Abstract
BackgroundThe Major Histocompatibility Complex (MHC) is a cluster of genes involved in the vertebrate immune system and includes loci with an extraordinary number of alleles. Due to the complex evolution of MHC genes, alleles from different loci within the same MHC class can be very similar and therefore difficult to assign to separate loci. Consequently, single locus amplification of MHC genes is hard to carry out in species with recently duplicated genes in the same MHC class, and multiple MHC loci have to be genotyped simultaneously. Since amplified alleles have the same length, accurate genotyping is difficult. Reference Strand-Mediated Conformational Analysis (RSCA), which is increasingly used in studies of natural populations with multiple MHC genes, is a genotyping method capable to provide high resolution and accuracy in such cases.FindingsWe adapted the RSCA method to genotype multiple MHC class II B (BLB) genes in black grouse (Tetrao tetrix), a non-model galliform bird species, using a 96-Capillary Array Electrophoresis, the MegaBACE™ 1000 DNA Analysing System (GE Healthcare). In this study we used fluorescently labelled reference strands from both black grouse and hazel grouse and observed good agreement between RSCA and cloning/sequencing since 71 alleles were observed by cloning/sequencing and 76 alleles by RSCA among the 24 individuals included in the comparison. At the individual level however, there was a trend towards more alleles scored with RSCA (1-6 per individual) than cloning/sequencing (1-4 per individual). In 63% of the pair-wise comparison, the identical allele was scored in RSCA as in cloning/sequencing. Nine out of 24 individuals had the same number of alleles in RSCA as in cloning/sequencing. Our RSCA protocol allows a faster RSCA genotyping than presented in many other RSCA studies.ConclusionsIn this study, we have developed the RSCA typing method further to work on a 96-Capillary Array Electrophoresis (MegaBACE™ 1000). Our RSCA protocol can be applied to fast and reliable screening of MHC class II B diversity of black grouse populations. This will facilitate future large-scale population studies of black grouse and other galliformes species with multiple inseparable MHC loci.
Highlights
The Major Histocompatibility Complex (MHC) is a cluster of genes involved in the vertebrate immune system and includes loci with an extraordinary number of alleles
In this study, we have developed the Reference Strand-Mediated Conformational Analysis (RSCA) typing method further to work on a 96-Capillary Array Electrophoresis (MegaBACETM 1000)
The objective of this study was to develop an RSCA approach further, for even higher throughput MHC genotyping on a 96-Capillary Array Electrophoresis in general, and for MHC genotyping in black grouse
Summary
The Major Histocompatibility Complex (MHC) is a cluster of genes involved in the vertebrate immune system and includes loci with an extraordinary number of alleles. MHC (Major Histocompatibility Complex) is a cluster of highly variable genes that plays an important role in the immune defence and which may evolve through different forms of balancing selection [1,2,3,4] This multigene family is highly interesting from an evolutionary perspective. Because of the complicated (and not fully understood) evolution of MHC genes there are pronounced differences in the genomic organization and number of the MHC loci between vertebrate lineages [13], especially between mammalian and non-mammalian species [12,14] For this reason it is a great challenge to accurately amplify MHC genes in non-model species. Many MHC studies in non-mammalian species are struggling with genotyping PCR products including more than two MHC alleles
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
