Fine Mapping Of The QTL For Ear Size On Pig Chromosome5and Preliminary Identification Of The Causative Gene

Ear size and erectness are important conformation characteristics of pig breeds. Moreover, ear is an important part of the auditory system. There have been many reports about congenital external ear diseases in human. In our previous study, we identified a significant QTL for ear size within an11.0-cM interval between SWR453and SW1987on SSC5through a genome-wide scan in a large scale White Duroc x Erhualian F2intercross. This QTL explained17.14%of the phenotypic variance.Here we fine-mapped this major QTL on SSC5to identify the causative gene for ear size. We developed4new microsatellite markers in the QTL interval and genotyped these markers across the White Duroc x Erhualian resource population. The QTL was then refined to an8.7cM interval between markers SSC5P3and SW2003. Subsequently, HMGA2, SOX5and PTHLH located in the8.7cM QTL region was selected as the positional candidate genes. Direct sequencing of4F1boars,70polymorphisms were detected within these genes and seven highly informative SNPs were selected and genotyped in the F2population by SNaPShot assays. Of the seven SNP, HMGA2SNP g.2836A>G showed the strongest association with ear size in the standard association test and marker-assisted association test (P=9.19E-46and1.92E-05, respectively). With the F-drop test, F value decreased by more than97%only when the genotypes of HMGA2g.2836A>G were included as a fixed effect. The haplotype-based association test showed that the phenotypic variance explained by HMGA2was similar to that explained by the QTL. However, the significant association between HMGA2g.2836A>G and ear size was only observed in the synthetic commercial Sutai line, but not in Sujiang and Suzhong pigs.To further improve the mapping resolution, the White Duroc x Erhualian intercross and a Sutai pig line were genotyped with Illumina porcine60k DNA chips. Genome-wide association (GWAS) results showed that21SNPs on SSC5were significantly associated with ear size in the White Duroc x Erhualian intercross, of which H3GA0016181, ALGA0031524and ASGA0025253were the three most significantly SNPs (corrected P value=0.0096). H3GA0016181and ASGA0025253were the most significant SNPs associated with ear size in the Sutai pig line (corrected P value=0.00049). The two SNPs located within the intergenic region between MSRB3and HMGA2and the intron region of HMGA2, respectively. The results from both Linkage disequilibrium and Linkage analysis(LDLA) and Linkage analysis (LA) showed that ALGA0031518and ALGA0031519were the most significantly associated SNP (P=1.656E-45and7.13E-49, respectively). Both SNPs were mapped to the intronic region of MSRB3. The most likely QTL positions mapped to a2.67Mb (ALGA0031489-ASGA0025275) or2.32Mb (ALGA0031500-ASGA0025287) region through LOD score dropoff of2of the most significant SNPs. We reconstructed the haplotypes of all19founder animals (2sires and17dams) in the2.32Mb refined QTL region with DualPHASE software. A haplotype of-1.28 Mb within the refined2.32Mb interval was shared by31Erhualian Q-bearing chromosomes. This shared haplotype is associated with an increase in ear size. Taken together, we refined the QTL region to the1.28Mb chromosome segment. This region encompasses6annotated genes included XPOT, TBC1D30, WIF1, LEMD3, MSRB3and HMGA2. RT-PCR was performed to detect expression levels of these genes in ear tissues. The results showed that except WIF1, all genes were expressed in the ear tissue. To further narrow the confidence interval of SSC5QTL,191SNPs in the1.28Mb critical interval were selected and genotyped in the slaughtered Sutai pig line and another well phenotyped Wujiang adult Sutai pig line. The association results showed that the most likely QTL position (LOD dropoff of2) located in a-925Kb region. This region encompasses4annotated genes included WIF1, LEMD3, MSRB3and HMGA2. Because WIF1was not expressed in the ear tissue, LEMD3, MSRB3and HMGA2is hence the most likely responsible gene for the major QTL.This study contributed to identify the causative gene and mutation underlying this major QTL.