次生物学重复。