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.

최근에 감에서 떫은맛을 조절하는 AST에 연관된 지역에서 분자 표지들이 개발되었다. 이중에서 sequence characterized amplified region (SCAR) marker는 5R region에 인접한 지역에서 개발되었다. 하지만 이 SCAR마커는 분석 방법이 다소 복잡하고 해석이 어려워 많은 교배실생을 분석할 경우에는 적합하지 않다. 우리는 5R 지역의 sequences에 기반하여 high-resolution melting (HRM)-based 분자 표지를 개발하였다. 개발된 HRM preimer set을 8개 품종의 단감 및 떫은감에 대해 적용한 결과 단감 품종에서는 직선을 나타낸 반면 떫은감 품종에서는 다양한 크기의 곡선으로 나타나서 차이를 확인할 수 있었다. 결과적으로 이번 연구에서 개발된 HRM primer set은 분자 표지를 활용한 감 품종 육성 연구에 매우 효율적으 로 활용될 수 있을 것으로 기대된다.

Background : Correct identification of Panax species is important to ensure food quality, safety, authenticity and health for consumers. This paper describes a high resolution melting (HRM) analysis based method using internal transcribed spacer (ITS) and 5.8S ribosomal DNA barcoding regions as target (Bar-HRM) to obtain barcoding information for the major Panax species and to identify the origin of ginseng plant. Methods and Results : A PCR-based approach, Bar-HRM was developed to discriminate among Panax species. In this study, the ITS1, ITS2, and 5.8S rDNA genes were targeted for testing, since these have been identified as suitable genes for use in the identification of Panax species. The HRM analysis generated cluster patterns that were specific and sensitive enough to detect small sequence differences among the tested Panax species. Conclusion : The results of this study show that the HRM curve analysis of the ITS regions and 5.8S rDNA sequences is a simple, quick, and reproducible method. It can simultaneously identify three Panax species and screen for variants. Thus, ITS1HRM and 5.8SHRM primer sets can be used to distinguish among Panax species.