Study On Differential Methylation Of Genes In Longissimus Dorsi Muscle Of Wannanhua Pig And Yorkshire Pig Based On MeDIP-Seq Technology

Represented by fat and muscle,meat quality traits,are important production economic indicators of animal farming industry.Different individual pigs vary widely in growth rate and skeletal muscle phenotype due to the breed,growth stage,living environment,and gender.In the present study,Wannanhua pigs(fat type)and Yorkshire pigs(lean)were selected to investigate the molecular and regulation mechanisms of DNA methylation in affecting the longissimus muscle development of different breeds.The main objective was to screen the candidate genes for the key functions,namely meat quality to provide a theoretical basis for the further study on pig intramuscular fat deposition.By MeDIP-Seq technique,genome-wide DNA methylation sequencing was obtained for the longissimus muscles from,3 Wannanhua pigs(WH-1,WH-2 and WH-3)and 3Yorkshire pigs in adulthood(YY-1,YY-2 and YY-3).The methylation patterns were analyzed and certificated.GO functional annotation and KEGG Pathway analysis were conducted to determine the candidate genes and regulatory pathways related to the formation and decomposition of fatty acids.The molecular mechanisms of DNA methylation in regulating the formation of meat quality traits were elucidated preliminarily.Then,the methylation levels identified by MeDIP-seq were validated by the pyrosequencing.By a conjoint analysis with MeDIP-seq and high-throughput transcriptome sequencing(RNA-seq),those genes presenting difference in both DNA methylation and RNA expression were identified.The main results obtained in this study:(1)A total of 33,179,625,37,610,867,and 32,924,118 effective resds were generated for WH-1,WH-2,and WH-3,respectively,and 31,634,309,31,056,478,and 27,457,277 effective resds for YY-1,YY-2,and YY-3,respectively.After the removal of low-quality reads,an average of 30.8 Gb of clean reads were generated from these 6 samples,with an effective reads ratio ranging from 98.84% to 98.96%.Then the total reads were mapped to the reference genome,of which 77.73%-85.65% were uniquely mapped according to Sscrofa 10.2.(2)By MeDIP-Seq sequencing application,the peak numbers generated were WH-1120,443,WH-2 206,080,WH-3 193,398,YY-1 194,507,YY-2 223,008,YY-3 171,769.From all peaks obtained,58,283 DMRs were determined,12086 of which were hypermethylated with the remaining of hypomethylated.(3)The results shows that the degree of methylation of the transcription start site(TSS)was less than the gene bady and the transcription termination site(TTS).The degree of methylation of peaks approximately follows: intergenic > introns?CDS > TSS?TTS >5 'UTR?3' UTR.(4)By analyzing the sequencing data,1425 differentially methylated genes were screened,447 of which were up-regulated and 978 of which down-regulated.(5)346 terms(P < 0.05)were found to be involved in 3 aspects: wherein 40 were related to the cellular components(11.56%);54 related to the molecular function(15.61%);and252related to the biological processes(72.83%).It was revealed that the main differences between the formation of meat quality traits for Wannanhua and Yorkshire pigs were due to the fatty acid metabolic process,fatty acid biosynthetic process,lipid metabolic process,lipid decomposition process,and the process of cellular protein metabolism.(6)KEGG Pathway analysis indicated that these methylated genes were enriched in 277 predicted pathways(P<0.05),and 24 terms involved in the PPAR signaling pathway,MAPK signaling pathway,adipocytokine signaling pathway,MAPK signaling pathway,glycerol metabolism,insulin signaling pathway,digestion and absorption of protein,and cAMP signaling pathways.(7)By a conjoint analysis with MeDIP-seq and RNA-seq,those genes presenting difference in both DNA methylation and RNA expression.There is a negative correlation relationship in 80 genes.RXRG?GPI?CPT2 and GABARAPL1 is signficantly negative correlation.(8)7 candidate genes fromrelated to meat quality traits were selected from DMGs and validated by the pyrosequencing.The results from pyrosequencing were in good agreement with that from MeDIP-seq and showed significant differences in the methylation patterns,Indicating the accuracy,reliability,and efficiency of MeDIP-seq sequencing.In summary,the present study conducted a comprehensive analysis of porcine epigenetics by examining genome-wide DNA methylation patterns from two pig breeds.It identified the candidate genes that may play a key role in fatty acid decomposition and intramuscular fat deposition and provided a theoretical basis for further study in molecular mechanism and meat quality traits of candidate genes in Chinese and foreign pig breeds.