Genome-wide Methylation And Transcriptome Study And Differential MiRNA Molecular Function Verification In Different Stages Of Chicken Embryo Leg Muscle Development

As an important part of China’s meat consumption structure,chicken’s growth rate is also the focus of research in poultry genetics and breeding.As an excellent local breed in Sichuan,Jiuyuan black chicken has tight meat quality,fine muscle fibers and well-developed leg muscles,but its growth rate,breed selection and breeding progress is slow.Therefore,analyzing the molecular regulation mechanism of the muscle growth and development of Jiuyuan black chicken is an important breakthrough for its molecular breeding improvement.In this study,we selected the leg muscles at different stages of embryonic development in Jiuyuan black chicken as the research materials,and used whole-genome methylation and transcriptome technology to study the DNA methylation level and RNA expression changes of leg muscles at different developmental stages.In order to screen of differentially methylated genes,differentially expressed genes and differentially expressed mi RNAs that regulate growth and development at different stages.Through the joint analysis of methylome and transcriptome data,study the differences in methylation-mediated gene expression and the regulation of mi RNA and target genes.Finally,perform a preliminary study on the functions of genes that may be related to muscle development in skeletal muscle satellite cells.It provides a theoretical basis for revealing the genetic mechanism of the growth and development of leg muscles in the embryonic period of Jiuyuan black chickens and the development mechanism of muscle fibers in high-quality local meat legs.The results are as follows:(1)Through the analysis of the morphological characteristics of the leg muscles in the embryonic stage of the Jiuyuan black chicken and the m RNA expression pattern of muscle development-related genes,it is speculated that the key period of leg muscle development in the embryonic stage is divided into: myoblast proliferation(E11),myotube period(E17)and muscle fiber period(D1).(2)Many differentially expressed genes and mi RNAs were detected during the development of leg muscles in the embryonic stage of Jiuyuan black chickens,among which a large number of differential mi RNAs,such as let-7 family,mi R-106-5p,mi R-206,mi R-133 family,etc.are involved in the regulation of leg muscle development in the early and mid embryonic stages..Further joint analysis found that there are more differential genes in the MAPK signaling pathway,Wnt signaling pathway,starch and sucrose metabolism,actin cytoskeleton regulation,and TGF-β signaling pathway enrichment,suggesting that these pathways may regulate the development of chicken embryonic leg muscles.Important signal pathway.(3)A single-base resolution methylation map of leg muscles in the embryonic stage of the Jiuyuan black chicken was drawn,which laid the foundation for exploring the DNA methylation regulation mechanism during the embryonic stage in Jiuyuan black chicken.The analysis of the distribution of basement regions and the biological functions of differentially methylated genes demonstrated the importance of promoter methylation regulation once again.At the same time,differentially methylated genes are significantly enriched in calcium signaling channels,Notch signaling pathways,Wnt signaling pathways,m TOR signaling pathways and MAPK signaling pathways are all closely related to growth and development.It is speculated that DNA methylation may regulate the growth and development of chicken embryonic leg muscles through these signaling pathways.(4)Through the association analysis between methylation and transcriptome,it is proved that the methylation intensity of the promoter region and the expression of genes are highly negatively correlated again,and DNA methylation may be affected muscle fiber development by regulating the expression of Myo G、FGF2、MEF2D、SMTN、CFL2 and TCF12 genes.(5)Studies have found that mi R-106-5p can promote the proliferation of chicken skeletal muscle satellite cells and inhibit their apoptosis;CFL2,as a negative regulator of chicken skeletal muscle satellite cell proliferation,promotes apoptosis;mi R-106-5p can target CFL2 to regulate cell function,and CFL2 may regulate the function of chicken skeletal muscle satellite cells through metabolism-related signal pathways.To sum up,this study explored the differences in methylation level,gene expression and mi RNAs expression in different stages of embryonic leg muscle development of Jiuyuan black chickens at the tissue and cell levels,and the influence of methylation on muscle development.The function research of the key regulatory genes and mi RNAs related to methylation will provide reference materials for the comprehensive analysis of the regulation mechanism of skeletal muscle muscle fiber development of high-quality local chickens,and provide a theoretical basis for further improving the growth and development speed of highquality local chickens.