Korean Journal of Environmental Biology Vol. 40 No. 2 (p.199-205)

Novel non-invasive molecular identification method for two tree frogs, Dryophytes suweonensis and Dryophytes japonicus, based on high resolution melting (HRM) analysis

키워드 :
endangered species,HRM analysis,eDNA,non-invasive,tree frogs


   1. Sample collection and genomicDNA extraction
   2. Sample preparation for eDNA analysis
   3. PCR primer design and DNA sequencing
   4. HRM analysis


Two tree frogs, Dryophytes suweonensis and Dryophytes japonicus, inhabiting Korea, are morphologically similar and share the same habitats. Therefore, they are identified mainly through their calls, especially for males. Dryophytes suweonensis is registered as an endangered (IUCN: EN grade) and protected species in South Korea. Thus, it is necessary to develop a method to rapidly identify and discriminate the two species and establish efficient protection and restoration plans. We identified significant genetic variation between them by sequencing a maternallyinherited mitochondrial 12S ribosomal DNA region. Based on the sequence data, we designed a pair of primers containing 7 bp differences for high resolution melting (HRM) analysis to rapidly and accurately characterize their genotypes. The HRM analysis using genomic DNA showed that the melting peak for D. suweonensis was 76.4±0.06°C, whereas that of D. japonicus was 75.0±0.05°C. The differential melt curve plot further showed a distinct difference between them. We also carried out a pilot test for the application of HRM analysis based on immersing D. suweonensis in distilled water for 30 min to generate artificial environmental DNA (eDNA). The results showed 1.10-1.31°C differences in the melting peaks between the two tree frog samples. Therefore, this HRM analysis is rapid and accurate in identifying two tree frogs not only using their genomic DNA but also using highly non-invasive eDNA.