Novel locus-specific primers for major histocompatibility complex class II alleles from glass frogs developed via genome walking


Major histocompatibility complex (MHC) genes can be used to study molecular evolution in wild vertebrate populations. Here, we used “genome walking” to develop primers for amplification of the second exon of a single expressed MHC class II locus in a glass frog, Espadarana prosoblepon. We tested the utility of the primers on two Panamian populations of E. prosoblepon. The MHC marker displayed high levels of allelic diversity (15 alleles/locus identified in each population) and evidence of positive selection in both populations. We also successfully amplified this marker from Sachatamia ilex, another centrolenid species. This marker can be used to assess the evolution of immune genes that may be associated with disease susceptibility in E. prosoblepon and closely related species in Central and South America.

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  1. Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, Slocombe R, Ragan MA, Hyatt AD, McDonald KR, Hines HB, Lips KR, Marantelli G, Parkes H (1998) Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proc Natl Acad Sci USA 95:9031–9036

    PubMed  Article  CAS  Google Scholar 

  2. Bernatchez L, Landry C (2003) MHC Studies in nonmodel vertebrates: what have we learned about natural selection in 15 years? J Evol Biol 16:363–377

    PubMed  Article  CAS  Google Scholar 

  3. Brown JH, Jardetzky TS, Gorga JC, Stern LJ, Urban RG, Strominger JL, Wiley DC (1993) Three-dimensional structure of the human class II histocompatibility HLA-DR1. Nature 364:33–39

    PubMed  Article  CAS  Google Scholar 

  4. Cottage A, Yang A, Maunders H, de Lacy R, Ramsay N (2001) Identification of DNA sequences flanking T-DNA insertions by PCR-walking. Plant Mol Biol Reptr 19:321–327

    Article  CAS  Google Scholar 

  5. Goudet J (1995) FSTAT (Version 1.2): a computer program to calculate F-statistics. J Hered 86:485–486

    Google Scholar 

  6. Guayasamin JM, Castroviejo-Fisher S, Trueb L, Ayarzaguena MR, Rada M, Vila C (2009) Phylogenetic systematics of glassfrogs (Amphibia: Centrolenidae) and their sister taxon Allophryne ruthveni. Zootaxa 2100:1–97

    Google Scholar 

  7. Hauswaldt SJ, Stuckas H, Pfautsch S, Tiedemann R (2007) Molecular characterization of MHC class II in a nonmodel anuran species, the fire-bellied toad Bombina bombina. Immunogenetics 59:479–491

    PubMed  Article  CAS  Google Scholar 

  8. Hughes A, Hughes M (1995) Natural selection on the peptide-binding regions of major histocompatibility complex molecules. Immunogenetics 42:233–243

    PubMed  Article  CAS  Google Scholar 

  9. IUCN (2012) IUCN Red list of threatened species. Version 2012.1. Accessed 05 July 2012

  10. Kiemnec-Tyburczy KM, Richmond JQ, Savage AE, Zamudio KR (2010) Selection, trans-species polymorphism and locus identification of major histocompatibility complex class IIβ alleles of new world ranid frogs. Immunogenetics 62:741–752

    PubMed  Article  CAS  Google Scholar 

  11. Klein J (1986) Natural history of the major histocompatibility complex. Wiley, New York

    Google Scholar 

  12. Lips KR, Brem F, Brenes R, Reeve JD, Alford RA, Voyles J, Carey C, Livo L, Pessier AP, Collins JP (2006) Emerging infectious disease and the loss of biodiversity in a neotropical amphibian community. Proc Natl Acad Sci USA 103:3165–3170

    PubMed  Article  CAS  Google Scholar 

  13. Radwan J, Biedrzycka A, Babik W (2010) Does reduced MHC diversity decrease viability of vertebrate populations? Biol Cons 143:537–544

    Article  Google Scholar 

  14. Raymond M, Rousset F (1995) GENEPOP (Version 1.2): population genetics software for exact tests and ecumenicism. J Hered 86:248–249

    Google Scholar 

  15. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    PubMed  Article  CAS  Google Scholar 

  16. Tong J, Bramson J, Kanduc D, Chow S, Sinha A, Ranganathan S (2006) Modeling the bound conformation of pemphigus vulgaris-associated peptides to MHC class II DR and DQ alleles. Immunome Res 2:1

    PubMed  Article  Google Scholar 

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We thank K.R. Lips for specimen collection and K.E. Tracy for assistance with PCR and cloning. Funding for this study was provided by National Science Foundation Grants (DEB-0815315 and DEB-1120249).

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Correspondence to Karen M. Kiemnec-Tyburczy.

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Kiemnec-Tyburczy, K.M., Zamudio, K.R. Novel locus-specific primers for major histocompatibility complex class II alleles from glass frogs developed via genome walking. Conservation Genet Resour 5, 109–111 (2013).

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  • Centrolenidae
  • Espadarana
  • Immunogenetics
  • Sachatamia