High-throughput simple sequence repeat (SSR) markers development for the kelp grouper (Epinephelus bruneus) and cross-species amplifications for Epinephelinae species

DOI: 10.4236/abb.2014.52016   PDF   HTML     4,057 Downloads   6,165 Views   Citations


The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).

Share and Cite:

Kubota, S. , Liu, Q. , Kessuwan, K. , Okamoto, N. , Sakamoto, T. , Nakamura, Y. , Shigenobu, Y. , Sugaya, T. , Sano, M. , Uji, S. , Nomura, K. and Ozaki, A. (2014) High-throughput simple sequence repeat (SSR) markers development for the kelp grouper (Epinephelus bruneus) and cross-species amplifications for Epinephelinae species. Advances in Bioscience and Biotechnology, 5, 117-130. doi: 10.4236/abb.2014.52016.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Heemstra, P.C. and Randall, J.E. (1993) FAO species catalogue, Vol. 16. Groupers of the world (Family Serranidae, Subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date. FAO Fisheries synopsis, 125, FAO, Rome.
[2] Morris, A.V., Roberts, C.M. and Hawkins, J.P. (2000) The threatened status of groupers (Epinephelinae). Biodiversity and Conservation, 9, 919-942.
[3] Marte, C.L. (2003) Larviculture of marine species in Southeast Asia: Current research and industry prospects. Aquaculture, 227, 293-304.
[4] Pierre, S., Gaillard, S., Prévot-D'Alvise, N., Aubert, J., Rostaing-Capaillon, O., Leung-Tack, D. and Grillasca, J.-P. (2008) Grouper aquaculture: Asian success and Mediterranean trials. Aquatic Conservation, 18, 297-308.
[5] Tupper, M. and Sheriff, N. (2008) Capture-based aquaculture of groupers. In: Lovatelli, A. and Holthus, P.F. Eds., Capture-Based Aquaculture. Global Overview, FAO, Rome, 217-253.
[6] Liu, Z.J. and Cordes, F.J. (2004) DNA marker technology and their applications in aquaculture genetics. Aquaculture, 238, 1-37.
[7] Chistiakov, D.A., Hellemans, B. and Volckaert, F.A.M. (2006) Microsatellites and their genomic distribution, evolution, function and applications: A review with special reference to fish genetics. Aquaculture, 255, 1-29.
[8] Zhu, Z.Y., Lo, L.C., Lin, G., Xu, Y.X. and Yue, G.H. (2005) Isolation and characterization of polymorphic microsatellites from red coral grouper (Plectropomus maculatus). Molecular Ecology Notes, 5, 579-581.
[9] Zeng, H.S., Ding, S.X., Wang, J. and Su, Y.Q. (2008) Characterization of eight polymorphic microsatellite loci for the giant grouper (Epinephelus lanceolatus Bloch). Molecular Ecology Resources, 8, 805-807.
[10] Yang, S., Wang, L., Zhang, Y., Liu, X.C., Lin, H.R. and Meng, Z.N. (2011) Development and characterization of 32 microsatellite loci in the giant grouper Epinephelus lanceolatus (Serranidae). Genetics and Molecular Research, 10, 4006-4011.
[11] Mokhtar, M.A.A., Normah, M.N., Kumar, S.V. and Baharum, S.N. (2011) Characterization of 10 novel microsatellite loci for the brown marbled grouper, Epinephelus fuscoguttatus (Serranidae). Genetics and Molecular Research, 10, 885-888.
[12] Renshaw, M.A., Nemeth, R.S. and Gold, J.R. (2012) Isolation of microsatellite markers from tiger grouper (Mycteroperca tigris) and characterization in yellowfin grouper (Mycteroperca venenosa), coney (Cephalopholis fulva), and red hind (Epinephelus guttatus). Conservation Genetics Resources, 4, 1049-1054.
[13] Na-Nakorn, U., Yashiro, R., Wachirachaikarn, A., Prakoon, W. and Pansaen, N. (2010) Novel microsatellites for multiplex PCRs in the humpback grouper, Cromileptes altivelis (Valenciennes, 1828), and applications for broodstock management. Aquaculture, 306, 57-62.
[14] An, H.S., Kim, J.W., Lee, J.W., Kim, S.K., Lee, B.I., Kim, D.J. and Kim, Y.C. (2012) Development and characterization of microsatellite markers for an endangered species, Epinephelus bruneus, to establish a conservation program. Animal Cells and Systems, 16, 50-56.
[15] Abdelkrim, J., Robertson, B.C., Stanton, J.-A.L. and Gemmell, N.J. (2009) Fast, cost-effective development of species-specific microsatellite markers by genomic sequencing. Biotechniques, 46, 185-192.
[16] Gardner, M.G., Fitch, A.J., Bertozzi, T. and Lowe, A.J. (2011) Rise of the machines-recommendations for ecologists when using next generation sequencing for microsatellite development. Molecular Ecology Resources, 11, 1093-1101. http://dx.doi.org/10.1111/j.1755-0998.2011.03037.x
[17] Gill, P., Kimpton, C., D’Aloja, E., Andersen, J.F., Bar, W., Brinkmann, B., Holgersson, S., Johnsson, V., Kloosterman, A.D., Lareu, M.V., Nellemann, L., Pfitzinger, H., Phillips, C.P., Schmitter, H., Schneider, P.M. and Stenersen, M. (1994) Report of the European DNA profiling group (EDNAP)–Towards standardization of short tandem repeat (STR) loci. Forensic Science International, 65, 51-59.
[18] Krenke, B.E., Tereba, A., Anderson, S.J., Buel, E., Culhane, S., Finis, C.J., Tomsey, C.S., Zachetti, J.M., Masibay, A., Rabbach, D.R., Amiott, E.A. and Sprecher, C.J. (2002) Validation of a 16-locus fluorescent multiplex system. Journal of Forensic Sciences, 47, 773-785.
[19] Barbará, T., Palma-Silva, C., Paggi, G.M., Bered, F., Fay, M.F. and Lexer, C. (2007) Cross-species transfer of nuclear microsatellite markers: Potential and limitations. Molecular Ecology, 16, 3759-3767.
[20] Nakamura, Y., Shigenobu, Y., Sugaya, T., Kurokawa, T. and Saitoh, K. (2013) Automated screening and primer design of fish microsatellite DNA loci on pyrosequencing data. Ichthyological Research, 60, 184-187.
[21] Benson, G. (1999) Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Research, 27, 573-580. http://dx.doi.org/10.1093/nar/27.2.573
[22] Rozen, S. and Skaletsky, H. (1999) PRIMER3 on the WWW for general users and for biologist programmers. Methods in Molecular Biology, 132, 365-386.
[23] Jurka, J. and Pethiyagoda, C. (1995) Simple repetitive DNA sequences from primates: Compilation and analysis. Journal of Molecular Evolution, 40, 120-126.
[24] Martins, W.S., Lucas, D.C.S., Neves, K.F.S. and Bertioli, D.J. (2009) WebSat-A web software for microsatellite marker development. Bioinformation, 3, 282-283.
[25] Wang, J., Yu, X., Zhao, K., Zhang, Y., Tong, J. and Peng, Z. (2012) Microsatellite development for an endangered bream Megalobrama pellegrini (Teleostei, Cyprinidae) using 454 sequencing. International Journal of Molecular Sciences, 13, 3009-3021.
[26] An, H.S., Lee, J.W. and Hong, S.W. (2012) Application of novel polymorphic microsatellite loci identified in the Korean Pacific abalone (Haliotis diversicolor supertexta (Haliotidae)) in the genetic characterization of wild and released populations. International Journal of Molecular Sciences, 13, 10750-10764.
[27] Zheng, X.H., Lu, C.Y., Zhao, Y.Y., Lee, C., Cao, D.C., Chang, Y.M., Liang, L.Q. and Sun, X.W. (2010) A set of polymorphic trinucleotide and tetranucleotide microsatellite markers for silver Crucian carp (Carassius auratus gibelio) and cross-amplification in Crucian carp. Biochemical Genetics, 48, 624-635.
[28] Iranawati, F., Jung, H., Chand, V., Hurwood, D.A. and Mather, P.B. (2012) Analysis of genome survey sequences and SSR marker development for Siamese mud carp, Henicorhynchus siamensis, using 454 pyrosequencing. International Journal of Molecular Sciences, 13, 10807-10827. http://dx.doi.org/10.3390/ijms130910807
[29] Saarinen, E.V. and Austin, J.D. (2010) When technology meets conservation: Increased microsatellite marker production using 454 genome sequencing on the endangered Okaloosa darter (Etheostoma okaloosae). Journal of Heredity, 101, 784-788.
[30] Kang, J.H., Park, J.Y. and Jo, H.S. (2012) Rapid development of microsatellite markers with 454 pyrosequencing in a vulnerable fish, the mottled skate, Raja pulchra. International Journal of Molecular Sciences, 13, 7199-7211. http://dx.doi.org/10.3390/ijms13067199
[31] Craig, M.T. and Hastings, P.A. (2007) A molecular phylogeny of the groupers of the subfamily Epinephelinae (Serranidae) with a revised classification of the Epinephelini. Ichthyological Research, 54, 1-17.
[32] Peakall, R., Gilmore, S., Keys, W., Morgante, M. and Rafalski, A. (1998) Cross-species amplification of soybean (Glycine max) simple sequence repeats (SSRs) within the genus and other legume genera: Implications for the transferability of SSRs in plants. Molecular Biology and Evolution, 15, 1275-1287.
[33] Reid, K., Hoareau, T.B. and Bloomer, P. (2012) Highthroughput microsatellite marker development in two sparid species and verification of their transferability in the family Sparidae. Molecular Ecology Resources, 12, 740-752. http://dx.doi.org/10.1111/j.1755-0998.2012.03138.x
[34] Carreras-Carbonell, J., Macpherson, E. and Pascual, M. (2008) Utility of pairwise mtDNA genetic distances for predicting cross-species microsatellite amplification and polymorphism success in fishes. Conservation Genetics, 9, 181-190. http://dx.doi.org/10.1007/s10592-007-9322-2

comments powered by Disqus

Copyright © 2020 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.