Comparative Analysis On Genome-wide Methylation And Transcriptome Of Different Coat Color Bashibai Sheep Skin

According to the different production direction of sheep,there are two main breeding directions:producing wool and producing meat.The stock of xinjiang sheep is about 26 million,most of which are coarse wool breeds.The coat color is mainly brown and red,but there are also black,cyan gray and white.The cashmere content is more than 55%,which has textile value.In this study,the red-brown and cyan gray phenotypes of 1-year bashba sheep from xinjiang were selected as the research objects.The whole genome methylation sequencing was used to detect the different methylation maps of skin tissues of the two groups of phenotypes,and the regulatory mechanism of DNA methylation on melanin production in the two groups of coat color was analyzed.At the same time,transcriptome sequencing was used to screen differentially expressed genes in two groups of phenotypic skin tissues,and the molecular mechanism of the regulation of two phenotypic genes on pigment deposition was analyzed.The results of whole genome methylation and transcriptome sequencing were analyzed to obtain the regulatory relationship between differentially methylated and differentially expressed genes,so as to further understand the molecular mechanism of DNA methylation affecting coat color generation on gene expression regulation.1.Genome-wide methylation analysis results showed that(1)the CG type methylation level of the two groups of phenotypes was high on the whole genome,and it was the main type of DNA methylation in sheep.The methylation level was low in CHH and CHG types,and there was no significant difference in methylation level and methylation density between the two groups.(2)all genes in the genomes of different functional areas of DNA methylation level,under the background of CG sequences within exons and last exons the highest level of DNA methylation,gene upstream area and the first exon DNA methylation in the lowest levels,and distal DNA methylation level is higher than in genes in under the background of CHG and CHH sequence,integral to the low level of DNA methylation,under the background of CHH sequence introns methylation level relative to a minimum.(3)There were a total of DMRs in the two groups.The CG type had the largest number of DMRs on the chromosome,5,391,and the length of DMRs was close,with the length of DMRs less than 300bp.DMR gene annotation results showed that in the Intergenic region,the number of differentially methylated genes in the body region and promoter in the CG sequence background was large,while the number of differentially methylated genes in the 5 ’UTR,1stExon and 3’ UTR regions was relatively small,and the number of differentially methylated genes in the 6 functional regions in the CHG and CHH sequences background was relatively small.(4)The functions of differential methylation-related genes GO and KEGG were mainly enriched in the positive regulation of canonical Wnt signaling pathway,Ras signaling pathway and Melanogenesis pathways.2.Transcriptome sequencing results showed that(1)There were 1977 differentially expressed genes,1298 up-regulated genes and 679 down-regulated genes in red-brown.(2)tThe functions of differentially expressed genes GO and KEGG are mainly concentrated in cell part,membrane,biological regulation,catalytic activity,signal sensor activity,Wnt signaling pathway,Ras signaling pathway,Melanogenesis.(3)The mRNA expression levels of TYRP1,DCT,PMEL,TYR,SLC45A2,MLANA and OCA2 genes in two groups of phenotypic skin tissues were verified.The expression levels of both genes were higher in the cyan gray group than in the red-brown group,which was consistent with the transcriptome sequencing results.3.Combined genome-wide methylation and transcription analysis showed that(1)5 expression levels(low,medium_low,medium,medium_high,high),upstream region and methylated water near genebody region decreased in sequence,indicating that gene expression of these genes is negatively regulated by DNA methylation in this functional region.In genebody area and downstream area high mRNA expression level corresponding to the high level of methylation and low mRNA expression level also corresponds to the high level of methylation,shows that genes in these two area,certain genes can be affected by methylation is regulation,and some gene is negative.(2)There were 113 up-regulated and down-regulated genes in the promoter region,79 up-regulated genes and 34 down-regulated genes in the two groups.DNA methylation and gene expression were negatively regulated in 55 and positively regulated in 58.4.CpG islands in the promoter regions of candidate genes WNT6,KIT,PMEL,DCT and RAB27A were predicted to exist in the promoter sequences of WNT6,KIT,PMEL and RAB27A.A total of 14 SNPs were detected in 5 gene promoter sequences of four groups of coat phenotypes(red-brown,black,white and cyan gray).The prediction results of transcription binding sites showed that DCT,PMEL and RAB27A genes may be regulated by Pax-6,Zic and p300 transcription factors.5.Differential methylation of TSPAN10,EGFL6,SDR16C5,SLC5A10,GPR160 genes was verified at the cell level.With the increase of SGI-1027 concentration,the expression levels of DNMT1,DNMT3A and DNMT3B were inhibited.The expression levels of EGFL6 gene and SDR16C5 gene gradually increased,while the expression levels of SLC5A10 gene and GPR160 gene generally showed a downward trend.It is preliminarily believed that the expression levels of EGFL6,SDR16C5 gene,SLC5A10 gene and GPR160 gene were regulated by DNA methylation6.The transcription factor Pax6 overexpression vector plasmid was transfected into melanocytes,and the expression levels of KIT,WNT6,RAB27A and PMEL genes were detected by fluorescence quantification,and the preliminary verification was made that Pax6 genes promoted the expression levels of KIT,WNT6,RAB27A and PMEL genes.Transcription factor Pax6 inhibited the apoptosis of melanocytes and promoted the increase of melanin content in melanocytes.