Korean Journal of Environmental Biology (환경생물)

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

  • Nakyung Yoo (Research Center for Endangered Species, National Institute of Ecology) ;
  • Keun-Yong Kim (AquaGenTech Co., Ltd) ;
  • Jung Soo Heo (AquaGenTech Co., Ltd) ;
  • Ju-Duk Yoon (Research Center for Endangered Species, National Institute of Ecology) ;
  • Keun-Sik Kim (Research Center for Endangered Species, National Institute of Ecology)
  • Received : 2022.05.03
  • Accepted : 2022.06.17
  • Published : 2022.06.30
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Abstract

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 maternally-inherited mitochondrial 12S ribosomal DNA region. Based on the sequence data, we designed a pair of primers containing 7bp 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℃, whereas that of D. japonicus was 75.0±0.05℃. 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℃ 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.

Keywords

Acknowledgement

This work was supported by a grant from the National Institute of Ecology (NIE), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIE-B-2022-45). We thank J.W. Yoo for support on the sample collection.

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