ISSN : 1225-3480
A multiplex PCR primers (the species-specific primers) set using molecular biological methods targeting four species of ark shells (Anadara kagoshimensis, Tegillarca granosa, Anadara broughtonii and Cucullaea labiata) distributed in Korea were built, and a rapid and accurate species identification method was developed. The species-specific primer was designed within the nucleotide sequence of about 573 bp corresponding to the cytochrome c oxidase subunit 1 (co1) gene region of mitochondrial DNA, and considering the single nucleotide polymorphism (SNPs) representing inter-species variation excluding intra-species variation, it was designed with an interval of 80-100 bp for each species. As a result, it was confirmed that species-specific bands were formed in the order of A. kagoshimensis (160 bp), T. granosa (245 bp), A. broughtonii (351 bp), and C. labiata (471 bp). In addition, in order to measure the limit of multiplex PCR amplification according to the concentration of genomic DNA (gDNA) and the number of PCR amplification repetitions, 15 experimental groups were divided and measured. As a result, when the number of amplification repeats was 25 cycles, the concentration of gDNA was detected down to a minimum of 1 ng/μl. Therefore, the multiplex PCR primer set for four species of ark shells developed in this study is expected to be of great help in accurately and rapidly identifying species in the field, it is thought that economic damage and academic errors caused by misidentification can be prevented.
Andre, C., Lindegarth, M., Jonsson, P.R. and Sundberg, P. (1999) Species identification of bivalve larvae using random amplified polymorphic DNA (RAPD): differentiation between Cerastoderma edule and C. lamarcki. Journal of the Marine Biological Association of the United Kingdom, 79(3): 563-565.
Avise, J.C. (2004) Molecular Markers, Natural History, and Evolution. 2nd edition. pp. 684, Sinauer Associates, Sunderland.
Bañón, R., Fernández, J., Trigo, J.E., Pérez-Dieste, J., Barros-García, D. and De Carlos, A. (2015) Range expansion, biometric features and molecular identification of the exotic ark shell Anadara kagoshimensis from Galician waters, NW Spain. Journal of the Marine Biological Association of the United Kingdom, 95(3): 545-550.
Boss, K.J. (1982) Mollusca. In; Synopsis and Classification of Living Organisms. pp. 1166. Mcgraw-Hill Book Company, New York.
Cho, K.H., Heo, S., Kim, H.R., Kim, J.H., Shin, I.S., Han, S.E., Kim, S.H. and Kim, D.H. (2010) Discrimination of Korean apple cultivars using combination of RAPD-SCAR markers. Horticultural Science & Technology, 28(5): 828-835.
Dieffenbach, C.W., Lowe, T.M. and Dveksler, G.S. (1993) General concepts for PCR primer design. Genome Research, 3: S30-S37.
Feng, Y., Li, Q. and Kong, L. (2015) Molecular phylogeny of Arcoidea with emphasis on Arcidae species (Bivalvia: Pteriomorphia) along the coast of China: Challenges to current classification of arcoids. Molecular phylogenetics and evolution, 85: 189-196.
Feng, Y., Li, Q., Yu, H. and Kong, L. (2017) Complete mitochondrial genome sequence of Cucullaea labiata (Arcoida: Cucullaeidae) and phylogenetic implications. Genes & Genomics, 39(8): 867-875.
Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3: 294-299.
Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symposium Series, 41: 95-98.
Jung, C.K., Oh, B.S., Kim, T.I. and Jin, Y.G. (2010) Effect of heating on the sex maturation and egg development of the ark shell, Scapharca subcrenata. The Korean Journal of Malacology, 26(3): 201-209. [in Korean]
Jung, S.W., Kim, H.J., Park, J.S., Lee, T.K., Shin, K., Jeong, S.Y., Hwange, S.Y. and Yoo, J.W. (2018) Planktonic bivalve larvae identification and quantification in Gomso Bay, South Korea, using next-generation sequencing analysis and microscopic observations. Aquaculture, 490: 297-302.
Kang, N.S., Kang, D.W., Kang, C.B., Kwak, M.S., Kwun, O.N., Kwon, H.J., Kim, K.M., Kim, M.S., Kim, I.H., Kim, J.H., Kim, H.N., Kim, H.J., Moon, H.W., Bae, S.S., Back, J.W., Shin, M.H., An, Y.R., Won, J.H., Lee, S.H., Lee, S.H., Lim, B.J., Jeong, D.W. and Jeong, H.K. (2021) National List of Marine Species II (Marine Invertebrates). pp. 523, National Marine Biodiversity Institute of Korea, Seocheon. [in Korean]
Kang, S.I., Sohn, S.K., Choi, K.S., Kim, K.H., Kim, Y.S., Lee, J.S., Heu, M.S. and Kim, J.S. (2020) Optimization of the Processing of Seasoning Sauce for Seasoned Broughton's Ribbed Ark Scapharca broughtonii Products Using Response Surface Methodology. Korean Journal of Fisheries and Aquatic Sciences, 53(3): 334-341.
Kim, H.J., Kang, J.S., Jung, S.W. and Park, Y.J. (2019) Changes in planktonic bivalve larvae of Tegillarca granosa and Anadara kagoshimensis in the Boseong coastal waters of South Korea. Korean Journal of Environmental Biology, 37(3): 351-361.
Kim, H.S., Seo, Y.B., Choi, S.S., Kim, J.H., Shin, J.Y, Yang, J.Y. and Kim, G.D. (2015) Development and validation of multiplex polymerase chain reaction to determine squid species based on 16s rRNA gene. Journal of Food Hygiene and Safety, 30(1): 43-50. [in Korean]
Kim, K.S. and Yoon, S.J. (2018) Development of Duplex-PCR Method for Rapid Identification of Hard-shelled Mussel. Mytilus Coruscus. Journal of Fisheries and Marine Sciences Education, 30(4): 1192-1199.
Koressaar, T. and Remm, M. (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics, 23: 1289-1291.
Koressaar, T., Lepamets, M., Kaplinski, L., Raime, K., Andreson, R. and Remm, M. (2018) Primer3_masker: integrating masking of template sequence with primer design software. Bioinformatics, 34: 1937-1938.
Ladoukakis, E.D. and Zouros, E. (2017) Evolution and inheritance of animal mitochondrial DNA: rules and exceptions. Journal of Biological Research-Thessaloniki, 24(1): 1-7.
Lee, J.S. (2013) Invertebrate Fauna of Korea-Bivalves I (Mollusca: Bivalvia: Pteriomorphia: Arcoida, Mytiloida). vol.19, no.2, pp. 130. National Institute of Biological Resources, Incheon.
Lee, J.S. (2016) List of Korean Mollusks. pp. 404. The Korean Journal of Malacology, Cheonan. [in Korean]
Lee, J.S. and Min, D.K. (2007) Korean Names of Foreign Shells Introduced to Korea. The Korean Journal of Malacology, 23(1): 105-154.
Lee, Y.W. and Hur, Y.S. (2017) A study on application of country of origin labelling system with fishery products. The institute of Business management, 40(2): 125-147. [in Korean]
Liu, Y.G., Kurokawa, T., Sekino, M., Tanabe, T. and Watanabe, K. (2013) Complete mitochondrial DNA sequence of the ark shell Scapharca broughtonii: an ultra-large metazoan mitochondrial genome. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 8(1): 72-81.
Meyer, C.P. and Paulay, G. (2005) DNA barcoding: error rates based on comprehensive sampling. PLoS biology, 3(12): e422.
Miura, T., Roisin, Y. and Matusmoto, T. (2000) Molecular phylogeny and biogeography of the nasute termite genus Nasutitermes (Isoptera: Termitidae) in the Pacific tropics. Molecular Phylogenetics and Evolution, 17(1): 1-10.
Nicol, D. (1950) Recent species of the prionodont pelecypod Cucullaea. Journal of the Washington Academy of Sciences, 40: 338-343.
Park, Y.C., Ahn, C.Y., Jin, S.O., Lim, J.Y., Kim, K.H., Lee, J.H., Cho, T.Y., Lee, H.J., Park, K.S. and Yoon, H.S. (2012) Identification of raw materials in processed meat products by PCR using species-specific primer. Journal of Food Hygiene and Safety, 27(1): 68-73. [in Korean]
Poutiers, J.M. (1998) Bivalves. FAO Species Identification Guide for Fishery Purposes. The Living Marine Resources of The Western Central Pacific volume 1. Seaweeds, Corals, Bivalves and Gastropods. pp. 158. Food and Agriculture Organization of The United Nations, Rome.
Razak, N.F.A., Supramaniam, C.V. and Zieritz, A. (2019) A dichotomous PCR-RFLP identification key for the freshwater mussels (Bivalvia: Unionida) of Peninsular Malaysia. Conservation Genetics Resources, 11(4): 457-464.
Rifai, N., Horvath, A.R., Wittwer, C.T. and Park, J. (2018) Principles and Applications of Molecular Diagnostics. pp. 422. 1st Edition. Elsevier Science, Amsterdam.
Rozas, J., Ferrer-Mata, A., Sánchez-DelBarrio, J.C., GN Sánchez-Gracia, A. (2017) DnaSP 6: DNA Sequence Polymorphism Analysis of Large Datasets. Molecular Biology and Evolution, 34: 3299-3302.
Souji, S. and Radhakrishnan, T. (2013) New Distribution Record and Taxonomy of Two Cucullaeidae Species (Bivalvia: Arcacea) from Thermal Beach, Thoothukudi, South East Coast of India. International Journal of Science and Research, 4(2): 939-943.
Strafella, P., Ferrari, A., Fabi, G., Salvalaggio, V., Punzo, E., Cuicchi, C., Santelli, A., Cariani, A., Tinti, F., Tassetti, A. and Scarcella, G. (2017) Anadara kagoshimensis (Mollusca: Bivalvia: Arcidae) in Adriatic Sea: morphological analysis, molecular taxonomy, spatial distribution, and prediction. Mediterranean Marine Science, 18(3): 443-453.
Suh, J.S., Kim, T.H., Shin, S.Y., Kahng, H.Y., Ahn, S.Y., Jung, J.S., Kim, Y.S. and Won, N.I. (2017) Geochemical Characteristics and Benthos Distribution in the Three Shellfish Farms in Suncheon Bay, Korea. Journal of Environmental Science International, 26(6): 691-710.
Sun, S.E., Kong, L., Yu, H. and Li, Q. (2015) The complete mitochondrial genome of Scapharca kagoshimensis (Bivalvia: Arcidae). Mitochondrial DNA, 26(6): 957-958.
Tanaka, T. and Aranishi, F. (2013) Mitochondrial DNA markers for PCR-based phylogenetic analysis of ark shells. Open Journal of Marine Science, 3(4): 182-189.
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22: 4673-4680.
Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B.C., Remm, M. and Rozen, S.G. (2012) Primer3-new capabilities and interfaces. Nucleic Acids Research, 40: e115.
Vannarattanarat, S., Zieritz, A., Kanchanaketu, T., Kovitvadhi, U., Kovitvadhi, S. and Hongtrakul, V. (2014) Molecular identification of the economically important freshwater mussels (Mollusca-Bivalvia- Unionoida) of Thailand: developing species-specific markers from AFLPs. Animal genetics, 45(2): 235-239.
Wiemers, M. and Fiedler, K. (2007) Does the DNA barcoding gap exist?-a case study in blue butterflies (Lepidoptera: Lycaenidae). Frontiers in zoology, 4(1): 1-16.
Yoo, S.K. (2000) Shallow Water Aquaculture. 2nd Edition. pp. 639. Guduk Publishing Co. Busan.
