Transgenerational Epigenetic and Phenotypic Inheritance Across Five Generations in Sheep

Despite two extensive reprogramming events during early embryogenesis and game-togenesis, epigenetic information can be passed to the next generations, which consti-tutes transgenerational epigenetic inheritance of phenotypes. Considering its utmost importance, there have been few studies focused on the transgenerational effects of di-etary interventions, such as methionine supplementation, in livestock. Using whole-genome bisulfite sequencing, we implemented a single-base resolution differen-tial methylation analysis for the F3 and F4 descendants of control vs. methio-nine-supplemented F0 twin-pair rams. Based on the results of our previous study on F0, F1, and F2 generations, we compared current results of 2,981 and 1,726 differen-tially methylated cytosines (DMCs) as well as 798 and 553 unique differentially meth-ylated genes (DMGs) in F3 and F4, respectively. We identified 41 DMGs that exhibited transgenerational epigenetic inheritance (TEI-DMGs) across four generations and 11 TEI-DMGs across five generations. Finally, we estimated the effect size of F0 diet group on F3 and F4 growth and fertility-related phenotypes, providing evidence for transgenerational effects of diet group accompanying inherited differentially methyl-ated genes. Here, for the first time using gene-level and phenotypic data, we demon-strate that a moderate dietary intervention can exert long-lasting transgenerational effects on offspring phenotypes extending beyond the F2 generation in sheep. Experimental population of F3 (five control vs. five treatment) and F4 (five control vs. five treatment) sheep were descended from F0 (five control vs. 5 treatment) rams that were supplemented with methionine + control diet vs. control diet. Whole genome bisulfite sequencing (WGBS) was performed on sperm samples to identify five generations of differentially methylated genes. Differential methylation cytosines in each generation was identified by a beta binomial regression method. Genomic positions and genes were annotated with Oar_rambouillet v1.0 genome assembly.

尽管在早期胚胎发生与配子发生过程中存在两次大规模的表观重编程事件，表观遗传信息仍可传递给子代，这一过程即为表型的跨代表观遗传（transgenerational epigenetic inheritance）。鉴于其极端重要性，目前针对家畜膳食干预（如甲硫氨酸补充）的跨代表观遗传效应的研究仍较为匮乏。本研究采用全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing）技术，对对照组与甲硫氨酸补充组F0代孪生公羊的F3、F4代后代开展单碱基分辨率的差异甲基化分析。结合本团队此前针对F0、F1及F2代的研究结果，我们对当前F3与F4代的分析数据进行对比：F3代共鉴定得到2981个差异甲基化胞嘧啶（differentially methylated cytosines, DMCs）与798个独特差异甲基化基因（differentially methylated genes, DMGs），F4代则分别包含1726个DMCs与553个DMGs。本研究共鉴定出41个可跨四代传递的跨代表观遗传差异甲基化基因（transgenerational epigenetic inheritance differentially methylated genes, TEI-DMGs），以及11个可跨五代传递的TEI-DMGs。最后，我们评估了F0代膳食组别对F3、F4代生长及繁殖相关表型的效应量，为膳食组别通过遗传差异甲基化基因产生跨代表观遗传效应提供了实验证据。本研究首次结合基因水平与表型数据，证实适度的膳食干预可对绵羊子代表型产生持久的跨代表观遗传效应，且该效应可延伸至F2代之后的子代。本研究的实验群体为：F3代（5只对照组与5只处理组）与F4代（5只对照组与5只处理组）绵羊，其亲本为分别饲喂甲硫氨酸补充日粮与基础日粮的F0代公羊（5只对照组与5只处理组）。我们对精子样本开展全基因组亚硫酸氢盐测序（WGBS），以鉴定五代群体的差异甲基化基因。各代的差异甲基化胞嘧啶均通过β二项式回归法进行鉴定。基因组位置与基因注释均基于Oar_rambouillet v1.0基因组组装版本完成。