Genotyping Of The Porcine Ryanodine Receptor1(RYR1) And Estrogen Receptor1(ESR1) Genes By High Resolution Melting (HRM) Approach

DNA mutations in the porcine ryanodine receptor gene (RYR1, C1843T) and the estrogen receptor1gene(ESR1, T1665G), have been widely used in marker-assisted selection (MAS) for the pig industry. These two well-characterized SNPs in RYR1and ESRI are responsible for porcine stress syndrome (PSS) and litter size, respectively. Here, we describe a high-efficiency method for genotyping of these two genes using the high-resolution melting (HRM) method in base of the exsiting PCR. In order to establish a reliable method with high accuracy and repeatability for determining the genotype, we then evaluated the repeatability and the accuracy of the HRM approach.We determined the genotypes of RYR1and ESR1genes for300pigs from three foreign pig breeds (Yorkshire, Landrace and Duroc) using the established HRM assay. We further determined the samples with inconsistent genotyping results between the HRM assay and PCR-RFLP method through direct sequencing analysis. The results showed the accuracy of RYR1genotype was99.6%, and ESRI genotype was99.3%by HRM assay, of which presented3.3and2.3percent higher than PCR-RFLP method respectively. The result indicated that the established HRM genotyping approach can distinguish accurately three different genotypes for both RYR1and ESRI genes. Besides, HRM genotyping approach exhibited high repeatability with very low Coefficient of variation (C.V) of Tm values among the intra-and inter-runs for both RYR1(C.Vintra-run≤0.08%, C.Vnter-run≤0.10%) and ESR genes (C.Vintra-run≤0.07%, C.Vinter-run≤0.11%).Test of accuracy and repeatability indicated we had successful constructed a HRM genotyping approach for RYR1and ESRI genes with high repeatability and up to99%accuracy, establishing a rapid, high-efficiency, cost-efficient, safe, comparable accuracy, reliable genotyping procedure. We had applied for two patents for rapid genotyping kits of RYR1and ESRI genes.