Chloroplast Microsatellite-Based High-Resolution Melting Analysis for Authentication and Discrimination of Ilex Species

Ilex species are important sources of high-quality raw plant materials for the production of drugs and functional foods. The precise identification of different species within the Ilex genus would greatly facilitate authentication and certification as well as forest resource monitoring in plantations. Combining DNA barcoding with high-resolution melting (HRM) analysis represents a robust strategy for species discrimination, as demonstrated in recent DNA barcoding studies. Here, using concatenated and aligned complete chloroplast genomes of different Ilex species, we conducted a sliding window analysis to identify regions of high nucleotide diversity (Pi). We optimized and validated the utility of PCR-based HRM coupled with microsatellite markers to discriminate among the four Ilex species, Ilex integra Thunb., Ilex rotunda Thunb., Ilex cornuta Lindl. and Paxton, and Ilex x wandoensis C.F. Mill and M. Kim, from wild populations in southwestern Korea. The marker trnS^UGA-psbZ produced clear melting patterns and distinct melting curve profiles for the four Ilex species using HRM analysis. We applied this protocol to commercially available Ilex accessions and consistently identified the correct species for all 15 accessions tested. Therefore, combining DNA barcoding with HRM analysis is a powerful method for identifying different species within the same genus, which could be used for quality control of raw materials in the functional food/medicinal plant industry.