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Characterization and locus-specific typing of MHC class I genes in the red-billed gull (Larus scopulinus) provides evidence for major, minor, and nonclassical loci

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Abstract

A major challenge facing studies of major histocompatibility complex (MHC) evolution in birds is the difficulty in genotyping alleles at individual loci, and the consequent inability to investigate sequence variation and selection pressures for each gene. In this study, four MHC class I loci were isolated from the red-billed gull (Larus scopulinus), representing both the first characterized MHCI genes within Charadriiformes (shorebirds, gulls, and allies) and the first full-length MHCI sequences described outside Galloanserae (gamebirds + waterfowl). Complete multilocus genotypes were obtained for 470 individuals using a combination of reference-strand conformation analysis and direct sequencing of gene-specific amplification products, and variation of peptide-binding region (PBR) exons was surveyed for all loci. Each gene is transcribed and has conserved sequence features characteristic of antigen-presenting MHCI molecules. However, higher allelic variation, a more even allele frequency distribution, and evidence of positive selection acting on a larger number of PBR residues suggest that only one locus (Lasc-UAA) functions as a major classical MHCI gene. Lasc-UBA, with more limited variation and PBR motifs that encompass a subset of Lasc-UAA diversity, was assigned a putative minor classical function, whereas the divergent and largely invariant binding-groove motifs of Lasc-UCA and -UDA are suggestive of nonclassical loci with specialized ligand-binding roles.

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Acknowledgments

We thank Kristen Choffe for her help in implementing the RSCA methodology, and three anonymous reviewers for their thoughtful comments regarding an earlier version of this manuscript. Funding was provided by a Marsden grant from the Royal Society of New Zealand (JAM and AJB), the Royal Ontario Museum Governors Fund and the Natural Sciences and Engineering Research Council of Canada (AJB), and a NSERC PGS-D scholarship (AC).

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Authors and Affiliations

  1. Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, ON, Canada, M5S 3B2

    Alison Cloutier & Allan J. Baker

  2. 10527 A Skyline Drive, Corning, NY, 14830, USA

    James A. Mills

  3. Department of Natural History, Royal Ontario Museum, 100 Queen’s Park, Toronto, ON, Canada, M5S 2C6

    Allan J. Baker

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  1. Alison Cloutier
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  2. James A. Mills
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Correspondence to Alison Cloutier.

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Supplemental Fig. S1

Reference-strand conformation analysis. a PCRs are used to amplify a FLR allele and the same region from a test sample. PCR products are shown as double-stranded DNA molecules, with different colours indicating different alleles. A star symbol represents fluorescence incorporated into one FLR strand. b PCR products are pooled, denatured, and cooled to allow re-annealing of complementary strands. Heteroduplex DNA molecules show bulges in regions of nucleotide sequence mismatch. c Hybridized DNA is separated by polyacrylamide gel electrophoresis under nondenaturing conditions. Molecules containing labelled reference strands are visualized, with relative mobility shifts resulting from the retardation of fragments due to mismatch bulges. (PDF 120 kb)

Supplemental Fig. S2

Exon–intron organization of red-billed gull MHCI genes compared to other avian taxa. Note that no information is available for Lasc-UDA exon and intron 1. Exons are drawn as shaded boxes, and introns as connecting lines. Sequence sources: duck (A. platyrhynchos Anpl-UAA, AY885227), goose (Anser sp. isolate G5, AY387655), chicken (G. gallus BF2, AL023516), quail (C. japonica Coja-B1, AB078884), turkey (Meleagris gallopavo class I antigen alpha chain 2, DQ993255). (PDF 162 kb)

Supplemental Fig. S3

Example of RSCA screening, showing mobility shifts caused by hybridization with FLR-UAA*03 (left panes) and FLR-UAA*04 (right panes) for the same individual. a Simultaneous screening of MHCI loci. b Lasc-UCA gene-specific RSCA. c Lasc-UDA gene-specific RSCA. d RSCA of cloned alleles, with identities indicated beneath sample plots. Grey shading indicates sizing bins used in allele assignment. Arrows indicate an allele not isolated during initial cloning experiments that requires validation by gene-specific PCR assays and segregation analyses (subsequently established as allele UAA*02). (PDF 193 kb)

Supplemental Fig. S4

Sequence logos of predicted PBR residues. X-axis residue numbering corresponds to Fig. 2, and residues predicted to contact peptide mainchain atoms are marked with asterisks. (PDF 245 kb)

Supplemental Fig. S5

Inferred interlocus gene conversion in red-billed gull MHCI genes. Nucleotide sequences are shown aligned to a majority rule consensus sequence. Predicted gene conversion tracts with simulation p values <0.05 are boxed in red (UAA–UBA), blue (between UAA, UCA, and UDA), and green (UBA–UDA). Additional homogenized tracts with p > 0.05 are boxed in grey. (PDF 151 kb)

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Cloutier, A., Mills, J.A. & Baker, A.J. Characterization and locus-specific typing of MHC class I genes in the red-billed gull (Larus scopulinus) provides evidence for major, minor, and nonclassical loci. Immunogenetics 63, 377–394 (2011). https://doi.org/10.1007/s00251-011-0516-x

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