Genome-Wide Methylation Differential Analysis And Functional Verification Of Candidate Genes In Valgus-Varus Deformity Broilers

With the continuous progress of modern breeding technology,fast-growing heavy broilers have realized faster growth rate and higher feed conversion rate.However,it also brings some animal welfare problems,especially leg disease in broiler breeding.Valgus-varus deformity（VVD）is one of the most common leg diseases in broiler breeding.VVD is a common long bone deformity in broilers and turkeys,was characterized by varus or valgus between the tibial tarsus and tarsometatarsus,which seriously affected the locomotion ability and meat quality of broilers.However,at present,the genetic etiology of VVD broiler is not clear,which restricts the genetic selection for this disease,and there is no effective control method.In this study,VVD of Cobb broiler chickens was taken as the research object.Whole genome methylation sequencing technology was used to construct methylation map,screen key pathogenic genes,and further verify function at the cellular level.The main results were as follows:Research 1: Whole genome DNA methylation of VVD broiler cartilage tissue.1.The cartilage tissue of 35-day-old Cobb broilers（3 broilers in VVD group and 3broilers in normal group）was used as material to conduct whole genome methylation sequencing,and a total of 219.8 Gb of data were obtained.2.Genome-wide methylation maps of broiler cartilage tissue in the VVD group and the normal group showed that,in CG sequence environment,more differential methylated regions（DMR）with high methylation level were screened in the VVD group,while more DMRs with low methylation level were screened in the normal group.The overall methylation level of VVD broilers was higher than that of the healthy group.3.By analyzing the whole-genome methylation sequencing data of broiler cartilage tissue in VVD group and normal group,4315 DMRs were screened.A total of 2326 differentially methylated genes（DMG）were identified from these DMRs,among which 1159 genes had DMRs with up-regulated methylation level,936 genes had DMRs with down-regulated methylation level,and 231 genes had two types of DMRs.These DMGs are mainly concentrated in cell development,cell differentiation,embryonic development and skeletal development.Research 2: Whole genome DNA methylation of VVD broiler leg muscle tissue.1.The leg muscle tissue of 35-day-old Cobb broilers（3 broilers in VVD group and 3broilers in normal group）was used as material for whole-genome methylation sequencing,and 217.9 Gb of data were obtained.2.The whole-genome methylation map of broiler leg muscle tissue in the VVD group and the normal group showed that relatively more DMRs with high methylation level were screened out in the normal group,while relatively more DMRs with low methylation level were screened out in the VVD group.3.By analyzing the whole-genome methylation sequencing data of broiler leg muscle tissue in the VVD group and the normal group,2609 DMRs were screened,and a total of 1555 DMGs were identified from these DMRs,among which 768 genes had DMRs with up-regulated methylation level,and 613 genes had DMRs with down-regulated methylation level.There were two types of DMRs in 174 genes.These DMGs are mainly concentrated in protein degradation,muscle atrophy,and dysfunction of skeletal muscle synthesis and decomposition.Research 3: Screening and functional verification of pathogenic genes in VVD broiler cartilage tissue.1.The results of joint analysis of the methylation data of VVD broiler cartilage tissue and transcriptome data showed that the methylation level of 2 Kb region before and after the gene was negatively correlated with the mRNA expression level in CG sequence environment.In CHG and CHH sequences,the methylation level of the 2Kb region before and after the gene was positively correlated with the mRNA expression level.The combined analysis of differentially methylated genes and differentially expressed genes showed that there were intersection of 63 genes.The results of protein interaction analysis of 63 genes showed that ESPL1,PRC1,MELK,KIF2 C,MK167,CDK1 and NCAPG proteins were interacting with each other.2.Correlation analysis showed that DNA methylation level in the promoter region of candidate gene ESPL1 was negatively correlated with mRNA expression level（r=-0.6073,P=0.2011）.The differential methylation region of the ESPL1 promoter region showed 19 potential transcription binding sites.3.The results of the effect of methyltransferase inhibitor 5-azactidine（5-AzaC）on chondrocyte activity showed that when different concentrations of 5-AzaC were added for 12 h,the chondrocyte activity was only inhibited in 1000 μmol/L 5-AzaC group（P<0.05）;At 24 h,the chondrocyte activity of the three experimental groups was inhibited,and the chondrocyte activity of the experimental group was significantly lower than that of the control group when the concentration of 5-AzaC was 10 μmol/L and 1000 μmol/L（P<0.01）.When the concentration of 5-AzaC was 10μmol/L,the chondrocyte activity of the experimental group was significantly lower than that of the control group（P<0.05）.At 36 h,the chondrocyte activity of the experimental group was higher than that of the control group,and the 10 μmol/L5-AzaC group reached a significant level（P<0.05）,the 1000 μmol/L 5-AzaC group reached a very significant level（P<0.05）,but the 100 μmol/L 5-AzaC group did not reach a significant level.At 48 h,chondrocyte activity in 1000 μmol/L 5-AzaC group was significantly lower than that in control group（P<0.05）.4.After adding 100 μmol/L methylation inhibitor for 12 h without affecting the activity of chondrocytes,the mRNA expression level of ESPL1 in experimental group was significantly higher than that in control group（P<0.01）,and the mRNA expression level of DNMT1 was significantly lower than that in control group（P<0.01）.The mRNA expression level of DNMT3 A was significantly lower than that of control group（P<0.05）.Research 4: Screening and functional verification of pathogenic genes in leg muscle tissue of VVD broilers.1.The results of joint analysis of the methylation data of VVD broiler leg muscle tissue and transcriptome data showed that the methylation level of 2 Kb region before and after the gene was negatively correlated with the mRNA expression level in CG sequence environment.In CHG and CHH sequences,the methylation level of the 2Kb region before and after the gene was positively correlated with the mRNA expression level.The results of protein interaction analysis of 147 genes showed that FOS,MYL9,HSPB1,FRAS1,GLI2,CACNA1 C and CNR1 genes were in the core position.2.Correlation analysis showed that DNA methylation levels of the two DMRs in the promoter region of candidate gene MYL9 were negatively correlated with mRNA expression levels（r₁=-0.7372,P₁=0.0945;r₂=-0.7448,P₂=0.0894）.The differential methylation region in the promoter region of MYL9 gene showed that transcription factors Sp1,Egr-1 and E2 may bind to the differential methylation region.3.The effect of methyltransferase inhibitor 5-AzaC on myoblast activity showed that when different concentrations of 5-AzaC were added for 12 h,24 h and 48 h,The activity of muscle cells in 100 μmol/L 5-AzaC group and 1000 μmol/L 5-AzaC group was significantly lower than that in control group（P<0.01）.The 10 μmol/L 5-AzaC muscle cells were not affected.At 36 h,the myoblast activity of the three experimental groups was significantly lower than that of the control group（P<0.01）.4.After 24 h treatment with 10 μmol/L methylation inhibitor without affecting the activity of myoblasts,the mRNA expression of MYL9 in experimental group was significantly higher than that in control group（P<0.01）,and the mRNA expression of DNMT1 and DNMT3 A in experimental group was significantly lower than that in control group（P<0.01）.