Preliminary Identcaton Of The Causative Gene And Mutation For QTL Inlfuencing Ear Size On Pig Chromosome5

The shape and size of ear are important conformation characteristics of pig breeds, and acrucial reason of auditory disease. There have been many reports about external earcongenital malformation in human, but the genetic mechanism is still unknown. As ananimal model for human disease research, deciphering the genetic mechanism of pig earcharacteristics are benefit for clinic research of human ear related disease. In our previousstudy, we indentified a significant QTL region for ear size within an11.0-cM (60cM-71cM) interval on SSC5, then refined the region to8.7-cM by adding4microsatellitemarkers. Finally, the QTL was fine mapped to a1.137Mb (32277440bp to33414008bp)interval by GWAS, LDLA and IBD analysis in White Duroc×Erhualian intercrossresource population and Sutai population using porcine whole genome high throughput60K SNP chip, this region include three annotated gene: WIF1, LEMD3, MSRB3andHMGA2.Based on this result, in this study, we further performed the Meta analysis on1,029F2individuals from White Duroc×Erhualian resource population and435Sutai pigs. TheQTL was fine-mapped to a632.722kb (32804318bp-33437040bp) region between themaker DRGA0005608and ASGA0025253, the most significant SNP is H3GA0016181(P=2.82×10-31), locating at intergenic region between MSRB3and HMGA2. We carried out thebioinformatic analysis to DNA sequence of1.137Mb region, and found two sequenceduplications. We confirmed that the duplications were not exist by absolute quantificationPCR analysis. We inferred that these duplications may be caused by sequence assembly.By using the results of porcine whole-genome resequencing, we found two CNVs withinMSRB3. We genotyped the two CNVs by relative quantitative PCR in Chinese indigenouspig breeds with different ear size, resource population with different QTL genotypes andSutai population. The results indicate that, in Chinese indigenous pig breeds, theindividuals with large and floppy ear had increased copy number at both CNVs, however,there was only1copy for pig breeds with small ear size. The CNV genotype of all of theF0resource population individuals were concordance with the QTL genotype, the CNVgenotypes in3and17F0parents were consistent with the QTL genotypes, and only3outof the24offsprings had the inconsistent genotypes between CNV and QTL. In all18F2individuals examined, only3individuals its CNV genotypes do not fit perfectly with QTLgenotypes. As there are also three genes (LEMD3, MSRB3and HMGA2) in632.722kbregion, to confirm which is the strong candidate causative gene, we analyzed theexpression of three candidate genes in ear tissue from4full-sib pairs of Sutai pigs withextreme phenotypes, the results indicate that HMGA2gene lowly expressed in pig eartissue, and the standard error of qRT-PCR is a little big. Moreover, gene expression levels were not perfectly consistent with the phenotype. The expression of MSRB3and LEMD3were not conformed to the phenotype. In order to verify the result of gene expression inlarge scale population, we collected105RNA ear samples from Sutai pigs, and performedgene expression analysis in three genes. The eQTL and QTT analysis were also performed.As the results, the strongest SNP related to HMGA2expression is32566461, locate atSSC5:33458644(Sscrofa10.2), in the intron of HMGA2(P=0.015), no eQTL wasmapped for MSRB3and LEMD3. In the QTT analysis, the expression of HMGA2(r=0.42,P=0.074), MSRB3(r=-0.20, P=0.68), LEMD3(r=-0.35, P=0.22) are not significantrelated with ear size. Then we analyzed the association of CNV with gene expression, andfound that, there is nearly no relation between CNV and gene expression of HMGA2andLEMD3. But significant association was found between the expression of MSRB3andCNV genotype. By integrating the results of Meta analysis, association of CNV with earsize, gene expression analysis, wu suggested that MSRB3maybe the causative geneinfluencing pig ear size and CNV within MSRB3is the candidate causative mutation forthis QTL. But we can’t exclude HMGA2as the candidate gene at present.The results from this study will contribute to indentify the QTN influencing pig ear sizeon SSC5.