Comparative Analysis On Genome-wide Methylation And RNA-seq In Longissimus Dorsi Muscle Between Dorper×Small Tailed Han Crossbred And Small Tailed Han Sheep

The Small Tail Han Sheep is one of the good breeding resources in China.It has the characteristics of strong fecundity,good resistance and stable genetic property,but its defects are low production performance.As people demand for higher yield and quality of mutton,the advantage of high fertility can not meet the demand of mutton sheep market consumption at present.Thus,some Small Tail Han sheep were crossbreeded by Dorper sheep,in order to improve production capacity and ensure the basic high reproductive performance,improve the quality of meat.Hybrids maintained the characteristics of high reproductive performance,good resistance of Small Tail Han sheep,and meat performance has been significantly improved.What functions genes involved in the regulation and influence the formation of meat traits in the process of improving the performance of meat.It is a research hotspot in the field of functional genomics in sheep.Dorper x Small Tail Han Crossbred and Small Tail Han Sheep were studied in this research,at first comparison has carried on production performance,the results found the average of weight for Dorper x Small Tail Han Crossbred Sheep were 66.35 g higher than Small Tail Han Sheep in growing stage,slaughter rate and net meat percentage higher than Small Tail Han Sheep 1.72% and 4.05%,respectively.Meat performance contrast found Dorper x Small Tail Han Crossbred Sheep tenderness obviously improved,intramuscular fat content,oleic acid content is better than that of Small Tail Han Sheep,showed obvious heterosis,hybrids inherited Dorper performance in the meat high force,good meat quality characteristics.In contrast with meat quality,on the basis of using the Me DIP-seq sequencingof Dorper x Small Tail Han Crossbred and Small Tail Han Sheep two groups of genome-wide methylation sequencing,a total of 23.08 Gb of data effectively,methylation gene screening to 808 differences.Compared with the Small Tail Han Sheep,the differentially methylated genes of the Dorper x Small Tail Han Crossbred Sheep are hypomethylated genes.The GO enrichment analysis found that the main enrichment of the differential methylation gene was in the combination of nucleotide,muscle activity,ATP binding,lixin complex,vesicle transport and cytoskeleton GO terms.The low methylation genes in the differential methylated regions were significantly enriched with the nucleotide binding,myoactivity,phospholipid binding,and cell connectivity of molecular function.KEGG enrichment analysis found that differences in 175 pathways,mainly concentrated in the phagosome,mucin type O-chitosan biosynthesis,fatty acid metabolism,PPAR signaling pathways,Wnt signaling pathways which influencing cell proliferation and differentiation,growth and development,lipid metabolism,involved 609 functional genes,which the phagosome,mucin in the biosynthesis pathway of O-chitosan enrichment of methylation gene related significant difference(p<0.05).The 12 differentially expressed genes,TGFB3,ACSL1,RYR1,ACOX2,PPARG2,NTN1,RIN2,MAPRE1,ADAMTS2,MYOM1,ZDHHC13,SH3PXD2 B,were choosed to analyze and found that genetic TGFB3 ACSL1 significant difference(p<0.05),RYR1,ACOX2,PPARG2,MAPRE1,ADAMTS2,MYOM1 and ZDHHC13 were significantly different(p<0.01).Dorper x Small Tail Han sheep compared with Small Tail Han sheep ACSL1,RIN2,ADAMTS2 gene expression level respectively,reduce,raise,lower,and the regular pattern of methylation differences to raise,lower,increases,the negative correlation indicating ACSL1,RIN2,ADAMTS2 gene expression differences may be due to methylation differences.Simultaneously,the study of transcriptome sequencing analysis between Dorper x Small Tail Han Crossbred Sheep and Small Tail Han Sheep were completed,which obtained 538 differentially expressed genes(p<0.05),there are 238 up-regulated genes and 300 down-regulated genes.Differentially expressed genes are mainlyenriched in 898 GO terms,of which 24 GO term significantly enriched(p<0.05),there are 15 biological processes,7 cell components,2 molecular function.63 genes were significantly enriched(p.adjust<0.05)in 7 KEGG pathways,including PPAR signaling pathways,fatty acid metabolism may be affect the growth and meat quality traits of sheep by lipid metabolisim.SCD,PLIN2,PLIN5,ACOX2,CPT1 B,FADS1,ACAA1,ACSL3 in the pathways play an important role may be major genes to affect the properties.Using fluorescence quantitative PCR to verify m RNA expression levels of CSRP3,MYOD1,MYL2,ABLIM1,ACTA1,MYH7,TPM3,FOS,JUN,ANKRD1,BMP4,ALDH5A1,CSRNP1,ARID5 B,WISP2,SOCS2,IGFBP5,SCD,CPT1 B,ACAA1 and ACSL3.The expression levels of above genes were consistent with the result of transcriptome.The genomic methylation and transcriptome analysis showed that the methylation of 22 DMGs were correlated with the expression levels of DEGs.Overlap between DNA methylation and gene expression of genes GO enrichment analysis showed that the difference of gene wereenriched in 76 GO terms,the two GO terms significantly enriched(p<0.05).ACOX2,ST3GAL3 and CDON were validated by quantitative PCR and the result in tissue were consistent with the transcriptome sequencing between Dorper x Small Tail Han Crossbred Sheep and Small Tail Han Sheep.The difference between CDON expression and methylation is contrary to each other.It is concluded that the regulation of CDON gene expression may be affected by methylation level.The relationship between DNA methylation and expression of DNMT1,ST3GAL3,ACSL1,OLAH,PPARG2,ALAS1,FRMD4 B,ADAMTS2,STRIP2 were verified in cell level by RG108 treatment.With the increase of RG108 concentration,the expression of DNMT1 was inhibited,and the expression levels of other genes changed.It is thought that the expression of genes may be regulated by methylation levels.Compared Dorper x Small Tail Han Crossbred Sheep with the performance of Small Tail Han Sheep,relying on the whole genome and transcriptome sequencing technology,methylation acquired a lot of DMRs and DEGs.The use of exogenousDNA methyltransferase inhibitors RG108 indirectly verified the related gene expression and the relationship between the methylation patterns.Results of growth,fleshy related genes for further mining,parsing mechanism of heterosis,exploration of the regulatory mechanism of meat quality traits form laid the theoretical foundation with obtaining the function of the gene expected to be used as genetic markers in the mutton genetic breeding and guide future improvement to promote the upgrade of the mutton sheep industry,which technological progress has great significance.