Genome-wide Methylation Regulation Analysis Of Skeletal Muscle Phenotypic Differences In Different Pig Breeds

The growth rate and skeletal muscle phenotype of different pig breeds show significantlydifference. In this study, to explore the molecular mechanisms of skeletal muscle development indifferent pig breeds, we selected the adult skeletal muscles of Tongcheng (obese-type), Landrace(lean-type) and Wuzhishan pigs (mini-pig) as the objects of our study. MeDIP-seq technology was usedto analyse the genome-wide methylation profiles of the adult skeletal muscle of Tongcheng, Landraceand Wuzhishan pigs. The regulatory role of methylation to skeletal muscle development had beenanalyzed. RNA-seq technology was used to analyse the gene expression profiles of the adult skeletalmuscle of Tongcheng, Landrace and Wuzhishan pigs. The gene expression pattern differences indifferent pig breeds were analyzed. Combined the MeDIP-seq and RNA-seq data, we analyzed theregulation role of methylation to the gene expression level, and further study the regulatory mechanismof methylation to the pig skeletal muscle development. At the same time, bisulfite sequencing PCR(BSP) and real-time quantitative PCR (qPCR) methods were used to validate the results ofhigh-throughput sequencing data.The results of MeDIP-seq showed that there were38,327,405(62.6%),41,130,862(67.18%)38431456(62.77%) reads could be uniquely mapped to the pig reference genome (Sscrofa10.2) inTongcheng, Landrace and Wuzhishan pigs, respectively. And these three pig breeds had similargenome-wide methylation profiles. We observed that methylation level correlates negatively withchromosome length (P=0.001) and positively with repeat elements density (P <0.05)and gene density(P <0.05). The uniquely mapped reads were mainly distributed in the repeat and intron regions, reads inthe repeat elements mainly concentrated in the SINE/tRNA-Glu and LINE/L1elements, accounted for79%of the total repeat reads. About8.35%,10.06%and8.79%of CpGIs were methylated inTongcheng, Landrace and Wuzhishan pigs, respectively. Compared these three pig breeds with eachother, there were5437differentially methylated genes (DMGs) between Tongcheng and Landrace pigs,5031DMGs between Tongcheng and Wuzhishan pigs,5884DMGs between Landrace and Wuzhishanpigs. Of which there were2203genes were differentially methylated among all these three pig breeds.These differentially methylated regions (DMRs) were mainly distributed in the CDS and intron regions.Ssc-mir-21had a DMR among all these three breeds. And ssc-mir-127had a DMR between Landraceand Tongcheng pigs and between Landrace and Wuzhishan pigs. Results of KEGG pathway analysisindicated that these DMGs were mainly involved in inositol phosphate metabolism, vascular smoothmuscle contraction, phosphatidylinositol signaling pathway and so on. The BSP results were consistentwith the MeDIP-Seq results, shown that our MeDIP-seq results were reliable.The results of RNA-seq showed that there were6,340,537(54.16%),6,490,599(55.57%),6,853,339(55.79%) reads could be uniquely mapped to the pig reference gene (Sscrofa10.2) inTongcheng, Landrace and Wuzhishan pigs, respectively. Compared these three pig breeds with eachother, there were43differentially expression genes (DEGs) between Tongcheng and Landrace pigs,92 DEGs between Tongcheng and Wuzhishan pigs,145DEGs between Landrace and Wuzhishan pigs.Four genes were differentially expressed in all these three pig breeds, including PDK4, UCP3, GLUL,NREP. Clustering analysis of gene expression patterns showed that Tongcheng and Landrace pigs had amore similar gene expression pattern than Wuzhishan pigs. GO and KEGG pathway analysis showedthat these DEGs significantly enriched in muscle development and fatty acid metabolism-relatedbiological processes and pathways. the qPCR results of14genes related to skeletal muscle developmentwere in good agreement with the RNA-seq data. The correlation coefficient were0.871(P <0.01).Combined the MeDIP-seq and RNA-seq results showed that there were8differentially methylatedand expressed gene between Tongcheng pigs and Landrace,23differentially methylated and expressedgene between Tongcheng and Wuzhishan pigs,50differentially methylated and expressed genebetween Landrace and Wuzhishan pigs. These differentially genes might be related to the pig musclegrowth and development and regulated by methylation.