Application of HRM analysis for specific DNA-based identification of the Hero green cultivar in Garcinia schomburgkiana

Garcinia schomburgkiana Pierre (“Madan”) is an economically important plant in Thailand widely used in food and cosmetic products. The Hero Green (HRG) cultivar has attracted commercial interest because of its large fruit size and suitability for industrial processing. However, morphological similarity among cultivars complicates accurate identification. This study aimed to develop a rapid DNA-based method for discrimination of the HRG cultivar using DNA sequencing and high-resolution melting (HRM) analysis. Chloroplast ( matK and rbcL ) and nuclear ITS regions were amplified and sequenced from HRG, Kiew Song La (KSL), and indigenous Madan varieties. Sequence analysis revealed limited variation in chloroplast markers, whereas the ITS1 region contained a unique single nucleotide polymorphism specific to HRG. Based on this variation, a novel primer pair targeting ITS1 was designed for HRM analysis. The HRM assay generated distinct and reproducible melting profiles that clearly differentiated HRG from KSL and indigenous varieties, with significant differences in melting temperature ( p = 0.0095). Principal Component Analysis and Hierarchical Cluster Analysis consistently separated HRG from other cultivars. The limit of detection (LOD) was determined to be 0.01 ng/μL. This HRM-based method provides a rapid, sensitive, and cost-effective approach for authentication of the HRG cultivar and may support quality control of Madan raw materials in food, herbal, and cosmetic industries.