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Selection, trans-species polymorphism, and locus identification of major histocompatibility complex class IIβ alleles of New World ranid frogs

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Abstract

Genes encoded by the major histocompatibility complex (MHC) play key roles in the vertebrate immune system. However, our understanding of the evolutionary processes and underlying genetic mechanisms shaping these genes is limited in many taxa, including amphibians, a group currently impacted by emerging infectious diseases. To further elucidate the evolution of the MHC in frogs (anurans) and develop tools for population genetics, we surveyed allelic diversity of the MHC class II β1 domain in both genomic and complementary DNA of seven New World species in the genus Rana (Lithobates). To assign locus affiliation to our alleles, we used a “gene walking” technique to obtain intron 2 sequences that flanked MHC class IIβ exon 2. Two distinct intron sequences were recovered, suggesting the presence of at least two class IIβ loci in Rana. We designed a primer pair that successfully amplified an orthologous locus from all seven Rana species. In total, we recovered 13 alleles and documented trans-species polymorphism for four of the alleles. We also found quantitative evidence of selection acting on amino acid residues that are putatively involved in peptide binding and structural stability of the β1 domain of anurans. Our results indicated that primer mismatch can result in polymerase chain reaction (PCR) bias, which influences the number of alleles that are recovered. Using a single locus may minimize PCR bias caused by primer mismatch, and the gene walking technique was an effective approach for generating single-copy orthologous markers necessary for future studies of MHC allelic variation in natural amphibian populations.

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Acknowledgments

We thank J. D. Austin and E. Rittmeyer for assistance with specimen collection. This study was supported by Population and Evolutionary Process National Science Foundation Grants DEB-0815315 (to KRZ) and DEB-0909013 (to KRZ and AES). Members of the Zamudio laboratory group made helpful comments on the earlier versions of this manuscript. The core facilities at Cornell University (Evolutionary Genetics Core Facility and Life Sciences Core Laboratories) provided the infrastructure for data collection.

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Authors and Affiliations

  1. Department of Ecology and Evolutionary Biology, Cornell University, E145 Corson Hall, Ithaca, NY, 14853, USA

    Karen M. Kiemnec-Tyburczy, Anna E. Savage & Kelly R. Zamudio

  2. U.S. Geological Survey, Western Ecological Research Center, San Diego, CA, 92101, USA

    Jonathan Q. Richmond

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  1. Karen M. Kiemnec-Tyburczy
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  2. Jonathan Q. Richmond
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Correspondence to Karen M. Kiemnec-Tyburczy.

Electronic supplementary material

Additional supporting material for this manuscript is provided online.

Online Resource 1

Locality data for the seven ranid species from which MHC class IIβ alleles were amplified (DOC 31 kb)

Online Resource 2

Text file containing anuran MHC class IIβ sequences used for detecting selection on amino acids (TXT 7 kb)

Online Resource 3

Nucleotide sequences of partial exon 2 and intron 2 obtained from gene walking and subsequent PCR. The reverse complement of the MHC-5R primer sequence is positioned above the alignment. The intron/exon boundary is indicated by the vertical line. The splice donor site is shaded, the dashed box corresponds to the annealing site of the primer MHC-5R, and “–” represent gaps. The boxes outline the sections of the last two sequences in the alignment, Xela-DAB (GenBank accesssion no. D50039) and Raca-D2B*01, that could not be aligned with the other ranid introns (GIF 185 kb)

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Kiemnec-Tyburczy, K.M., Richmond, J.Q., Savage, A.E. et al. Selection, trans-species polymorphism, and locus identification of major histocompatibility complex class IIβ alleles of New World ranid frogs. Immunogenetics 62, 741–751 (2010). https://doi.org/10.1007/s00251-010-0476-6

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