DNA methylation dynamics during cotton ovule and fiber development [Bisulfite-Seq]

DNA methylation is essential for plant and animal development. In plants, methylation occurs at CG, CHG, and CHH (H = A, C or T) sites. CHH methylation is established by the small RNA-directed DNA methylation (RdDM) pathway. Cotton is an allotetraploid consisting of two progenitor genomes, and each cotton fiber is a rapidly-elongating cell from the ovule epidermis. Here we show that inhibiting DNA methylation impairs fiber development. Genome-wide bisulfite -, mRNA-, and small RNA-sequencing analyses reveal that CHH hypermethyaltion through RdDM in euchromatin is associated with expression changes of nearby genes in ovules. The ovule-derived fiber cells not only maintain euchromatic CHH hypermethylation, but also generate additional heterochromatic CHH hypermethylation independent of RdDM. Moreover, CHG and CHH methylation in promoter and transcribed regions contribute to the expression bias of homoeologous genes in the allotetraploid cotton. This epigenetic and expression dynamics of developmental regulation could provide a molecular basis for natural selection and domestication of plants and animals. Overall design: Libraries for MethylC-seq with two replicates, small RNA-seq with three replicates and mRNA-seq with two replicates were constructed using leaves, ovules at 0 DPA and 14 DPA, fibers at 14 DPA of Gossypium hirsutum cv. TM-1. These libraries were sequenced using Hiseq 2500.

DNA甲基化（DNA methylation）对动植物发育至关重要。在植物中，甲基化修饰发生于CG、CHG及CHH（H代表A、C或T）位点。CHH甲基化由小RNA导向的DNA甲基化（small RNA-directed DNA methylation, RdDM）通路介导建立。棉花为异源四倍体物种，包含两套祖先基因组，而每一根棉纤维均为源自胚珠表皮的快速伸长细胞。本研究发现，抑制DNA甲基化会损害棉纤维的正常发育。全基因组亚硫酸氢盐测序、mRNA测序及小RNA测序分析显示，常染色质中通过RdDM通路产生的CHH超甲基化，与胚珠内邻近基因的表达变化显著相关。源自胚珠的纤维细胞不仅维持常染色质的CHH超甲基化状态，还可产生不依赖RdDM通路的额外异染色质CHH超甲基化修饰。此外，启动子区与转录区域内的CHG及CHH甲基化，会影响异源四倍体棉花中部分同源基因的表达偏倚。这种调控发育过程的表观遗传与表达动态特征，可为动植物的自然选择与驯化提供分子基础。
总体实验设计：以陆地棉（Gossypium hirsutum cv. TM-1）的叶片、开花后0天（0 DPA）和开花后14天（14 DPA）的胚珠、开花后14天的棉纤维为实验材料，构建了含2个生物学重复的MethylC-seq文库、3个生物学重复的小RNA测序文库以及2个生物学重复的mRNA测序文库，随后采用HiSeq 2500测序平台完成测序。