Comparative Analyses Of The Whole Methylome And Transcriptome Of Muscle Of Broilers And Laying Hens At Embryonic Developmental Stages

After the animal is born,the number of muscle fibers is almost constant,indicating that the postnatal muscle development is closely related to the developmental pattern of muscle tissue in the embryonic stage.The main muscle fibers of birds are formed at the sixth day of incubation whereas the secondary muscle fibers are mainly formed during the 12-16 days of embryonic development.The differentiation of muscle fibers is mainly completed at about 3/4 of the entire incubation period.Muscle development in animal embryos is a complex biological process regulated by cell cycle,protein factor interaction and related gene expression,as well as multiple signaling pathways.In the post-genome era,epigenetic modifications such as DNA methylation and non-coding RNA were found to regulate skeletal muscle myogenesis.After long-term artificial breeding,layers and broilers exhibits significant difference in muscle growth.Due to the similar genetic background,layers and broilers are ideal materials for comparative study of muscle development.At present,although several studies in the epigenetic regulation mechanism of skeletal muscle growth in chicken embryos has been reported,they mainly study using the single omics level.The mechanism of interactional regulation between DNA methylation and lncRNA has rarely been reported.In this study,ROSS-308 broilers and WhiteLoghorn layers were used as the comparative study models.Single-resolution whole-genome methylation and RNA-seq were performed using the breast muscles of chick at different developmental time points（E10,E13,E16 and E19）.Association analysis between methylome and transcriptome was performed and the molecular regulatory network was constructed,aiming at deciphering the regulation mechanism of epigenetic modification on skeletal muscle growth in chicken embryos.There are several main conclusions below:（1）The layers and broilers exhibit morphological differences during the embryonic development.The broilers have the bigger body size and the more number of muscle fibers compared to the layers.（2）Among 24 samples of WGBS libraries,the average sequencing depth is 30.3×.A total of 3.4 billion reads were generated and 71.99%of the clean reads was aligned on the reference genome of chicken.More than 75%genomic region was sequenced at least 10×.（3）The differential methylated regions between two chicken lines are mainly distributed in intron and intergenic regions and most of the differential methylated regions are hypo-methylated for broilers.Taking the CG context which is predominate in animal as an example,the hypo-methylated regions account for80.53%,83.65%,66.22%and 82.66%of the differential methylated regions at 4developmental time points,respectively.（4）The differential methylated genes are mainly enriched in muscle development and nerve development related biological processes such as muscle cell development,muscle organ development,neuromuscular process controlling balance and positive regulation of nervous system.（5）A total of 20656 lncRNA was predicetd in the RNA-seq,of which most are intergenic lncRNA（lincRNA,63.6%）and some are Antisense-lncRNA（20.9%）.The lncRNAs show the higher methylation level around the transcription start site（TSS）and lower expression level,compared to the known coding genes.（6）Through classifying the genes by their expression level,the methylation level of TSS region of genes was found to have a negative correlation with the mRNA expression.Besides,the similar negative correlation was found for lncRNAs between their TES region and expression level.（7）The identified lncRNA MyH1-AS shows a high negative correlation with the DMR in its intron region(ρ=-0.75,P<10^-4)and has a high positive correlation with its predicted target gene MyH1E(ρ=0.94,P<10^-5).The WGCNA analysis reveals that this lncRNA may participate in the embryonic muscle development.（8）MyH1-AS Silencing in skeletal muscle satellite cells for 24h resulted in the downregulation of mRNA and protein expression level of muscle related genes like MyoD1,MyH3 and MyoG,indicating that lncRNA play a part in growth of muscle satellite cells in chicken.In summary,the lower methylation level on the genomic regions may contribute to the faster muscle development of the broilers compared to the layers.LncRNA MyH1-AS is influenced by DNA methylation and participate in embryonic skeletal muscle development in chicken.