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Conservation Genetics Resources

, Volume 2, Supplement 1, pp 181–185 | Cite as

New primers for the amplification and sequencing of nuclear loci in a taxonomically wide set of reptiles and amphibians

  • Catarina PinhoEmail author
  • Sara Rocha
  • Bruno M. Carvalho
  • Susana Lopes
  • Sofia Mourão
  • Marcelo Vallinoto
  • Tuliana O. Brunes
  • Célio F. B. Haddad
  • Helena Gonçalves
  • Fernando Sequeira
  • Nuno Ferrand
Technical Note

Abstract

We report new primers for the amplification and sequencing of 11 nuclear markers in squamate reptiles and anuran amphibians (five in squamates, six in anurans). Ten out of the 11 loci are introns (three of which are linked) that were amplified using an exon-primed, intron-crossing (EPIC) PCR strategy, whereas an eleventh locus spans part of a protein-coding gene. Squamate and anuran primers were initially developed for Lacerta schreiberi (Squamata: Lacertidae) and Pelodytes spp. (Anura: Pelodytidae), respectively. Cross-species amplification of the squamate markers was evaluated in four genera representing two additional families, whereas for anurans three genera corresponding to three additional families were tested. Three out of the five loci were successfully sequenced in all squamate taxa tested. Cross-amplification of the six anuran markers had lower, but still significant, success. We predict these markers will be of great utility for both population genetics and phylogenetic studies.

Keywords

Anura Squamata Nuclear sequence markers Introns 

Notes

Acknowledgments

This work was partially financed by Fundação para a Ciência e a Tecnologia (FCT) research projects POCI/BIA-BDE/60911/2004, PTDC/BIA-BDE/69769/2006 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and FCT joint project CAPES/FCT 244/09. CP, HG and FS are supported by post-doctoral fellowships SFRH/BPD/28869/2006, SFRH/BPD/26555/2006 and SFRH/BPD/27134/2006, respectively and SR is supported by pre-doctoral grant SFRH/BD/17541/2004, all from FCT. BMC and TOB are supported by the research grants (BI/PTDC/BIA-BDE/69769/2006 and BI/PTDC/BIA-QOR/71492/2006, respectively). MV is supported by a post-doctoral grant CAPES 1362-07-0. CFBH thanks Fundação de Amparo à Pesquisa do Estado de S. Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico for financial support. We further thank Raquel Godinho and Miguel Tejedo for providing tissue samples.

References

  1. Avise JC (2000) Phylogeography: the history and formation of species. Harvard University Press, CambridgeGoogle Scholar
  2. Ballard JWO, Whitlock MC (2004) The incomplete natural history of mitochondria. Mol Ecol 13:729–744CrossRefPubMedGoogle Scholar
  3. Dolman G, Phillips B (2004) Single copy nuclear DNA markers characterized for comparative phylogeography in Australian Wet Tropics rainforest skinks. Mol Ecol Notes 4:185–187CrossRefGoogle Scholar
  4. Edwards SV, Beerli P (2000) Perspective: gene divergence, population divergence, and the variance in coalescence time in phylogeographic studies. Evolution 54:1839–1854PubMedGoogle Scholar
  5. Gibbons JW, Scott DE, Ryan TJ, Buhlmann KA, Tuberville TD, Metts BS, Greene JL, Mills T, Leiden Y, Poppy S, Winne CT (2000) The global decline of reptiles, déjà vu amphibians. Bioscience 50:653–666CrossRefGoogle Scholar
  6. Godinho R, Mendonça B, Crespo EG, Ferrand N (2006) Genealogy of the nuclear b-fibrinogen locus in a highly structured lizard species: comparison with mtDNA and evidence for intragenic recombination in the hybrid zone. Heredity 16:454–463CrossRefGoogle Scholar
  7. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  8. Hoegg S, Vences M, Brinkmann H, Meyer A (2004) Phylogeny and comparative substitution rates of frogs inferred from sequences of three nuclear genes. Mol Biol Evol 21:1188–1200CrossRefPubMedGoogle Scholar
  9. Hudson RR, Kaplan NL (1985) Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111:147–164PubMedGoogle Scholar
  10. Irwin DE (2002) Phylogeographic breaks without geographic barriers to gene flow. Evolution 56:2383–2394PubMedGoogle Scholar
  11. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452CrossRefPubMedGoogle Scholar
  12. Martínez-Solano I, Gonçalves H, Arntzen JW, García-París M (2004) Phylogenetic relationships and biogeography of midwife toads (Discoglossidae: Alytes). J Biogeogr 31:603–618CrossRefGoogle Scholar
  13. Paulo S, Dias C, Bruford MW, Jordan WC, Nichols RA (2001) The persistence of Pliocene populations through the Pleistocene climatic cycles: evidence from the phylogeography of an Iberian lizard. Proc R Soc B 268:1625–1630CrossRefPubMedGoogle Scholar
  14. Pinho C, Ferrand N, Harris DJ (2006) Reexamination of the Iberian and North African Podarcis (Squamata: Lacertidae) phylogeny based on increased mitochondrial DNA sequencing. Mol Phylogenet Evol 38:266–273CrossRefPubMedGoogle Scholar
  15. Rosenblum EB, Hoekstra HE, Nachman MW (2004) Adaptive reptile color variation and the evolution of the Mc1r gene. Evolution 58:1794–1808PubMedGoogle Scholar
  16. Saint KM, Austin CC, Donnellan SC, Hutchinson MN (1998) C-mos, a nuclear marker useful for squamate phylogenetic analysis. Mol Phylogenet Evol 10:259–263CrossRefPubMedGoogle Scholar
  17. Sánchez-Herraíz MJ, Barbadillo LJ, Machordom A, Sanchiz B (2000) A new species of pelodytid frog from the Iberian Peninsula. Herpetologica 56:105–118Google Scholar
  18. Stephens M, Smith NJ, Donnelly P (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68:978–989CrossRefPubMedGoogle Scholar
  19. Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, Waller RW (2004) Status and trends of amphibian declines and extinctions worldwide. Science 306:1783–1786CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Catarina Pinho
    • 1
    Email author
  • Sara Rocha
    • 1
    • 2
    • 3
  • Bruno M. Carvalho
    • 1
  • Susana Lopes
    • 1
  • Sofia Mourão
    • 1
  • Marcelo Vallinoto
    • 1
    • 4
  • Tuliana O. Brunes
    • 1
  • Célio F. B. Haddad
    • 5
  • Helena Gonçalves
    • 1
  • Fernando Sequeira
    • 1
  • Nuno Ferrand
    • 1
    • 2
  1. 1.CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos. Campus Agrário de VairãoUniversidade do PortoVairãoPortugal
  2. 2.Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade do PortoPortoPortugal
  3. 3.Departamento de Bioquímica, Genética e Inmunología, Facultad de BiologíaUniversidad de VigoVigoSpain
  4. 4.Universidade Federal do ParáBragançaBrazil
  5. 5.Departamento de Zoologia, Instituto de BiociênciasUniversidade Estadual PaulistaRio ClaroBrazil

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