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. Overall design: 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.