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

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.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.Natural genetic variation is the basic material for evolution, and to study its origin and persistence at the molecular level is required if we are to understand how species and populations evolved as a response to selection. The major histocompatibility complex (MHC) is one of the most polymorphic regions in the vertebrate genome [1,2]. Classical MHC genes (class I and II) encode for cell surface molecules that present self and non-self antigens to T-cells and therefore play an essential role for the recognition of pathogens invading the body [3]. MHC genes are also involved in mate choice decisions in several species [4]. The high polymorphism of MHC class I and II genes is reflected at three levels: (i) the presence of several gene loci, often as a result of recent duplication (ii) a high total number of alleles (iii) an exceptionally high nucleotide diversity at the sequence level between a