Korean Journal of Ecology and Environment (생태와환경)

The Korean Society of Limnology (한국수생태학회)
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Efficiency Comparison of Environmental DNA Metabarcoding of Freshwater Fishes according to Filters, Extraction Kits, Primer Sets and PCR Methods

분석조건별 담수어류의 환경 DNA 메타바코딩 효율 비교: 필터, 추출 키트, 프라이머 조합 및 PCR 방법

  • Kim, Keun-Sik (Research Center for Endangered Species, National Institute of Ecology) ;
  • Kim, Keun-Yong (AquaGenTech Co., Ltd) ;
  • Yoon, Ju-Duk (Research Center for Endangered Species, National Institute of Ecology)
  • 김근식 (국립생태원 멸종위기종복원센터) ;
  • 김근용 (아쿠아진텍(주)) ;
  • 윤주덕 (국립생태원 멸종위기종복원센터)
  • Received : 2021.09.09
  • Accepted : 2021.09.15
  • Published : 2021.09.30
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Abstract

Environmental DNA (eDNA) metabarcoding is effective method with high detection sensitivity for evaluating fish biodiversity and detecting endangered fish from natural water samples. We compared the richness of operational taxonomic units(OTUs) and composition of freshwater fishes according to filters(cellulose nitrate filter vs. glass fiber filter), extraction kits(DNeasy2® Blood & Tissue Kit vs. DNeasy2® PowerWater Kit), primer sets (12S rDNA vs. 16S rDNA), and PCR methods (conventional PCR vs. touchdown PCR) to determine the optimal conditions for metabarcoding analysis of Korean freshwater fish. The glass fiber filter and DNeasy2® Blood & Tissue Kit combination showed the highest number of freshwater fish OTUs in both 12S and 16S rDNA. Among the four types, the primer sets only showed statistically significant difference in the average number of OTUs in class Actinopterygii (non-parametric Wilcoxon signed ranks test, p=0.005). However, there was no difference in the average number of OTUs in freshwater fish. The species composition also showed significant difference according to primer sets (PERMANOVA, Pseudo-F=6.9489, p=0.006), but no differences were observed in the other three types. The non-metric multidimensional scaling (NMDS) results revealed that species composition clustered together according to primer sets based on similarity of 65%; 16S rDNA primer set was mainly attributed to endangered species such as Microphysogobio koreensis and Pseudogobio brevicorpus. In contrast, the 12S rDNA primer set was mainly attributed to common species such as Zacco platypus and Coreoperca herzi. This study provides essential information on species diversity analysis using metabarcoding for environmental water samples obtained from rivers in Korea.

메타바코딩을 이용한 환경 DNA 분석은 검출 감도가 높아 어류의 생물다양성 평가 및 멸종위기종의 검출에 유용한 기술이다. 이번 연구는 메타바코딩을 이용해 우리나라 담수어류를 대상으로 높은 검출 효율을 보일 수 있는 적합한 분석방법을 확인하기 위해 4가지 분석조건별, 즉 필터(cellulose nitrate filter, glass fiber filter), 추출 키트(DNeasy® Blood & Tissue Kit, DNeasy® PowerWater Kit), 프라이머 조합(12S rDNA, 16S rDNA) 그리고 PCR 방법(conventional PCR, touchdown PCR)로 나타나는 Operational Taxonomic Units(OTUs) 수와 종 조성을 비교하였다. Glass fiber filter와 DNeasy® Tissue & Blood Kit를 이용해 추출한 시료는 12S rDNA와 16S rDNA 프라이머 조합에서 담수어류 OTUs가 가장 많이 검출되었다. 모든 분석조건 중 프라이머 조합에서만 조기어강(Class Actinopterygii) 평균 OTUs 수에서 통계적으로 유의한 차이를 보였고(Non-parametric Wilcoxon Signed Ranks Test, p=0.005), 담수어류 평균 OTUs 수는 유의하지 않았다. 종 조성 비교 결과 역시 프라이머 조합에서 유의한 차이를 보였고(PERMANOVA, Pseudo-F=6.9489, p=0.006), 나머지 분석조건에서는 유의한 차이를 보이지 않았다. NMDS 분석 결과 종 조성은 유사도 65% 기준에서 프라이머 조합에 따라 묶였고, 16S rDNA 프라이머 세트는 주로 멸종위기종인 모래주사(Microphysogobio koreensis), 꼬치동자개(Pseudogobio brevicorpus)가 기여하였고, 12S rDNA 프라이머 세트는 주로 일반종인 피라미(Zacco platypus), 꺽지(Coreoperca herzi) 등이 기여한 것으로 나타났다. 본 연구는 국내 하천에서 채취한 시료에 대한 메타바코딩을 이용한 종 다양성 분석의 기초정보를 제공한다.

Keywords

Acknowledgement

본 논문은 환경부의 재원으로 국립생태원의 지원을 받아 수행하였습니다(NIE-기반연구-2021-45).

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