Large-scale MHC class II genotyping of a wild lemur population by next generation sequencing

Elise Huchard,Susanne Schliehe-Diecks,Alice Baniel,Peter M. Kappeler Peter,Christina Albrecht,Markus Brameier,Christian Roos

doi:10.1007/s00251-012-0649-6

Elise Huchard, Susanne Schliehe-Diecks + Show 5 more

Open Access

https://doi.org/10.1007/s00251-012-0649-6

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Abstract

The critical role of major histocompatibility complex (MHC) genes in disease resistance, along with their putative function in sexual selection, reproduction and chemical ecology, make them an important genetic system in evolutionary ecology. Studying selective pressures acting on MHC genes in the wild nevertheless requires population-wide genotyping, which has long been challenging because of their extensive polymorphism. Here, we report on large-scale genotyping of the MHC class II loci of the grey mouse lemur (Microcebus murinus) from a wild population in western Madagascar. The second exons from MHC-DRB and -DQB of 772 and 672 individuals were sequenced, respectively, using a 454 sequencing platform, generating more than 800,000 reads. Sequence analysis, through a stepwise variant validation procedure, allowed reliable typing of more than 600 individuals. The quality of our genotyping was evaluated through three independent methods, namely genotyping the same individuals by both cloning and 454 sequencing, running duplicates, and comparing parent–offspring dyads; each displaying very high accuracy. A total of 61 (including 20 new) and 60 (including 53 new) alleles were detected at DRB and DQB genes, respectively. Both loci were non-duplicated, in tight linkage disequilibrium and in Hardy–Weinberg equilibrium, despite the fact that sequence analysis revealed clear evidence of historical selection. Our results highlight the potential of 454 sequencing technology in attempts to investigate patterns of selection shaping MHC variation in contemporary populations. The power of this approach will nevertheless be conditional upon strict quality control of the genotyping data.

Highlights

Major histocompatibility complex (MHC) molecules are cell surface glycoproteins that play a critical role in the vertebrate immune system by binding “self” and “non-self” antigenic peptides and presenting them to T-lymphocytes, which initiate an immune response against pathogens
A recombination occurring between the end of the DRB fragment (4 nucleotides before the start of the DRB reverse primer) and the reverse DQB specific primer led to chimeric sequences associating the DRB forward primer with the DQB reverse primer, in roughly 155,000 (22 %) out of 700,000 reads
In line with previous work on the MHC class II genes in the grey mouse lemur we found extensive allelic diversity at both DRB and DQB genes following a large-scale, high-resolution genotyping effort

Summary

Introduction

Major histocompatibility complex (MHC) molecules are cell surface glycoproteins that play a critical role in the vertebrate immune system by binding “self” and “non-self” antigenic peptides and presenting them to T-lymphocytes, which initiate an immune response against pathogens. Because nonsynonymous substitutions are typically concentrated in the antigen-binding sites (ABS) of the MHC molecules, the extent of allelic polymorphism is Immunogenetics (2012) 64:895–913 thought to reflect parasite-driven selection (Hughes and Hughes 1995; Hughes and Nei 1988). Individual MHC molecules can only bind a subset of antigens, and gene duplications and polymorphism at the ABS are thought to reflect adaptations enabling individuals to respond to a greater variety of antigens. MHC has attracted interest from biologists across various fields, including immunology and genetics, and reproductive and evolutionary biology as well as conservation biology

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