Korean Journal of Ichthyology (한국어류학회지)

The Ichthyological Society of Korea (한국어류학회)
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Species-specific Marker Development for Environmental DNA Assay of Endangered Bull-head Torrent Catfish, Liobagrus obesus

멸종위기어류 퉁사리의 환경 DNA 분석을 위한 종 특이 마커 개발

  • 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)
  • 윤봉한 (순천향대학교 생명과학과) ;
  • 김용휘 (순천향대학교 생명과학과) ;
  • 성무성 (순천향대학교 생명과학과) ;
  • 한호섭 (순천향대학교 생명과학과) ;
  • 한정호 (한국수자원공사 물환경개선처) ;
  • 방인철 (순천향대학교 생명과학과)
  • Received : 2022.08.16
  • Accepted : 2022.09.28
  • Published : 2022.09.30
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Abstract

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.

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.

Keywords

Acknowledgement

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.

References

  1. Chae, B.S., H.B. Song and J.Y. Park. 2019. A field guide to the freshwater fishes of Korea. LG Evergreen Foundation, Seoul, Republic of Korea, 355pp.
  2. Chang, C.H., F. Li, K.T. Shao, Y.S. Lin, T. Morosawa, S.M. Kim, H.Y. Koo, W. Kim, J.S. Lee, S. He, C. Smith, M. Reichard, M. Miya, T. Sado, K. Uehara, S. Lavoue, W.J. Chen and R.L. Mayden. 2014. Phylogenetic relationships of Acheilognathidae (Cypriniformes: Cyprinoidea) as revealed from evidence of both nuclear and mitochondrial gene sequence variation: evidence for necessary taxonomic revision in the family and the identification of cryptic species. Mol. Phylogenet. Evol., 81: 182-194. https://doi.org/10.1016/j.ympev.2014.08.026.
  3. Ficetola, G.F., C. Miaud, F. Pompanon and P. Taberlet. 2008. Species detection using environmental DNA from water samples. Biol. Lett., 4: 423-425. http://doi.org/10.1098/rsbl.2008.0118.
  4. Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser., 41: 95-98.
  5. Han, M.S., K.S. Choi and M.H. Ko. 2020. Has the Restoration Project of Pseudopungtungia tenuicorpa (Pisces: Cyprinidae) in the Jojongcheon Stream, Hangang River Failed?. Korean J. Ichthyol., 32: 182-190. https://doi.org/10.35399/ISK.32.3.8
  6. Ji, Y., L. Ashton, S.M. Pedley, D.P. Edwards, Y. Tang, A. Nakamura, R. Kitching, P.M. Dolman, P. Woodcock, F.A. Edwards, T.H. Larsen, W.W. Hsu, S. Benedick, K.C. Hamer, D.S. Wilcove, C. Bruce, X. Wang, T. Levi, M. Lott, B.C. Emerson and D.W. Yu. 2013. Reliable, verifiable and efficient monitoring of biodiversity via metabarcoding. Ecol. Lett., 16: 1245-1257. https://doi.org/10.1111/ele.12162.
  7. Jo, T., A. Fukuoka, K. Uchida, A. Ushimaru and T. Minamoto. 2020. Multiplex real-time PCR enables the simultaneous detection of environmental DNA from freshwater fishes: a case study of three exotic and three threatened native fishes in Japan. Biol. Invasions., 22: 455-471. https://doi.org/10.1007/s10530-019-02102-w.
  8. Jo, T., S. Ikeda, A. Fukuoka, T. Inagawa, J. Okitsu, I. Katano, D. Hideyuki, N. Katsuki, I. Hidetaka and T. Minamoto. 2021. Utility of environmental DNA analysis for effective monitoring of invasive fish species in reservoirs. Ecosphere, 12: e03643. https://doi.org/10.1002/ecs2.3643.
  9. Kim, H.S., H. Yang and Y.K. Hong. 2012. Spawning site characters in the natural environment of bull-head torrent catfish, Ligbagrus obesus (Siluriformes: Amblycipitidae) in the Gosan stream, Mangyeong river water system, Korea. Korean J. Ichthyol., 24: 183-190.
  10. Kim, I.S. and J.Y. Park. 2002. Freshwater Fishes of Korea. Kyohak Publishing Co. Ltd., Seoul, Korea, 466pp.
  11. Kim, M.J., Y. Hong and H.Y. Kim. 2014. Authentication of Foods from Animal Origins using Gene Analysis. Food Science and Industry, 47: 13-19. https://doi.org/10.23093/FSI.2014.47.2.13
  12. Kwon, K.R., S.I. Baek and S.H. Choi. 2009. Identification of Fel ursi and Cattle and Pig Bile Juices by species-specific PCR and PCR-RFLP. J. Pharmacopuncture, 12: 13-20. https://doi.org/10.3831/KPI.2009.12.1.013.
  13. Laramie, M.B., D.S. Pilliod and C.S. Goldberg. 2015. Characterizing the distribution of an endangered salmonid using environmental DNA analysis. Biol. Conserv., 183: 29-37. https://doi. org/10.1016/j.biocon.2014.11.025.
  14. ME(Ministry of Environment). 2006. Studies on the genetic diversity, artificial propagation and ex situ restoration of a threatened national monument fish Hemibarbus mylodon. Soonchunhyang University, Asan, Korea, 520pp.
  15. ME (Ministry of Environment). 2011. Culture and restoration research of endangered freshwater fish (four species include Liobagrus obesus). Soonchunhyang University, Asan, Korea, 359pp.
  16. ME (Ministry of Environment). 2017. Conservation and management laws of wildlife (amendment of enforcement regulations)(Law No. 10977).
  17. ME (Ministry of Environment). 2019. A study on conservation plan of endangered freshwater fish (Microphysogobio rapidus, Pseudobagrus brevicorpus). Soonchunhyang University, Asan, Korea, 214pp.
  18. ME (Ministry of Environment). 2020. Research on the ecological characteristic and conservation of endangered species(Kichulchoia brevifasciata, Gobiobotia macrocephala, Microphysogobio rapidus and Pseudobagrus brevicorpus). Research Center for Endangered Species, National Institute of Ecology, Yeongyang, Korea, 313pp.
  19. MLTM (Ministry of Land, Transport and Maritime Affairs). 2010. Culture and restoration of endangered species in the major four river drainages. Soonchunhyang University, Asan, Korea, 489pp.
  20. MLTM (Ministry of Land, Transport and Maritime Affairs). 2011. Culture and restoration of endangered species in the major four river drainages II. Soonchunhyang University, Asan, Korea, 363pp.
  21. MLTM (Ministry of Land, Transport and Maritime Affairs). 2012. Culture and restoration of endangered species in the major four river drainages III. Soonchunhyang University, Asan, Korea, 423pp.
  22. NIBR(National Institute of Biological Resources). 2019. Red data book of Republic of Korea, Volume 3. Freshwater fishes. Ministry of Environment, National Institute of Biological Resources, Incheon, Korea, 250pp.
  23. NIE (National Institute of Ecology). 2019. Specific monitoring of distribution of Gobiobotia naktongensis(endangered species level 1) in Geum River watershed. Research Center for Endangered Species, National Institute of Ecology, Yeongyang, Korea, 89pp.
  24. Piggott, M.P., S.C. Banks, B.T. Broadhurst, C.J. Fulton and M. Lintermans. 2020. Comparison of traditional and environmental DNA survey methods for detecting rare and abundant freshwater fish. Aquat Conserv., 31: 173-184. https://doi.org/10.1002/ aqc.3474.
  25. Rozas, J., A. Ferrer-Mata, J.C. Sanchez-DelBarrio, S. Guirao-Rico, P. Librado, S.E. Ramos-Onsins and A. Sanchez-Gracia. 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol. Biol. Evol., 34: 3299-3302. https://doi.org/10.1093/molbev/msx248.
  26. Sakata, M.K., N. Maki, H. Sugiyama and T. Minamoto. 2017. Identifying a breeding habitat of a critically endangered fish, Acheilognathus typus, in a natural river in Japan. Sci. Nat., 104: 1-8. https://doi.org/10.1007/s00114-017-1521-1.
  27. Son, Y.M. and H.K. Byeon. 2004. Ecological characteristics and inhabitation condition of Liobagrus obesus. In: Proc. of Fall Academic Symposium of the Ichthyological Society of Korea, pp. 29-46.
  28. Son, Y.M. and I.Y. Choo. 1988. Ecological studies of catfish, genus Liobagrus from Korea. Korea J. Limnol., 21: 243-251.
  29. Son, Y.M., I.S. Kim and I.Y. Choo. 1987. A new species of torrent catfish, Liobagrus obesus from Korea. Korean J. Limnol., 20: 21-29.
  30. Taboada, L., A. Sanchez and C.G. Sotelo. 2017. A new real-time PCR method for rapid and specific detection of ling (Molva molva). Food Chem., 228:469-475. https://doi.org/10.1016/j.foodchem.2017.01.117.
  31. Takahara, T., T. Minamoto and H. Doi. 2013. Using environmental DNA to estimate the distribution of an invasive fish species in ponds. PloS One, 8: e56584. https://doi.org/10.1371/journal.pone.0056584.
  32. Thompson, J.D., D.G. Higgins and T.J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22: 4673-4680. https://doi.org/10.1093/nar/22.22.4673.
  33. Thomsen, P.F. and E. Willerslev. 2015. Environmental DNA-An emerging tool in conservation for monitoring past and present biodiversity. Biol. Conserv., 183: 4-18. https://doi.org/10.1016/j.biocon.2014.11.019.
  34. Xu, W., M. Fu, M. Huang, X. Cui, Y. Li, M. Cao, L. Wang, X. Xiong and X. Xiong. 2021. Duplex real-time PCR combined with melting curve analysis for rapid detection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). J. Food. Compos. Anal., 97: 103765. https://doi.org/10.1016/j.jfca.2020.103765.
  35. Ye, J., J. Feng, S. Liu, Y. Zhang, X. Jiang and Z. Dai. 2016. Identification of four squid species by quantitative real-time polymerase chain reaction. Mol. Cell. Probes., 30: 22-29. https://doi.org/10.1016/j.mcp.2016.01.001.