Genes of the major histocompatibility complex (MHC) code for cell surface proteins essential for adaptive immunity. They show the most outstanding genetic diversity in vertebrates, which has been connected with various fitness traits and thus with the long-term persistence of populations. In this study, polymorphism of the MHC class II DRB locus was investigated in chamois with Single-Strand Conformation Polymorphism (SSCP)/Sanger genotyping and Ion Torrent S5 next-generation sequencing (NGS). From eight identified DRB variants in 28 individuals, five had already been described, and three were new, undescribed alleles. With conventional SSCP/Sanger sequencing, we were able to detect seven alleles, all of which were also detected with NGS. We found inconsistencies in the individual genotypes between the two methods, which were mainly caused by allelic dropout in the SSCP/Sanger method. Six out of 28 individuals were falsely classified as homozygous with SSCP/Sanger analysis. Overall, 25% of the individuals were identified as genotyping discrepancies between the two methods. Our results show that NGS technologies are better performing in sequencing highly variable regions such as the MHC, and they also have a higher detection capacity, thus allowing a more accurate description of the genetic composition, which is crucial for evolutionary and population genetic studies.

• Klíčová slova
• Ion Torrent, Rupicapra rupicapra, major histocompatibility complex, next-generation sequencing,
• Publikační typ
• časopisecké články MeSH

The major histocompatibility complex (MHC) plays a central role in the adaptive immune response and is the most polymorphic gene family in vertebrates. Although high-throughput sequencing has increasingly been used for genotyping families of co-amplifying MHC genes, its potential to facilitate early steps in the characterisation of MHC variation in nonmodel organism has not been fully explored. In this study we evaluated the usefulness of de novo transcriptome assembly in characterisation of MHC sequence diversity. We found that although de novo transcriptome assembly of MHC I genes does not reconstruct sequences of individual alleles, it does allow the identification of conserved regions for PCR primer design. Using the newly designed primers, we characterised MHC I sequences in the bank vole. Phylogenetic analysis of the partial MHC I coding sequence (2-4 exons) of the bank vole revealed a lack of orthology to MHC I of other Cricetidae, consistent with the high gene turnover of this region. The diversity of expressed alleles was characterised using ultra-deep sequencing of the third exon that codes for the peptide-binding region of the MHC molecule. High allelic diversity was demonstrated, with 72 alleles found in 29 individuals. Interindividual variation in the number of expressed loci was found, with the number of alleles per individual ranging from 5 to 14. Strong signatures of positive selection were found for 8 amino acid sites, most of which are inferred to bind antigens in human MHC, indicating conservation of structure despite rapid sequence evolution.

• MeSH
• alely MeSH
• Arvicolinae genetika   MeSH
• DNA primery MeSH
• exony MeSH
• fylogeneze MeSH
• genetická variace MeSH
• genotyp MeSH
• geny MHC třídy I * MeSH
• hlavní histokompatibilní komplex genetika   MeSH
• multigenová rodina MeSH
• myši MeSH
• transkriptom * MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA primery MeSH