High-resolution melting (HRM) analysis is a simple, fast, and inexpensive real-time polymerase chain reaction (PCR)-based method used to identify genetic variation between populations and detect single-nucleotide polymorphisms (SNPs) in nucleic acid sequences. HRM is a powerful technique that detects the differences between SNP allele melting temperatures by using a fluorescent dye inserted into the duplex deoxyribonucleic acid (DNA) structure. Prior to performing HRM analysis, optimizing the primer design, PCR mixture, and software settings is essential to obtain accurate and reliable results. In this chapter, we describe a detailed SNP genotyping method that includes primer design and the analysis of the shapes and positions of the melt curve of the luminescence intensity of the fluorescent dye attached to amplified DNA using software of qPCR instruments. This protocol is applicable for genotyping germplasm, genetic mapping, and marker-assisted breeding in plants.
Keywords: Genetic variation; HRM; High-resolution melting analysis; Real-time PCR; SNP genotyping; Single-nucleotide polymorphism.
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