Conservation Genetics Resources

, Volume 4, Issue 1, pp 15–17 | Cite as

Isolation and PCR-multiplex genotyping of 18 novel microsatellite markers for the threatened southern pygmy perch (Nannoperca australis)

  • Daniel C. Carvalho
  • Michael P. Hammer
  • Luciano B. BeheregarayEmail author
Technical Note


A set of 18 microsatellites was developed for the freshwater fish southern pygmy perch (SPP) Nannoperca australis (Percichthyidae). SPP is an endangered species endemic to South-eastern Australia. Applying a next generation DNA sequencing approach, we obtained 12,725 sequences containing putative microsatellite motifs using 1/8 of a shot-gun pyrosequencing reaction. Twenty-three microsatellite motifs with enough flanking sequence were selected for primer design. All 18 microsatellite loci were successfully genotyped using three multiplex reactions in 38 specimens of SPP rescued from the wild and kept in captivity. The number of alleles per locus varied from 3 to 15 alleles per locus (mean = 7.6) and the observed heterozygosity ranged from 0.368 to 0.921 (mean = 0.648). Evidence for null alleles was observed only for one locus (Nau33). No deviation from Hardy–Weinberg equilibrium or linkage disequilibrium between pairs of loci were detected. These polymorphic markers will prove useful for an ongoing conservation-breeding program of SPP aimed at minimizing kinship in captivity and for studies on landscape and restoration genetics.


Next Generation DNA sequencing Endangered species Percichthyidae Microsatellites Captive breeding program Conservation 



Funding for this study was provided by the Australian Research Council (LP100200409 to Beheregaray, Harris & Adams). Additional support was received by Department of Environment and Natural Resources, SA Museum, SA Murray-Darling Basin NRM Board, PIRSA Fisheries and Native Fish Australia (SA). We thank Mike Gardner for assistance with data analysis and Mariana Bertelli for laboratory assistance. DC is grateful to CAPES, an institution from the Brazilian Ministry of Education, for his postdoctoral fellowship (# 4095-09-0).


  1. Beheregaray LB, Sunnucks P (2000) Microsatellite loci isolated from Odontesthes argentinensis and the O. perugiae species group and their use in other South American silverside fish. Mol Ecol 9(5):629–631. doi: 10.1046/j.1365-294x.2000.00882.x PubMedCrossRefGoogle Scholar
  2. Carvalho D, Beheregaray L (2011) Rapid development of microsatellites for the endangered neotropical catfish Conorhynchus conirostris using a modest amount of 454 shot-gun pyrosequencing. Conserv Genet Resour 3:373–375Google Scholar
  3. Cook BD, Ma J, Hillyer MJ, Song SD, Hughes JM (2006) Polymorphic microsatellite markers in an Australian freshwater fish, southern pygmy perch Nannoperca australis. Mol Ecol Notes 6(3):907–908. doi: 10.1111/j.1471-8286.2006.01395.x CrossRefGoogle Scholar
  4. Cook BD, Bunn SE, Hughes JM (2007) Molecular genetic and stable isotope signatures reveal complementary patterns of population connectivity in the regionally vulnerable southern pygmy perch (Nannoperca australis). Biol Conserv 138(1–2):60–72. doi: 10.1016/j.biocon.2007.04.002 CrossRefGoogle Scholar
  5. Faircloth BC (2008) Msatcommander: detection of microsatellite repeat arrays and automated, locus-specific primer design. Mol Ecol Resour 8(1):92–94. doi: 10.1111/j.1471-8286.2007.01884.x PubMedCrossRefGoogle Scholar
  6. Hall A, Higham J, Hammer M, Brice C, Zampatti B (2009) Drought action plan for South Australian murray-darling basin threatened freshwater fish populations 2009–2010; rescue to recovery. AdelaideGoogle Scholar
  7. Hammer M (2001) Molecular systematics and conservation biology of the southern pygmy perch Nannoperca australis (Günther, 1861) (Teleostei: Percichthyidae) in south-eastern Australia. B. Sc. (Honours) thesis, Adelaide UniversityGoogle Scholar
  8. Hammer M, Wedderburn S, van Weenan J (2009) Action plan for South Australian freshwater fishes. Native Fish Australia (SA) Inc., Adelaide. p 206Google Scholar
  9. Holleley CE, Geerts PG (2009) Multiplex manager 1.0: a cross-platform computer program that plans and optimizes multiplex PCR. Biotechniques 46 (7):511–517. doi: 10.2144/000113156
  10. Lintermans M (2007) Fishes of the Murray-Darling basin: an introductory guide. Canberra, Murray-Darling Basin CommissionGoogle Scholar
  11. Margulies M, Egholm M, Altman WE et al (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437(7057):376–380. doi: 10.1038/nature03959 PubMedGoogle Scholar
  12. Meglécz E (2007) Microfamily (version 1): a computer program for detecting flanking-region similarities among different microsatellite loci. Mol Ecol Notes 7(1):18–20. doi: 10.1111/j.1471-8286.2006.01537.x CrossRefGoogle Scholar
  13. Rice WR (1989) Analyzing tables of statistical tests. Evolution 43(1):223–225. doi: 10.2307/2409177 CrossRefGoogle Scholar
  14. Rousset F (2008) Genepop ‘007: a complete re-implementation of the genepop software for windows and linux. Mol Ecol Resour 8(1):103–106. doi: 10.1111/j.1471-8286.2007.01931.x PubMedCrossRefGoogle Scholar
  15. Rozen S, Skaletsky H (2000) Primer3 on the www for general users and for biologist programmers. Methods Mol Biol 132:365–386. doi: 10.1385/1-59259-192-2:365 PubMedGoogle Scholar
  16. Unmack PJ, Hammer M, Adams M, Dowling TE (in press). A phylogenetic analysis of pygmy perches (Teleostei: Percichthyidae) with an assessment of the major historical influences on aquatic biogeography in southern Australia. Syst BiolGoogle Scholar
  17. Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) Micro-checker: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4(3):535–538. doi: 10.1111/J.1471-8286.2004.00684.X CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Daniel C. Carvalho
    • 1
    • 2
  • Michael P. Hammer
    • 3
  • Luciano B. Beheregaray
    • 1
    Email author
  1. 1.Molecular Ecology Laboratory, School of Biological SciencesFlinders UniversityAdelaideAustralia
  2. 2.Laboratório de Genética Animal, Escola de VeterináriaUniversidade Federal de Minas GeraisMinas GeraisBrazil
  3. 3.Evolutionary Biology UnitSouth Australian MuseumNorth Terrace, AdelaideAustralia

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