Distinct DNA methylomes of moso bamboo in the phase transition from vegetative to reproductive growth. Distinct DNA methylomes of moso bamboo in the phase transition from vegetative to reproductive growth

In mammals, it is well known that DNA methylation has important roles during aging. However, age-related DNA methylation changes during phase transitions largely remain unclear in plants. Moso bamboo needs a very long time to transition from vegetative to floral phase. To comprehensively investigate the influence of DNA methylation on aging, we present here a distinctive single-base-resolution DNA methylation profile using both high-throughput bisulfite sequencing (BS-Seq) and single-molecule nanopore-based DNA sequencing, which covers the long period of vegetative growth and transition to flowering of moso bamboo’s development. We discovered that CHH methylation gradually accumulated from vegetative to reproductive growth in a time-dependent fashion. Importantly, DMR (differential DNA methylation) correlating with chronological aging occurred preferentially at both transcription start sites (TSS) and transcription termination sites (TTS). Genes with CG methylation changes showed enrichment of gene ontology (GO) categories in ‘vegetative to reproductive phase transition of meristem’. In addition, In combination with mRNA sequencing (RNA-Seq) revealed that DNA methylation in promoter, intron, and exon has different roles in regulating gene expression. Finally, circular RNA sequencing (circRNA-Seq) revealed that flanking introns of circRNAs show hypermethylation. Further analysis revealed that these flanking introns were enriched in LTR-retrotransponsons. Together, observations in this study provide insights about dynamic DNA methylation and circRNAs landscape with chronological ages, which paves the way to further study the impact of epigenetic factors on flowering in moso bamboo. Overall design: In this study we used Oxford Nanopore Technologies (ONT) to directly identify 5mC by detection of electrical current signals for three week leaf and flower leaf, respectively.

在哺乳动物中，DNA甲基化（DNA methylation）在衰老过程中发挥重要作用已是学界共识。然而，植物在发育阶段转变过程中与年龄相关的DNA甲基化变化仍尚未被充分阐明。毛竹（Moso bamboo）从营养生长向开花阶段的转变需要极长的时间。为全面探究DNA甲基化对衰老的影响，本研究通过高通量亚硫酸氢盐测序（high-throughput bisulfite sequencing, BS-Seq）与单分子纳米孔DNA测序（single-molecule nanopore-based DNA sequencing），构建了覆盖毛竹营养生长全周期及向开花转变过程的单碱基分辨率DNA甲基化图谱。研究发现，CHH甲基化（CHH methylation）随时间进程从营养生长向生殖生长逐渐累积，呈现时间依赖性。尤为重要的是，与时序衰老相关的差异甲基化区域（differential DNA methylation, DMR）优先富集于转录起始位点（transcription start sites, TSS）与转录终止位点（transcription termination sites, TTS）。携带CG甲基化变化的基因，其基因本体（gene ontology, GO）注释显著富集于"分生组织从营养生长向生殖生长转变"功能条目。此外，结合mRNA测序（mRNA sequencing, RNA-Seq）的分析结果显示，启动子、内含子与外显子区域的DNA甲基化在调控基因表达中发挥着不同的作用。最后，环状RNA测序（circular RNA sequencing, circRNA-Seq）分析显示，环状RNA的侧翼内含子呈现高甲基化状态；进一步分析发现，这些侧翼内含子显著富集于LTR逆转录转座子（LTR-retrotransponsons）。本研究的一系列观测结果揭示了随时序年龄变化的动态DNA甲基化与环状RNA调控图景，为进一步探究表观遗传因素（epigenetic factors）对毛竹开花过程的影响奠定了坚实基础。整体实验设计：本研究使用牛津纳米孔技术公司（Oxford Nanopore Technologies, ONT）的测序平台，通过检测电流信号直接识别5-甲基胞嘧啶（5mC），分别对三周龄叶片与花器官叶片进行测序。