Studies On The Genome-wide DNA Methylation Changes And Transcriptomic Profile Of Leg Muscle In Jinghai Yellow Chicken

The early growth pattern,especially the age of peak growth,of broilers affects the time to market and slaughter weight,which in turn affect the profitability of the poultry industry.However,the underlying mechanisms regulating chicken growth and development have rarely been studied.DNA methylation is a one of the most common epigenetic modifications in eukaryotic organisms,and plays a crucial role in regulating genes' expression on the level of transcription or after transcription,which in turn has an impact on the growth of animals.At present,the epigenetic regulatory mechanism of early growth in quality broiler has rarely been reported.Therefore,in this study,Jinghai Yellow chickens at 4 weeks of age(slowgrowing stage),12 weeks of age(fast-growing stage),and 16 weeks of age(growth retardation/on-sale stage)were selected to investigate the genome-wide gene expression changes,DNA methylome variation and the regulatory mechanism of DNA methylation on the expression of growth-related genes.The RNA-Seq and BS-Seq technology combined with bioinformatic tools were used in this study.The results of this study are useful for understanding the mechanisms regulating the development of leg muscle and the pattern of chicken growth.The main results were as follows:1.Three nonlinear growth models were used to fit growth curves of Jinghai Yellow chicken,using the growth data of 300 Jinghai Yellow chicken from 0 to 25 weeks of age.It was found that the growth curve resembled an S-curve.And the age and body weight at the inflexion point of growth calculated by the logistic model were respectively 11.48 weeks and 796.68 g.2.Nine cDNA libraries,three from each growth stage(including 4,12,and 16 weeks of age)were sequenced by RNA-Seq technology.More than 8 Gb data was obtained for each sample.A total of 15,699 expressed genes were detected in leg muscles,of which 14,432 genes were commonly expressed in all three groups.And 59,724 and 633 differentially expressed genes(DEGs)were respectively detected in the comparisons of M4W vs 12W,M4W vs 16W and M12W vs M16W(FC?2,FDR<0.05).Among these DEGs,MYOD1,FBX032,CEBPB,and FOXO3 4 transcription factors,and a series of genes involved in the somatotropic axis,such as GH,IGF2BP1,IGF2BP2,IGF2BP3,IGFBP3,IGFBP5,and IGFBP7 were found,which have been reported to be closely related to growth.3.Go and KEGG functional enrichment analysis showed that the DEGs were significantly enriched in the growth-related biological processes,such as cell proliferation and differentiation,cell migration,cell adhesion and junction,cell growth,skeletal muscle fiber development,muscle organ development,and skeletal muscle cell differentiation(P<0.05).Some well-known candidate genes of growth including MYOD1,IGFBP3,IGFBP5,IGFBP7 and MYH10 were found in these GO terms.Among the significantly enriched pathways(P<0.05),five pathways related to growth and development were found:extracellular matrix-receptor interaction,focal adhesion,tight junction,insulin signaling pathway,and regulation of the actin cytoskeleton.A total of 42 DEGs assigned to these pathways,such as MYH10,FGF2,FGF16,FN1 and MAPK9.These pathways and genes were suggested to play important roles in early growth of Jinghai Yellow chicken and the development of chicken leg muscle.4.This study established a single-base resolution DNA methylation profile of the leg muscle in Jinghai Yellow chicken by BS-Seq technique and revealed the chicken methylome feature:The methylation of cytosines mainly occurred in the CG context,and only a small part of methylcytosines occurred in the CHG and CHH context.No sequence preference was found in the mCG-flanking regions,however,an adenine was always present after the non-CG methylcytosine site.The methylation degree in mCG significantly decreased to a lower level around the transcriptional start site(TSS)of the genes,and increased to a stable high level in the genebody.However,the non-CG methylcytosine sites have a completely opposite methylation pattern.The analysis on the methylation level of different functional elements showed that the methylation levels in the promoter regions and CpG islands were the lowest,however,CDS regions and exons showed the highest methylation levels.DNA methylation density and the CG methylation level showed large variations across each chromosome.5.The analysis on the DNA methylation difference between the three growth stages showed that 2141,2260 and 1550 differentially methylated regions(DMRs)were respectively detected in the comparisons of M4W vs 12W,M4W vs 16W and M12W vs M16W(FDR<0.05).And 1022,1055 and 797 DMR-associated genes were respectively obtained in the three comparisons,which were called differentially methylated genes(DMGs).Of all DMGs,263,239 and 229 genes were differentially methylated in promoter regions.6.Functional enrichment analysis on the DMGs in promoter regions indicated that these genes were significantly enriched in growth-related biological processes,such as regulation and negative regulation of transforming growth factor beta receptor signaling pathway,positive regulation of cell migration and proliferation,regulation of cell differentiation and proliferation,skeletal system development,actin cytoskeleton organization and cell adhesion(P<0.05).And five growth-related pathways were found in the significantly enriched KEGG pathways(P<0.05),which were TGF-beta signaling pathway,Cell cycle,Lysosome,Ribosome and Cell adhesion molecules(CAMs),including BMP4 and SMAD4 genes.7.Correlation analysis between DNA methylation and gene expression showed that most of the DNA methylations in promoter regions of genes were negatively correlated with mRNA expression.And we detected 26 differentially methylated genes in promoter regions which were differentially expressed as well.Among these genes,20 genes showed a significant negative correlation between changes in the methylation levels in promoter region and gene expression,including two well-known growth-related genes:IGF2BP1 and IGF2BP3.The bisulfite sequencing PCR(BSP)approach was used to confirm the methylation level of IGF2BP1 and IGF2BP3 in the promoter regions.It was showed that the result of BSP approach was consistent with the BS-Seq technology.Combining with the expression levels of these two genes,the results from BSP and BS-Seq methods both showed a negative correlations between the methylation levels in the promoter region and gene expression.Therefore,it was inferred that the DNA methylation in promoter regions of IGF2BP1 and IGF2BP3 may inhibit their expression,and in turn play crucial roles in regulating the growth and development of Jinghai Yellow Chicken.