Browse > Article
http://dx.doi.org/10.35399/ISK.34.3.7

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

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)
Publication Information
Korean Journal of Ichthyology / v.34, no.3, 2022 , pp. 208-217 More about this Journal
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.
Keywords
Liobagrus obesus; environmental DNA; real-time PCR; species-specific marker;
Citations & Related Records
Times Cited By KSCI : 3  (Citation Analysis)
연도 인용수 순위
1 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.   DOI
2 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.   DOI
3 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.   DOI
4 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.
5 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.
6 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.
7 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.
8 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.   DOI
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.   DOI
12 ME (Ministry of Environment). 2011. Culture and restoration research of endangered freshwater fish (four species include Liobagrus obesus). Soonchunhyang University, Asan, Korea, 359pp.
13 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.   DOI
14 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.   DOI
15 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.
16 ME (Ministry of Environment). 2017. Conservation and management laws of wildlife (amendment of enforcement regulations)(Law No. 10977).
17 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.   DOI
18 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.   DOI
19 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.   DOI
20 ME (Ministry of Environment). 2019. A study on conservation plan of endangered freshwater fish (Microphysogobio rapidus, Pseudobagrus brevicorpus). Soonchunhyang University, Asan, Korea, 214pp.
21 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.   DOI
22 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.   DOI
23 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.
24 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.   DOI
25 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.
26 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.   DOI
27 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.   DOI
28 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.   DOI
29 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.
30 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.
31 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.
32 Son, Y.M. and I.Y. Choo. 1988. Ecological studies of catfish, genus Liobagrus from Korea. Korea J. Limnol., 21: 243-251.
33 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.
34 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.   DOI
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.   DOI