Large-scale genotyping of highly polymorphic loci by next-generation sequencing: how to overcome the challenges to reliably genotype individuals?

Studying the different roles of adaptive genes is still a challenge in evolutionary ecology and requires reliable genotyping of large numbers of individuals. Next-generation sequencing (NGS) techniques enable such large-scale sequencing, but stringent data processing is required. Here, we develop an easy to use methodology to process amplicon-based NGS data and we apply this methodology to reliably genotype four major histocompatibility complex (MHC) loci belonging to MHC class I and II of Alpine marmots (Marmota marmota). Our post-processing methodology allowed us to increase the number of retained reads. The quality of genotype assignment was further assessed using three independent validation procedures. A total of 3069 high-quality MHC genotypes were obtained at four MHC loci for 863 Alpine marmots with a genotype assignment error rate estimated as 0.21%. The proposed methodology could be applied to any genetic system and any organism, except when extensive copy-number variation occurs (that is, genes with a variable number of copies in the genotype of an individual). Our results highlight the potential of amplicon-based NGS techniques combined with adequate post-processing to obtain the large-scale highly reliable genotypes needed to understand the evolution of highly polymorphic functional genes.

Contrasting patterns of selection acting on MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota).

The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.