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Open Access Methodology article

RSCA genotyping of MHC for high-throughput evolutionary studies in the model organism three-spined stickleback Gasterosteus aculeatus

Tobias L Lenz1*, Christophe Eizaguirre1, Sven Becker12 and Thorsten BH Reusch3

Author Affiliations

1 Department of Evolutionary Ecology, Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plön, Germany

2 Canada Centre for Inland Waters, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6, Canada

3 Leibniz-Institute of Marine Sciences (IFM-GEOMAR), Evolutionary Ecology of Marine Fishes, Düsternbrooker Weg 20, 24105 Kiel, Germany

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BMC Evolutionary Biology 2009, 9:57  doi:10.1186/1471-2148-9-57

Published: 16 March 2009

Abstract

Background

In all jawed vertebrates, highly polymorphic genes of the major histocompatibility complex (MHC) encode antigen presenting molecules that play a key role in the adaptive immune response. Their polymorphism is composed of multiple copies of recently duplicated genes, each possessing many alleles within populations, as well as high nucleotide divergence between alleles of the same species. Experimental evidence is accumulating that MHC polymorphism is a result of balancing selection by parasites and pathogens. In order to describe MHC diversity and analyse the underlying mechanisms that maintain it, a reliable genotyping technique is required that is suitable for such highly variable genes.

Results

We present a genotyping protocol that uses Reference Strand-mediated Conformation Analysis (RSCA), optimised for recently duplicated MHC class IIB genes that are typical for many fish and bird species, including the three-spined stickleback, Gasterosteus aculeatus. In addition we use a comprehensive plasmid library of MHC class IIB alleles to determine the nucleotide sequence of alleles represented by RSCA allele peaks. Verification of the RSCA typing by cloning and sequencing demonstrates high congruency between both methods and provides new insight into the polymorphism of classical stickleback MHC genes. Analysis of the plasmid library additionally reveals the high resolution and reproducibility of the RSCA technique.

Conclusion

This new RSCA genotyping protocol offers a fast, but sensitive and reliable way to determine the MHC allele repertoire of three-spined sticklebacks. It therefore provides a valuable tool to employ this highly polymorphic and adaptive marker in future high-throughput studies of host-parasite co-evolution and ecological speciation in this emerging model organism.