The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1–10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy–Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni’s correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.
References
[1]
Jeong, C.H. A review of taxonomic studies and common names of rajid fishes (Elasmobranchii, Rajidae) from Korea. Koren J. Ichthyol 1999, 11, 198–210.
[2]
Choi, Y.; Kim, J.H.; Park, J.Y. Marine Fishes of Korea; Kyo_Hak Publishing: Seoul, Korea, 2002.
[3]
Ishihara, H. Studies on the Systematics and Resources of the Skates (Rajidae) in the North Pacific Ocean. Ph.D. Dissertation, Tokyo University, Tokyo, Japan, 1990.
[4]
Yeon, I.J.; Hong, S.H.; Cha, H.K.; Kim, S.T. Feeding habitats of Raja pulchra in the Yellow Sea. Bull. Natl. Fish. Res. Dev. Inst. Korea 1999, 57, 1–11. (In Korean with English abstract).
[5]
Antonenko, D.V.S.; Solomatov, F.; Balanov, A.A.; Kim, S.T.; Kalchugin, P.V. Occurrence of Skate Raja pulchra (Rajidae, Rajiformes) in Russian waters of the sea of Japan. J. Ichthyol 2011, 51, 426–431.
[6]
Ishihara, H. The Skates and Rays of the Western North Pacific: An Overview of Their Fisheries, Utilisation and Classification. In Elasmobranchs as Living Resources: Advances in Biology, Ecology, Systematic and the Status of Fisheries; Pratt, H.L., Gruber, S.H., Taniuchi, T., Eds.; NOAA Technical Report NMFS 90; U.S. Department of Commerce: Springfield, VA, USA, 1990.
[7]
Fisheries information service. Ministry for Food, Agriculture; Forestry and Fisheries: Gwacheon, Korea, 2009. Available online: http://www/fips.go.kr , accessed on 6 March 2012.
[8]
Ishihara, H.; Wang, Y.; Tanaka, S.; Nakaya, K.; Jeong, C.-H. Raja pulchra. IUCN Red List of Threatened Species. Version 2011.2, Available online: http://www.iucnredlist.org , accessed on 11 February 2012.
[9]
Chistiakov, D.A.; Hellemans, B.; Volckaert, F.A.M. Microsatellites and their genomic distribution, evolution, function and applications: A review with special reference to fish genetics. Aquaculture 2006, 255, 1–29.
[10]
Jarne, P.; Lagoda, P.J.L. Microsatellites from molecules to populations and back. Trends Ecol. Evol 1996, 11, 424–429.
[11]
Hamilton, M.; Pincus, E.L.; di Fiore, A.; Fleischer, R.C. Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites. Biotechniques 1999, 27, 500–507.
[12]
Zane, L.; Bargelloni, L.; Patarnello, T. Strategies for microsatellite isolation: A review. Mol. Ecol 2002, 11, 1–16.
[13]
Kircher, M.; Kelso, J. High-throughput DNA sequencing-concepts and limitations. BioEssays 2010, 32, 524–536.
[14]
Scaglione, D.; Acquadro, A.; Portis, E.; Taylor, C.A.; Lanteri, S.; Knapp, S.J. Ontology and diversity of transcript-associated microsatellites mined from a globe artichoke EST database. BMC Genomics 2009, 10, 1–17.
[15]
Abdelkrim, J.; Robertson, B.C.; Stanton, J.-A.L.; Gemmell, N.J. Fast, cost effective development of species-specific microsatellite markers by genomic sequencing. Biol. Tech 2009, 46, 185–191.
[16]
Yu, J.N.; Won, C.; Jun, J.; Lim, Y.W.; Kwak, M. Fast and cost-effective mining of microsatellite markers using NGS technology: An example of a Korean Water Deer Hydropotes Inermis Argyropus. PLoS One 2011, 6, doi:10.1371/journal.pone.0026933.
[17]
Setsuko, S.; Uchiyama, K.; Sugai, K.; Yoshimaru, H. Rapid development of microsatellite markers for Pandanus boninensis (Pandanaceae) by pyrosequencing technology. Am. J. Bot 2012, 99, e33–e37.
[18]
McEwen, J.R.; Vamosi, J.C.; Rogers, S.M. Rapid isolation and cross-amplification of microsatellite markers in Plectritis congesta (Valerianaceae) with 454 sequencing. Am. J. Bot 2011, 98, e369–e371.
[19]
Perry, J.C.; Rowe, L. Rapid microsatellite development for water striders by next-generation sequencing. J. Hered 2010, 102, 125–129.
[20]
Castoe, T.A.; Poole, A.W.; Gu, W.; de Konig, A.P.J.; Daza, J.M.; Smith, E.N.; Pollock, D.D. Rapid identification of thousands of copperhead snake microsatellite loci from modest amounts of 454 shotgun genome sequence. Mol. Ecol. Resour 2010, 10, 341–347.
[21]
Saarinen, E.V.; Austin, J.D. When technology meets conservation: Increased microsatellite marker production using 454 genome sequencing on the endangered Okaloosa Darter (Etheostoma okaloosae). J. Hered 2010, 101, 784–788.
[22]
Wang, J.; Yu, X.; Zhao, K.; Zhang, Y.; Tong, J.; Peng, Z. Microsatellite development for an endangered bream Megalobrama Pellegrini (Teleostei, Cyprinidae) using 454 sequencing. Int. J. Mol. Sci 2012, 13, 3009–3021.
[23]
Greenley, A.P.; Muguia-Vega, A.; Saenz-Arroyo, A.; Micheli, F. New tetranucleotide microsatellite loci in pink abalone (Haliotis corrugata) isolated via 454 pyrosequencing. Conserv. Genet. Resour 2012, 4, 265–268.
[24]
Gardner, M.G.; Fitch, A.J.; Bertozzi, T.; Lowe, A.J. Rise of the machines—Recommendations for ecologists when using next generation sequencing for microsatellite development. Mol. Ecol. Resour 2011, 11, 1093–1101.
Zane, L.; Bargelloni, L.; Patarnello, T. Strategies for microsatellite isolation: A review. Mol. Ecol 2002, 11, 1–16.
[27]
Csencsics, D.; Brodbeck, S.; Holderegger, R. Cost-effective, species-specific microsatellite development for the endangered Dwarf Bulrush (Typha minima) using next-generation sequencing technology. J. Hered 2010, 101, 789–793.
[28]
Malausa, T.; Gilles, A.; Meglécz, E.; Blanquart, H.; Duthoy, S.; Costedoat, C.; Dubut, V.; Pech, N.; Castagnone-Sereno, P.; Délye, C.; et al. High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries. Mol. Ecol. Resour 2011, 11, 638–644.
[29]
Carvalho, D.C.; Beheregaray, L.B. Rapid development of microsatellites for the endangered Neotropical catfish Conorhynchus conirostris using a modest amount of 454 shot-gun pyrosequencing. Conserv. Genet. Resour 2011, 3, 373–375.
[30]
Zalapa, J.E.; Cuevas, H.; Zhu, H.; Steffan, S.; Senalik, D.; Zeldin, E.; McCown, B.; Harbut, R.; Simon, P. Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences. Am. J. Bot 2012, 99, 193–208.
[31]
Hoffman, J.I.; Nichols, H.J. A novel approach for mining polymorphic microsatellite markers. PLoS One 2011, 6, doi:10.1371/journal.pone.0023283.
[32]
Griffiths, A.M.; Sims, D.W.; Cotterell, S.P.; Nagar, A.E.; Ellis, J.R.; Lynghammar, A.; McHugh, M.; Neat, F.C.; Pade, N.G.; Queiroz, N.; et al. Molecular markers reveal spatially segregated cryptic species in a critically endangered fish, the common skate (Dipturus batis). Proc. R. Soc. B 2010, 277, 1497–1503.
[33]
Barbará, T.; Palma-Silva, C.; Paggi, G.M.; Bered, F.; Fay, M.F.; Lexer, C. Cross-species transfer of nuclear microsatellite markers: Potential and limitations. Mol. Ecol 2007, 16, 3759–3767.
[34]
Olivatti, A.M.; Boni, T.A.; Silva-Júnior, N.J.; Resende, L.V.; Gouveia, F.O.; Telles, M.P. Heterologous amplification and characterization of microsatellite markers in the Neotropical fish Leporinus friderici. Genet. Mol. Res 2011, 10, 1403–1408.
[35]
Kang, J.H.; Kim, Y.K.; Park, J.Y.; An, C.M.; Nam, M.M.; Byun, S.G.; Lee, B.I.; Lee, J.H.; Choi, T.J. Microsatellite analysis as a tool for discriminating an interfamily hybrid between olive flounder and starry flounder. Genet. Mol. Res 2011, 10, 2786–2794.
[36]
Delmas, C.E.; Lhuillier, E.; Pornon, A.; Escaravage, N. Isolation and characterization of microsatellite loci in Rhododendron ferrugineum (Ericaceae) using pyrosequencing technology. Am. J. Bot 2011, 98, e120–e122.
[37]
Rozen, S.; Skaletsky, H.J. Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols: Methods in Molecular Biology; Krawets, S., Misener, S., Eds.; Humana Press: Totowa, NJ, USA, 2000; pp. 365–386.
[38]
Basic Local Alignment Search Tool (BLAST), Available online: http://ncbi.nlm.nih.gov/blast , assessed on 9 October 2011.
[39]
Excoffier, L.; Lischer, H.E.L. Arlequin suite ver. 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol. Ecol. Resour. 2010, 10, 564–567.
[40]
Raymond, M.; Rousset, F. Genepop (version 1.2), population genetics software for exact test and ecumenicism. J. Hered 1995, 86, 248–249.
[41]
Van Oosterhout, C.; Hutchinson, W.F. Micro-checker: Software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 2004, 4, 535–538.
[42]
Goudet, J. FSTAT (version 1.2): A computer program to calculate F-statistics. J. Hered 1995, 86, 485–486.
