The Identification And Mechanism Of The Causative Mutation For QTL Influencing Ear Size On Pig Chromosome 5

The ear type and size are important conformation characteristics of pigs. It is diverse in different breeds. Although many studies about microtia in human have been reported previously, the genetic mechanism have not been deciphered. Pig is an important animal model for studying human microbia because the physiological characteristics are very similar with human. The research on genetic dissection of pig ear type is helpful for breeding and provide clinical experience for human microtia studies. Several studies identified significant QTL for ear size on pig chromosome (SSC) 5, while the causative gene and mutation had not been characterized. In our previous studies, the QTL interval was fine mapped to 632.722 kb using 60K SNP chip in White Duroc x Erhualian resource population and Sutai population, and three candidate genes, LEMD3, MSRB3 and HMGA2 locate within this interval.Based on the previous study, we conducted GWAS using high-throughput porcine 60K SNP chip in three populations of Laiwu, Erhualian and DLY commercial population.As a result, the most significantly associated SNPs in Laiwu pigs were ASGA0025241,ASGA0025245, H3GA0016181 and ALGA0031527. In Erhualian pigs, the peak SNP was ALGA0040227 on SSC7 and we did not found significant SNPs on SSC5. The top SNPs in DLY pigs were ASGA0025245 and ASGA0025241. And then, we performed a meta-GWAS analysis in those three populations and previously used two populations. As a result,we identified genome-wide significant SNPs on SSC 1,4, 5, 7, 13 and X,the most significant SNP was ASGA0025245 on SSC5. We defined the QTL confidence interval using LOD drop off 2 and r2?0.8 from the strongest SNP. As a result,the confidence intervals of QTL in the F2, Sutai, Laiwu and DLY were 471.75 kb, 1.09 Mb, 1.11 Mb and 709.61 kb, respectively. We defined the QTL interval to 471.75 kb (32.97 Mb - 33.44 Mb)through combining the overlapped region of the four QTL. We further conducted the haplotype sharing analysis. The haplotypes were constructed in 4 Erhualian, 6 Minzhu, 3 Wuzhishan and 3 Tibetan pigs with 118 SNPs in a 1.18-Mb QTL region. As a result, we found that the large-eared pigs shared a 345.54-kb (32.76 - 33.10 Mb) and a 235.29-kb(32.52 - 32.76 Mb) region. Through combining the GWAS and haplotype shared results,the empirical QTL region was defined to 138.05 kb (32.97 - 33.10 Mb). This region covered the exon 2-7 of MSRB3 gene and 15.14 kb downstream of MSRB3, So MSRB3 gene was the most likely causative gene.We totally found six alternative splicing transcripts by analyzing the mRNA of MSRB3, but did not find any missense mutations. We also analyzed the expression profile of the six transcripts of MSRB3 gene, HMGA2 and LEMD3 gene in the ear tissue QQ and qq piglets. However, we did not find any differential expression. But we really found the differential protein level between QQ and qq individuals in western blot and immunohistochemistry analysis. We took the advantage of whole genome resequencing datas to find the structural variations in QTL region, we found a 38.4-kb duplication which overlapped with the last two exons of MSRB3 gene in all F0 Erhualian sows. The CNV was genotyped by using qPCR. We found that the genotypes of CNV was completely concordant with the QTL genotype and phenotype, but we did not find any other variants that were concordance with QTL genotype. This result suggested that, the CNV was the causative mutation of this QTL. When the CNV was fixed in the GWAS model, the GWAS signal disappeared, suggesting the CNV explained all of the QTL effect. Compared with the peak SNPs in GWAS, the CNV explained more phenotypic variation in Sutai, Laiwu and DLY. As we found differential expression of MSRB3 protein, but not in the mRNA level between QQ and qq individuals, it indicated that the translation was inhibited.Interestingly, we found miR-584 that locate at the 3' downstream of CNV and has the binding site in 3'UTR of MSRB3. We discovered the expression of miR-584 in different QTL genotype individuals, and found that its expression level was corresponded to the protein expression level. We screened all the variants between CNV and miR-584. Except CNV, we did not find any other variants that consistent with QTL genotype and phenotype.So, we concluded that the MSRB3 was the causative gene for QTL on SSC5, and the CNV overlapped with MSRB3 was the causative mutation. The findings from this study provided novel insights into the genetic mechanism for human microtia and associated diseases.