Genome-wide Association Studies For Porcine Ear Size And Exploration Of Major Genes

Ear size and erectness exhibit remarkable diversity in pig breeds, and these traits play importantroles in pig breed standards. It is considered as a conformation characteristic for distinguishing pigbreeds, especially in Chinese local pig breeds. Besides, pig breeds could be regarded as perfect aimalmodels for human Microtia study. The quantitative trait loci(QTL) for porcine ear size was previouslyreported to mainly focus on SSC5 and SSC7. Recently, a missense mutation, G32 E, in PPARD in theQTL interval on SSC7 was identified as the causative mutation for ear size. However, on account of thelarge interval of QTL, the major gene on SSC5 has not been identified. In this study, a Genome-WideAssociation Study(GWAS) was carried out to screen the major candidate genes, and then the candidateswere analysed for identifying the major gene and causative mutations.In this study, an intercross population was constructed from the large-eared Minzhu, anindigenous Chinese pig breed, and the western commercial Large White pig to examine the geneticbasis of ear size diversity. A GWAS was performed to detect SNPs significantly associated with ear size.Thirty-five significant SNPs defined a 10.78-Mb(30.14-40.92 Mb) region on SSC5. Further, combininglinkage disequilibrium and haplotype sharing analysis, a reduced region of 3.07-Mb was obtained.Finally, by using a selective sweep analysis, a critical region of about 450-Kb interval containing twoannotated genes LEMD3 and WIF1 was refined in this work. Functional analysis indicated that bothrepresent biological candidates for porcine ear size, with potential application in breeding programs.The two genes could also be used as novel references for further study of the mechanism underlyinghuman microtia.To identify the major gene and causative mutations for ear size on SSC5, preliminary studies werecarried out with genes of WIF1, LEMD3 and HMGA2. Since the full-length m RNAs of porcine WIF1,LEMD3 and HMGA2 were not available in the databases, we firstly cloned full-length c DNAs of thesegenes by using RACE. The length of WIF1, LEMD3 and HMGA2 c DNAs was 2338 bp, 4843 bp and2998 bp. To detect the polymorphisms of these three genes, sequence amplification and alignment wereperformed to screen all exons and promoter regions, and a total of 23 polymorphisms were identified(11 in WIF1, 3 in LEMD3 and 9 in HMGA2). Association of these detected SNPs with ear size wasassessed in two experimental populations(Large White × Minzhu F2 population and Beijing Black pigpopulation). A total of 13 SNPs(10 in WIF1 and 3 in LEMD3) were found significantly associated withear size(P < 0.01) in both populations, other SNPs were not found in Beijing Black pig population.Tissue expression patterns of these genes showed that WIF1 expressed extremely high in earcartilage; differential expressions were analysed in ear cartilages of large-eared Erhualian pigs andmiddle-eared Large White pigs, both in m RNA level and protein level, and only found WIF1 expressedsignificantly different both in m RNA level(P<0.05) and protein level(P<0.01) in the two pig breeds.So, WIF1 was more likely to be the major gene for ear size, its function mechanism needs furtherverification.Together, in this study, a significant region associated with ear size was mapped on SSC5, then wefine-mapped this region and found WIF1 and LEMD3 to be the major candidate genes. Our researchmay have important theoretical significance on major gene exploration after GWAS, and can providemolecular markers for germplasm resources appraisal of pig breeds. The findings of this research alsoprovided a good model and reference for molecular regulation mechanism of ear diversity in human andother animals.