Identification And Study Of Phenotypes And Susceptibility Genes In Broilers Affected By Valgus-varus Deformity

Leg disorders are the major economic and welfare problem in broiler industry.The etiology of leg deformity is a combination of multiple factors,which the conventional methods are not effective to resolve this problem.However,the prevalence of the leg disorders has been reduced by genetic selection.VVD(valgus-varus deformity)is a common the long-bone disorder in broilers and is characterized by valgus or varus angulation of the intertarsal joint.The experimental material is Hubbard broilers in our study.First,based on a case/control design with VVD birds and normal birds.The bone quality,component and bone morphometry of VVD broilers and these indexes of normal broilers were compared.Then,we identified susceptibility genes which associated with VVD according to GWAS and RNA-seq.The chicken FGF10 and FGF12 coding sequence were amplified,bioinformatics analysis and expression patterns,respectively.This study provides a reference for pathogenesis of VVD and leg health in broilers.The main results are as follows:Research 1:Difference analysis of growth performance,bone traits and serum lipid content between normal broilers and VVD broilers1.This experiment confirmed that the type of leg disease was VVD according to clinical diagnosis and anatomical examination.2.This experiment investigates that the incidence of VVD among the birds at a commercial facility in Tangshan China was 1.75%(n=52,000),distributed about 1:1,between females and males.The incidence of left legs is basically the same as the incidence of right legs.Approximately 97.9%of affected birds were classified as the "valgus" type.And only 2.1%of affected birds were classified as the "varus" type.The incidence of unilateral limbs is 17.6%and bilateral abnormality is 82.4%.3.Compared with normal birds,body weight,shank length and girth of VVD birds were significantly lower(P<0.05).The tibia and metatarsal bone index were significantly higher in VVD birds than the bone index of normal birds(P<0.05).And,the bone mineral density of VVD birds were significantly lower(P<0.05).Serum Ca,CHO and LDL-C levels were significantly higher in VVD birds(P<0.05).Serum P,ALP and HDL-C levels were significantly lower in VVD birds(P＜0.05).4.Histopathological examination showed that the trabecular structure in VVD birds was relatively sparse and disruptive.Compared with normal birds,the trabecular bone area was lower and the number of chondrocytes was increased in VVD birds.And,calcification disorders or delayed of chondrocytes in VVD birds.Research 2:Transcriptome sequencing and screening susceptibility gene associated with VVD1.Six cDNA libraries were constructed and transcriptome sequencing was performed using the leg cartilage of 3 normal broilers and 3 VVD broilers.A total of 78.39 GB data was obtained.2.A total of 231 differentially expressed mRNA were obtained by transcriptome data.Among these,134 mRNAs were significantly up-regulated and 97 were significantly down-regulated.Meanwhile,23 differentially expressed IncRNAs were screened,9 IncRNAs were significantly up-regulated and 14 were significantly down-regulated.3.Subsequently,the target genes of differentially expressed lncRNA were predict ed by cis and trans.And functional annotation was performed on the differentially expressed mRNA and differentially expressed lncRNA target genes.It was found that these genes were enriched in the signaling pathways associated with bone and vascular development,including P53,MAPK,toll-like receptor,JAK-STAT,Hedgehog and PPAR.4.We constructed lncRNA-mRNA interaction network analysis.We found that FGF10,FGF12,BMP3 and DENND4A genes may be related to the occurrence of VVD.Compared with normal broilers,the relative expression levels of BMP3 genes decreased n leg cartilage of VVD birds.And the relative expression levels of DENND4A genes increased in leg cartilage of VVD birds5.At the same time,8 randomly selected mRNAs or lncRNAs were verified by RT-qPCR,and the results were consistent with the trend of transcriptome sequencing results.The results indicated that transcriptome data were authentic and reliable.Research 3:GWAS analysis and the integration of GWAS and transcriptome to screen VVD susceptibility genes1.A total of 126 normal broilers and 122 VVD broilers were used to evaluate their walking ability.The results showed that the gait scores of VVD broilers were basically between 3-5.2.We constructed 248 GBS sequencing libraries.After data quality control,15 unqualified individual data were filtered.The overall coefficient of variation of the remaining 233 individual sequencing data was 24.6%.PCA analysis results showed that there was no obvious population stratification in the population structure of the experimental chickens,that indicated that genetic background of these birds was similar.3.Through the processing of sequencing data,a total of 272,814 SNPs was finally obtained.The mixed linear model was used to correlation analysis with the results of VVD leg disease and gait score.The results showed that no significantly correlated SNP sites were found.4.The combined analysis of genes in the upstream and downstream 500K of top 20 SNP and transcriptome sequencing differentially expressed genes showed that PTH1R,GARS,HOXA2,BMP6 and ASCC1 were closely related to bone development or bone deformity.Research 4:Cloning and bioinformatics and tissue distribution analysis of FGF10 and FGF12 gene1.In this experiment,the CDS sequences of FGF10 and FGF12 were cloned.The full length of FGF10 mRNA was 950bp,and the full length of CDS was 639bp,encoding 212 amino acid residues.The mRNA sequence of FGF12 gene in chicken was 825bp,and the CDS region was 732bp,encoding 243 amino acid residues.2.Tissue distribution results indicated that FGF10 and FGF12 were igh-abundance expression in the bursa of fabricius and low-abundance expression in leg muscle.Compared with normal birds,the expression levels of FGF10 and FGF12 were higher in leg cartilage of VVD birds(P<0.01).3.Bioinformatics analysis of FGF10 and FGF12 amino acid sequences showed that chicken FGF10 and FGF12 had typical functional domains,FGF10 had a transmembrane spiral region,but FGF12 had no transmembrane domain.There are three secondary structures of FGF10 and FGF12 proteins in chickens which namely Alpha helix,Extended strand,Random coil,the predicted results of protein tertiary structure are consistent with those of secondary structure.4.Phylogenetic analysis showed that FGF10 and FGF12 had close evolutionary relationship.5.After successful overexpression FGF10 gene,the content of collagen Ⅱ was significantly higher in chondrocytes(P<0.05).