Marine Biotechnology

, Volume 5, Issue 5, pp 505–517 | Cite as

Genetic Diversity and Molecular Markers of the Tropical Abalone (Haliotis asinina) in Thailand

  • S. Klinbunga
  • P. Pripue
  • N. Khamnamtong
  • N. Puanglarp
  • A. Tassanakajon
  • P. Jarayabhand
  • I. Hirono
  • T. Aoki
  • P. Menasveta
Original Contributions


Genetic diversity of abalone in Thailand, Haliotis asinina, H. ovina, and H. varia, was analyzed by polymerase chain reaction (PCR) of 18S and 16S rDNAs, with randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). Species-specific RAPD markers were found in each abalone species. Restriction analysis of 18S (nuclear) ribosomal DNA with Alu I, Taq I, and Hae III and 16S (mitochondrial) rDNA with Bam HI, Eco RI, Hae III, and Alu I gave 12 and 13 digestion patterns, respectively. A total of 49 composite haplotypes were found. A dendogram obtained by the unweighted pair-group method with arithmetic mean, constructed from divergence between pairs of composite haplotypes, revealed reproductively isolated gene pools of these abalone and indicated that H. asinina and H. ovina are genetically closer than H. varia. When H. varia was discovered owing to small sample sizes, geographic heterogeneity analysis and FST estimate indicated clear genetic differentiation between H. ovina originating from the Andaman Sea (west) and the Gulf of Thailand (east, P < 0.0001), whereas partial differentiation was observed between the Philippines and the remaining H. asinina samples (P < 0.0021). The amplified 16S rDNAs of individuals representing composite haplotypes found in this study were cloned and sequenced. A neighbor-joining tree constructed from sequence divergence of 16S rDNA accurately allocated those sequences according to species origins of abalone. Species-specific PCR based on 16S rDNA polymorphism was successfully developed in H. asinina and H. varia but not in H. ovina.


abalone PCR RAPD genetic diversity population differentiation 



We thank the National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), and Department of Biochemistry, Faculty of Science, Chulalongkorn University, for providing facilities required by our experiments. This work is supported by the Thailand Research Funds (TRF) project 4320015 awarded to S.K. and A.T. and by the JSPS to S.K.


  1. Avise, J.C. 1994Molecular Markers, Natural History and Evolution.Chapman and HallLondon, U.K.Google Scholar
  2. Brown, L.D. 1995Genetic evidence for hybridisation between Haliotis rubra and H. laevigata. Mar Biol1238993.Google Scholar
  3. Dower, W.J., Miller, J.F., Ragsdale, C.W. 1988High efficiency transformation of E. coli by high voltage electroporation.Nucleic Acids Res1661276145.PubMedGoogle Scholar
  4. Felsenstein, J. 1993PHYLIP (Phylogenetic Inference Package) Version 3.5c.Department of Genetics, University of WashingtonSeattleGoogle Scholar
  5. Geiger, D.L. 1998Recent genera and species of the family Haliotidae Rafinesque, 1815 (Gastropoda: Vetigastropoda).Nautilus11185116.Google Scholar
  6. Hamm, D.E., Burton, R.S. 2000Population genetics of black abalone, Haliotis cracheradii, along the central California coast.J Exp Mar Biol Ecol254235247.CrossRefPubMedGoogle Scholar
  7. Heath, D.D., Iwama, O.K., Delvin, R.H. 1993PCR primed with VNTR core sequences yields species-specific patterns and hypervariable probes.Nucleic Acids Res2157825785.Google Scholar
  8. Huang, B.X., Peakall, R., Hanna, P.J. 2000Analysis of genetic structure of blacklip abalone (Haliotis rabra) populations using RAPD, minisatellite and microsatellite markers.Mar Biol136207216.Google Scholar
  9. Jarayabhand, P., Paphavasit, N. 1996A review of the culture of tropical abalone with special reference to Thailand.Aquaculture140159168.Google Scholar
  10. Jarayabhand, P., Yom-La, R., Popongviwat, A. 1998Karyotypes of marine molluscs in the family Haliotidae found in Thailand.J Shellfish Res17761764.Google Scholar
  11. Kimura, M. 1980A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences.J Mol Biol16111120.Google Scholar
  12. Klinbunga, S., Sodsuk, S., Penman, D., McAndrew, B.J. 1996An improved protocol for total DNA isolation and visualization of mtDNA RFLP(s) in tiger prawn, Penaeus monodon.Thai J Aquat Sci33641.Google Scholar
  13. Klinbunga, S., Boonyapakdee, A., Pratoomchat, B. 2000Genetic diversity and species-diagnostic markers of mud crabs (genus Scylla) in eastern Thailand determined by RAPD analysis.Mar Biotechnol2180187.Google Scholar
  14. Klinbunga, S., Siludjai, D., Wudthijinda, W., Tassanakajon, A., Jarayabhand, P., Menasaveta, P. 2001Genetic heterogeneity of the giant tiger shirmp (Penaeus monodon) in Thailand revealed by RAPD and mitochondrial DNA RFLP analyses.Mar Biotechnol3428438.Google Scholar
  15. Klinbunga, S., Khamnamtong, N., Tassanakajon, A., Puanglarp, N., Jarayabhand, P., Yoosukh, W. 2002Molecular genetic identification tools for three commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand.Mar Biotechnol52736.Google Scholar
  16. Lee, Y.H., Vacquier, V.D. 1995Evolution and systematics in Haliotidae (Mollusca: Gastropoda): inferences from DNA sequences of sperm lysin.Mar Biol124267278.Google Scholar
  17. Maniatis, T., Frisch, E.F., Sambrook, J. 1982Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory PressCold Spring Harbor, N.Y.Google Scholar
  18. McElroy, D., Moran, P., Bermingham, E., and Kornfield, I. (1991). REAP; The Restriction Enzyme Analyses Package, Version 4.0Google Scholar
  19. Muchmore, A.E., Moy, G.W., Swanson, W.J., Vacquier, V.D. 1998Direct sequencing of genomic DNA for characterization of a satellite DNA in five species of Eastern Pacific abalone.Mol Mar Biol Biotechnol716.Google Scholar
  20. Naganuma, T., Hisadome, K., Shiraishi, K., Kojima, H. 1998Molecular distinction of two resemblant abalones, Haliotis discus discus and Haliotis discus hannai by 18S rDNA sequences.J Mar Biotechnol65961.Google Scholar
  21. Nateewathana, A., Bussarawit, S. 1988Abundance and distribution of abalones along the Andaman Sea coast of Thailand.Kasetsart J (Natural Sci)22815.Google Scholar
  22. Nei, M. 1987Molecular Evolutionary Genetics.Columbia University PressNew York, N.Y.Google Scholar
  23. Nei, M., Li, W. 1979Mathematical model for studying genetic variation in terms of restriction endonucleases.Proc Natl Acad Sci USA7652695273.PubMedGoogle Scholar
  24. Nei, M., Tajima, F. 1981DNA polymorphism detectable by restriction endonucleases.Genetics97145163.PubMedGoogle Scholar
  25. Palumbi, S.R., Martin, A., Pomano, S., McMillan, W.O., Grabowski, G. 1991The Simple Fool’s Guide to PCR, Version 2.Zoology DepartmentUniversity of Hawaii.Google Scholar
  26. Raymond, M., Rousset, F. 1995GENEPOP (Version 1.2): population genetic software for exact tests and ecumenicism.J Hered86248249.Google Scholar
  27. Rice, W.R. 1989Analyzing tables for statistical tests.Evolution41223225.Google Scholar
  28. Roff, D.A., Bentzen, P. 1989The statistical analysis of mitochondrial DNA polymorphisms: χ2 and the problems of small samples.Mol Biol Evol6539545.PubMedGoogle Scholar
  29. Saitou, N., Nei, M. 1987The neighbor-joining method: a new method for reconstructing phylogenetic trees.Mol Biol Evol4406425.PubMedGoogle Scholar
  30. Shepherd, S.A., Lowe, D., Partington, D. 1992Studies of southern Australian abalone (genus Haliotis), 13: larval dispersal and recruitment.J Exp Mar Biol Ecol164247260.Google Scholar
  31. Singhagraiwan, T., Doi, M. 1993Seed production and culture of a tropical abalone, Haliotis asinina Linne’.Department of Fisheries, Ministry of Agriculture and CooperativesThailand.Google Scholar
  32. Small, M.P., Chapman, R.W. 1997Intraspecific variation in the 16S ribosomal gene of Crassostrea virginica.Mol Mar Biol Biotechnol6189196.Google Scholar
  33. Sneath, P.H.A., Sokal, R.R. 1973Numerical Taxonomy.W.H. Freeman and CoSan Franciso, Calif.Google Scholar
  34. Sweijd, N.A., Bowie, R.C.K., Lopata, A.L., Marinaki, A.M., Harley, E., Cook, P.A. 1998A PCR technique for forensic, species-level identification of abalone tissue.J Shellfish Res17889895.Google Scholar
  35. Thompson, J.D., Higgin, D.G., Gibson, T.J. 1994CLUSTAL W: improving the sensitivity of progressive multiple sequence weighting, position-specific penalties and weight metric choices.Nucleic Acids Res2246734680.PubMedGoogle Scholar
  36. Tookwinas, S., Leknim, V., Donyadol, Y., Predalampabut, Y., and Paengmark, P. (1986). A survey of species and distribution of abalone (Haliotis spp) in Surat Thani, Nakhon Si Thammarat and Songkla. Technical Report No. 1/1986, NICA.Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • S. Klinbunga
    • 1
  • P. Pripue
    • 2
  • N. Khamnamtong
    • 1
  • N. Puanglarp
    • 1
  • A. Tassanakajon
    • 3
  • P. Jarayabhand
    • 4
    • 5
  • I. Hirono
    • 6
  • T. Aoki
    • 6
  • P. Menasveta
    • 1
    • 5
  1. 1.Marine Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC)National Science and Technology Development Agency, 113 Paholyothin Rd., Klong 1, Klong Luang, Patthumthani 12120Thailand
  2. 2.Program of Biotechnology, Faculty of ScienceChulalongkorn University, Bangkok 10330Thailand
  3. 3.Department of Biochemistry, Faculty of ScienceChulalongkorn University, Bangkok 10330Thailand
  4. 4.Aquatic Resources Research InstituteChulalongkorn University, Bangkok 10330Thailand
  5. 5.Department of Marine Science, Faculty of ScienceChulalongkorn University, Bangkok 10330Thailand
  6. 6.Department of Aquatic BiosciencesTokyo University of Fisheries, Konan 4-5-7, Tokyo 108-8477Japan

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