Study On Genetic Markers Associated With Inguinal And Scrotal Hernia In Pigs

Inguinal and scrotal hernias,which occur in the weak areas of the inguinal canal or the processus vaginalis,are some of the most frequent congenital disorders observed in pigs,and they may cause severe economic loss in the pig breeding industry.Hernia development is caused by both multiple genes and environmental factors,but the genetic mechanisms of inguinal/scrotal hernia are poorly understood.Based on the previous research of specific-locus amplified fragment sequencing(SLAF-seq)on 120(59 cases and 61 controls)full and half sib pigs,we performed a genome-wide association study(GWAS)to identify genetic loci underlying variations in inguinal/scrotal hernias.The main results were as follow:1.The 120 samples were divided into three classes(group 1:diseased and healthy individuals;group 2:diseased Yorkshire,healthy Yorkshire,diseased Landrace,healthy Landrace,diseased crossbred and healthy crossbred;group 3 diseased French lines,healthy French lines,diseased American lines and healthy American lines)for further statistical analysis with the farmCPU model.In order to filter the associated results,two different thresholds were selected by farmCPU.A total of 21 SNPs and 9 candidate genes(AUH,TMPRSS3,UBASH3A,PTPN20B,VWA3B,NOL10,TENM3,PLCG2 and NUAK1)were confirmed by p.threshold=0.01/N(where N is the number of total SNPs);6 SNPs and 3 other candidate genes(DEGSI,MEPE and PRKCE)were identified by p.threshold =1E-05.Both the results were significantly(P<0.01)associated with inguinal/scrotal hernia and tested by QQ plot.2.To validate the SNPs associated with inguinal/scrotal hernias,we chosed the most strongly associated genes(DEGS1,PLCG2,PRKCE,NUAK1 and TENM3)that were shown to be related to cell apoptosis in previous studies for replication of the association signal.The association results were as follow:The genotypes and alleles of PRKCE were showed significant differences in all individuals and the French Yorkshire(P<0.05);the genotypes and alleles of DEGS1 were showed no differences in all kinds of groups(P>0.05);the genotypes of NUAK1 were showed significant differences in French individuals and the French Landrace(P<0.05);the genotypes of PLCG2 were showed significant differences in all individuals(P<0.05);the genotypesof TENM3 were showed significant differences in French individuals,French Landrace and crossbred(P<0.05)and extremely remarkable in American and all the individuals(P<0.05).3.According to the prediction of transcription factors binding site,TENM3 gene was selected as candidate gene for enriching the scientific research of inguinal/scrotal hernia.Firstly,with the forecast website NNPP,we cloned TENM3 gene including the core promoter sequences and constructed its luciferase reporter plasmid.Through double luciferase assays of TENM3 deletions,the fragment of TENM3-2 showed the highest activity(P<0.01)and confirmed as the core promoter region of TENM3.Then the recombinant pGL3-Basic vector constructed with TENM3-2 and different genotypes(G/T)sequences were used to detect the fluorescent value by method of relative activity of luciferase.Through the dual luciferase report system,luciferase activity of pGL3-P2-G was significantly higher than pGL3-P2-T(P<0.05).After transfection of siRNA-RUNX2,the numbers of apoptotic cells showed no significantly difference,compared to control group(P>0.05),but the mRNA and protein level of TENM3 gene were both significantly decreased(P<0.05).The results indicated that the different genotypes could affect the binding of transcription factor RUNX2 and the SNP of TENM3 gene,And the transcription factor RUNX2 may has inhibition effect on transcriptional activity of TENM3...