Research The Methylation And Transcriptome Patterns In Broiler Chicken’s Breast And Leg Muscle By BS-seq And RNA-seq Methods

Broiler chicken have the main growth characteristics that breast muscle development faster than leg muscle,and the percentage of breast muscle was higher than that of leg muscle at the same time.The process of muscle growth is a complex process which was affected by multiple gene regulation.In this study,we used the broiler chicken as the research objectives and studied the molecular mechanism of skeletal muscle growth and differentiation.This experiment used the genome-wide methylation of single-base resolution sequencing technology ande the transcriptome sequencing technology to explor the breast muscle and leg muscle growth characteristics and differences of brioler chicken in gene expression.The results of this study are as follows:1 Obtain the single-base methylation pattern of the breast muscle and leg muscle,through the genome-wide methylation bisulfite sequencing,Both of the breast and leg muscle are characterized by the highest degree in CG methylation,while the CHH and CHG were low degree of methylation.The cytosine as the sequence like CAT was in higher methylation level in CHH state bases,and the cytosine as the sequence like CTG was in higher methylation level in CHG state bases.Under the environment of CG cytosine methylation has tissue specificity,while the Cytosine methylation of CHH CHG have no tissue specificity.Breast muscle methylation level had high frequency distribution in 0.6-0.8,and leg muscle methylation level had high frequency distributiontissue in 0.3-0.4;2 Get the two organization of genome-wide methylation distribution in each gene function area,three states of CG CHH CHG in genome methylation level distribution of each functional area.Results are characterized by the promoter methylation level sequence upstream area is lower,and the inner regions of the gene methylation level is higher;3 Analyze the two differences in methylation region of the organization,and the difference in methylation on chromosome distribution.Difference in methylation on chromosome evenly distributed,which was not appear to chromosomes structure change of methylation area,the length of difference methylation fragment was mainly distributed in the range of 300 bp.the difference methylation region has no chromosome preference;4 We annotated the differences methylation gene,a total of 8990 genes has annotation,769 of the gene promoter region in the methylation state;compareing the breast and leg muscle,the degree of methylation decline has 453 genes;as for the promoter methylation status,the breast muscle gene promoter methylation level higher than leg muscle;Analysis showed that the main function was carried out on the difference in methylation enrichment occurred in protein and cellular function;5 Though the transcriptome sequencing,we obtained 17,108 transcription of chiken skeletal muscle,among the has 4247 new transcript,as the gene annotation obtained 8639 genes;breast muscle detected 17,922,229 splice sites,while the leg muscle detected 15,293,178 splice sites,all of the splice sites can be divied into five form,an the main form was exon skipping;6 We detected 893 differentially expressed genes in breast and leg muscle tissue,including 524 gene increased in leg muscle,and the other 369 genes was lower expression;Analysis of differentially expressed genes in the GO term to get the high expression of genes in the process the result shown that some of the differenct gene with high expression was enrichment in cell components,Lower expression genes was enrichment in molecular function;KEGG analysis found that leg muscle increased gene significantly enriched in muscle contraction signal pathway,And gene expression of downgrade main enrichment on the biosynthesis of amino acids and amino acid metabolic pathways;7 Joint analysis of methylation and transcription,get CG methylation level had a negative correlation(r=-0.674,p=4.312×10-3)with the expression of genes;when the CG methylation level below 0.1,The gene expression was the highest,As CG methylation level increases,the expression of genes gradually decline;8 By analyzing the differences between the gene functional domain methylation and gene expression,There are 82 differentially expressed genes in the promoter region with differences methylation regions;comparing the breast muscle and leg muscle,have differentially expressed 25 genes with low methylation level;has 578 differentially expressed genes with gene body methylation;as the impact of methlytion,it affect the Leg muscle gene expression was smaller than ithe effection on breast muscle gene expression,Show the methylation in leg muscle regulation function is less than the regulation function in the breast muscle;9 The correlation between methylation and the difference of gene expression was not significant(P=0.4878)when the DMR in gene body,At the same time the DMR in promoteris is also not significant correlation with gene expression(P=0.791;10 Get 17 genes which had significantly differences in the methylation and gene expression,Four genes CD9 HSPB7 LMOD2 Wnt9A were significantly enriched in muscle growth related pathways;detected the expression pattern of these four genes in laying chicken and brioler chickens in the different time point,four genes expression in laying chicken and brioler chicken had significant differences trends;In conclusion,this study carried out the functional genomics research on the brioler chicken skeletal muscle by DNA methylation sequencing and mRNA sequencing,though analysis the two omics results,received breast and leg muscle character difference of candidate genes,Laid the foundation for the study of muscle growth and development,at the same time provides a molecular local breeds breeding research train of thought.