Genotyping strategy matters when analyzing hypervariable major histocompatibility complex‐Experience from a passerine bird

Abstract

Genotyping of classical major histocompatibility complex (MHC) genes is challenging when they are hypervariable and occur in multiple copies. In this study, we used several different approaches to genotype the moderately variable MHC class I exon 3 (MHCIe3) and the highly polymorphic MHC class II exon 2 (MHCIIβe2) in the bluethroat (Luscinia svecica). Two family groups (eight individuals) were sequenced in replicates at both markers using Ion Torrent technology with both a single‐ and a dual‐indexed primer structure. Additionally, MHCIIβe2 was sequenced on Illumina MiSeq. Allele calling was conducted by modifications of the pipeline developed by Sommer et al. (BMC Genomics, 14, 2013, 542) and the software AmpliSAS. While the different genotyping strategies gave largely consistent results for MHCIe3, with a maximum of eight alleles per individual, MHCIIβe2 was remarkably complex with a maximum of 56 MHCIIβe2 alleles called for one individual. Each genotyping strategy detected on average 50%–82% of all MHCIIβe2 alleles per individual, but dropouts were largely allele‐specific and consistent within families for each strategy. The discrepancies among approaches indicate PCR biases caused by the platform‐specific primer tails. Further, AmpliSAS called fewer alleles than the modified Sommer pipeline. Our results demonstrate that allelic dropout is a significant problem when genotyping the hypervariable MHCIIβe2. As these genotyping errors are largely nonrandom and method‐specific, we caution against comparing genotypes across different genotyping strategies. Nevertheless, we conclude that high‐throughput approaches provide a major advance in the challenging task of genotyping hypervariable MHC loci, even though they may not reveal the complete allelic repertoire.

Lack of statistical power as a major limitation in understanding MHC‐mediated immunocompetence in wild vertebrate populations

“…Compared to other gene systems, the combination of the highly dynamic evolution of the MHC system (occurrence of many duplicated genes, high rates of recombination and gene conversion, and high levels of polymorphism) and errors generated during PCR and sequencing make it difficult to discriminate artefacts from true MHC alleles (Babik, ; Burri, Promerova, Goebel, & Fumagalli, ). However, the advent of high‐throughput sequencing technology coupled with recent filtering procedures specifically designed for the MHC system have considerably helped to gain reliable genotypes (Babik, Taberlet, Ejsmond, & Radwan, ; Biedrzycka, Sebastian, Migalska, Westerdahl, & Radwan, ; Ferrandiz‐Rovira, Bigot, Allaine, Callait‐Cardinal, & Cohas, ; Gaigher et al, ; Grogan, McGinnis, Sauther, Cuozzo, & Drea, ; Lighten et al, ; Oomen, Gillett, & Kyle, ; Promerová et al, ; Rekdal, Anmarkrud, Johnsen, & Lifjeld, ; Sebastian, Herdegen, Migalska, & Radwan, ; Sommer et al, ; Stutz & Bolnick, ). Such technology also enables the sequencing of thousands of samples in parallel, which can drastically improve sample sizes for association studies.…”

Extra‐pair mating in a passerine bird with highly duplicated major histocompatibility complex class II: Preference for the golden mean

“…Only the male with the highest number of offspring in the respective nest was included when there were two EPMs genotyped for one nest (relevant in four nests in this data set). All the statistical analyses were conducted in r (R Core Team, , see Rekdal, Anmarkrud, Lifjeld, & Johnsen, for scripts and input data), while the amino acid distances were calculated by mega7 (Kumar, Stecher, & Tamura, ). The distance to the population average number of PSS alleles was square root transformed to endeavor normality in all tests using this variable.…”

“…All DNA samples were amplified in duplicates using the primers MHCIIFihy-E2CF and MHCIIFihy-E2CR (Canal, Alcaide, Anmarkrud, & Potti, 2010) and the sample indexing setup described by Fadrosh et al (2014). Details regarding PCR conditions and thermal profile are presented elsewhere (Rekdal et al, 2018). The amplicons were sequenced on an Illumina MiSeq instrument using v3 chemistry.…”

“…In this study, we investigated the significance of MHC‐based mate choice in a passerine species with highly duplicated MHC. The bluethroat ( Luscinia svecica , Linnaeus, 1758) provides an excellent study system as it is among the bird species with the highest intraindividual MHCII diversity known to date (minimum 28 loci; Rekdal, Anmarkrud, Johnsen, & Lifjeld, ), and has an extensive extra‐pair mating system (i.e., about 50% of the nests have extra‐pair offspring; Johnsen & Lifjeld, ). Intriguingly, immunogenetic benefits of extra‐pair copulations have indeed been suggested for this species: Johnsen, Andersen, Sunding, and Lifjeld () and Fossøy, Johnsen, and Lifjeld () found a higher immune response in extra‐pair offspring than in both their maternal and paternal half‐siblings.…”

“…In this study, we investigated the significance of MHC-based mate choice in a passerine species with highly duplicated MHC. The bluethroat (Luscinia svecica, Linnaeus, 1758) provides an excellent study system as it is among the bird species with the highest intraindividual MHCII diversity known to date (minimum 28 loci; Rekdal, Anmarkrud, Johnsen, & Lifjeld, 2018), and has an extensive extra-pair mating system (i.e., about 50% of the nests have extra-pair offspring; Johnsen & Lifjeld, 2003).…”