Analysis Of Anal Atresia Trait In Pigs By Genome-wide Association Study And Functional Candidate Gene Strategy

Anal atresia is a congenital disease that due to the abnormal prenatal development of the anorectum and the affected indivaduls cannot excrete intestinal content because of the failure of an anal opening at the surface of body.In this study,We used FarmCPU model to screen the significantly associated SNPs with the porcine anal atresia trait based on the 60K SNP chip genotyping data from 98 control,10 affected and 2 carrier indivaduls.Combining with functional candidate gene methods,we chose three genes（PTPRB,BMP6 and ANKRD6）as candidate genes to explore their involvement in the progress of porcine anal atresia from genetic mutation and gene expression studies.The main results we obtained as following:（1）By using FarmCPU model we got 46 SNPs which were significantly associated with the porcine anal atresia trait(P<10^-7),and 31 SNPs form the FarmCPU model were selected for further genotyping in another bigger population by GT-seq.We found the allele frequency from six SNPs were significantly different between the case and control groups,and three out of them also showed significantly different genotype frequency.Then we screened three candidate genes（PTPRB,BMP6 and ANKRD6）combined with functional candidate gene methods for further study.（2）Two synonymous mutations in the coding region of PTPRB were found by direct PCR sequencing.The tissue distribution profile showed this gene was expressed by multiple tissues.And this gene also did not show any significant expression difference in the rectum between the case and control groups by qPCR and immunohistochemistry.（3）We found one synonymous mutation on BMP6 gene.The tissue distribution profile shows that BMP6 expressed in multiple tissues,and its expression quantity were not significantly different in the rectum between the case and control groups by qPCR and immunohistochemical.（4）We cloned twelve ANKRD6 transcripts,with big difference among their translated amino acids.We found two synonymous and three nonsynonymous mutations on this gene but the allele frequency and genotype frequency for the three nonsynonymous mutations are not significantly different between the case and control groups.The tissue distribution profile showed that ANKRD6 expressed in many tissues,and its expression quantity were not significantly different in the rectum between the case and control groups.Conclusions:The genetic mechanism of porcine anal atresia is complexed,and our research obtained multiple significantly associated SNPs by GWAS and GT-seq.Combining with functional candidate gene methods,we chose three genes for further study and our results can provide references for porcine anal atresia genetic mechanism.