Novel locus-specific primers for major histocompatibility complex class II alleles from glass frogs developed via genome
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TECHNICAL NOTE
Novel locus-specific primers for major histocompatibility complex class II alleles from glass frogs developed via genome walking Karen M. Kiemnec-Tyburczy • Kelly R. Zamudio
Received: 24 July 2012 / Accepted: 6 August 2012 / Published online: 24 August 2012 Ó Springer Science+Business Media B.V. 2012
Abstract 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. Keywords Centrolenidae Espadarana Immunogenetics Sachatamia
Genes of the major histocompatibility complex (MHC) commonly undergo adaptive evolution because they encode proteins that make physical contact with foreign peptides (Brown et al. 1993; Tong et al. 2006) and initiate immune responses (Klein 1986). Associations between disease susceptibility and MHC genotypes in natural wildlife populations (Bernatchez and Landry 2003) suggest
GenBank accession numbers will be provided upon acceptance for publication. K. M. Kiemnec-Tyburczy (&) K. R. Zamudio Department of Ecology and Evolutionary Biology, Cornell University, E145 Corson Hall, Ithaca, NY 14853, USA e-mail: [email protected]
that quantifying MHC genetic diversity may be used to predict how different populations may respond to emerging immunogenetic challenges (Radwan et al. 2010). In this study, we generated primers to amplify an MHC class II locus from a glass frog Espadarana prosoblepon, a stream-breeding frog that ranges from Honduras to the Pacific lowlands of Colombia and Ecuador (Guayasamin et al. 2009). Although the current ICUN status of E. prosoblepon is ‘‘Least Concern’’ (IUCN 2012), it is a focal species in ongoing ecological studies assessing the impact of an emerging fungal pathogen and has shown substantial declines in abundance due to pathogen introduction (Lips et al. 2006; Berger et al. 1998). Primer development consisted of three major steps: (1) amplifying expressed alleles from E. prosoblepon, (2) obtaining flanking intronic sequence from these alleles, and (3) designing and testing primer specificity at the population level. First, we extracted total RNA from the intestinal tissue of two individuals from Cana, Panama preserved in RNAlater (Invitrogen, Carlsbad, CA) using TRIzolÒ (Invitrogen) and generated complementary DNA (cDNA) from RNA using the ImProm-IITM reverse transcription system (Promega, Ma
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