한국어류학회지 (Korean Journal of Ichthyology)

  • 제34권3호
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  • Pages.208-217
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  • 2022
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  • 1225-8598(pISSN)
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  • 2288-3371(eISSN)
한국어류학회 (The Ichthyological Society of Korea)
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멸종위기어류 퉁사리의 환경 DNA 분석을 위한 종 특이 마커 개발

Species-specific Marker Development for Environmental DNA Assay of Endangered Bull-head Torrent Catfish, Liobagrus obesus

  • 윤봉한 (순천향대학교 생명과학과) ;
  • 김용휘 (순천향대학교 생명과학과) ;
  • 성무성 (순천향대학교 생명과학과) ;
  • 한호섭 (순천향대학교 생명과학과) ;
  • 한정호 (한국수자원공사 물환경개선처) ;
  • 방인철 (순천향대학교 생명과학과)
  • Yun, Bong Han (Department of Biology, Soonchunhyang University) ;
  • Kim, Yong Hwi (Department of Biology, Soonchunhyang University) ;
  • Sung, Mu Sung (Department of Biology, Soonchunhyang University) ;
  • Han, Ho-Seop (Department of Biology, Soonchunhyang University) ;
  • Han, Jeong-Ho (Water Environmental Improvement Dept., Korea Water Resources Corporation) ;
  • Bang, In-Chul (Department of Biology, Soonchunhyang University)
  • 투고 : 2022.08.16
  • 심사 : 2022.09.28
  • 발행 : 2022.09.30
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초록

멸종위기어류 퉁사리 Liobagrus obesus를 대상으로 종 특이 프라이머 한 쌍과 프로브를 제작하여 하천수 시료에서 추출된 환경 DNA로부터 퉁사리를 검출할 수 있는 실시간 PCR 분석방법을 개발하고자 하였다. 퉁사리 종 특이 프라이머와 프로브는 미토콘드리아 DNA의 cytochrome b (cytb) 유전자 영역 내에서 국내에 서식하는 65종의 담수어류 간에 단일염기다형성 부위를 고려하여 비교한 후 제작하였다. 실시간 PCR 분석에서 제작한 프라이머 및 프로브는 국내에 서식하는 65종의 담수어류 gDNA를 이용한 특이성 검증 결과, 퉁사리 gDNA에서만 양성으로 나타나 높은 특이성을 보였다. 퉁사리 gDNA의 연속 희석 농도를 이용한 검출한계 분석에서는 0.2 pg까지 검출이 가능한 것으로 나타나 높은 감도를 보였다. 이후, 제작한 프라이머 및 프로브를 사용하여 금강 중·상류 유역의 8개 지점에서 확보한 하천수 시료를 대상으로 실시간 PCR 분석을 수행한 결과, 5개 지점에서 퉁사리의 cytb 유전자가 검출되었으며, 해당 검출 지점들은 현장 조사 당시에 퉁사리가 채집된 3개 지점을 모두 포함하였다. 따라서, 본 연구에서 개발한 퉁사리의 종 특이 프라이머와 프로브를 이용한 실시간 PCR 분석 방법은 하천수 채수로 확보한 환경 DNA로부터 퉁사리의 cytb 유전자를 검출할 수 있어 기존 서식지 모니터링과 더불어 잠재적인 신규 서식지 발굴에 활용될 수 있을 것으로 판단된다.

We wanted to develop a real-time PCR assay capable of detecting Liobagrus obesus in environmental DNA (eDNA) extracted from freshwater samples using a pair of species-specific primers and probe for the endangered fish, L. obesus. The species-specific primers and probe were designed in consideration of single nucleotide polymorphisms between 65 species of freshwater fish living in the Republic of Korea within the cytochrome b (cytb) gene of mitochondrial DNA. The species-specific primers and probe, in the real-time PCR assay, showed high specificity as only the L. obesus genomic DNA (gDNA) was found to be positive in the specificity verification using 65 species gDNA of freshwater fish in the Republic of Korea. In addition, in the detection limit analysis using the serial dilution concentrations of L. obesus gDNA, it was found that it was possible to detect up to 0.2 pg, showing high sensitivity. Afterwards, using the species-specific primers and probe, real-time PCR assay was performed on freshwater samples obtained from 8 stations in the mid-upper basin of Geum River. As a result, the cytb gene of L. obesus was detected in total 5 stations including all 3 stations where this species was collected at the time of field survey. Therefore, the species-specific primers and probe developed in present study, and the real-time PCR assay using them, can accurately detect the cytb gene of L. obesus from eDNA samples, which can be utilized to monitor the existing habitats of this species and to discover potential new habitats.

키워드

과제정보

실험 설계에 도움을 주신 국립생태원 멸종위기종복원센터의 김근식 박사님께 감사의 말씀을 전해드립니다. 본 연구는 한국수자원공사 (과제명: 댐 유역 하천의 멸종위기어류 정밀모니터링 및 복원방안 연구 용역)와 순천향대학교의 연구비 지원을 받아 수행되었습니다.

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