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Genotyping of black grouse MHC class II B using Reference Strand-Mediated Conformational Analysis (RSCA)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Population and Conservation Biology. (Jacob Höglund)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Population and Conservation Biology.
2011 (English)In: BMC Research Notes, ISSN 1756-0500, Vol. 4, no 183Article in journal (Refereed) Published
Abstract [en]


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. 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.


We 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.


In 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.

Place, publisher, year, edition, pages
2011. Vol. 4, no 183
National Category
Research subject
Population Biology
URN: urn:nbn:se:uu:diva-159185DOI: 10.1186/1756-0500-4-183OAI: oai:DiVA.org:uu-159185DiVA: diva2:447849
Available from: 2011-10-13 Created: 2011-09-23 Last updated: 2013-06-12Bibliographically approved
In thesis
1. European Black Grouse: MHC Genetic Diversity and Population Structure
Open this publication in new window or tab >>European Black Grouse: MHC Genetic Diversity and Population Structure
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Black grouse Tetrao tetrix is a bird species composed of large, continuous as well as severely reduced and fragmented populations, making it an optimal species to investigate how genetic diversity is affected by habitat fragmentation. I have focused on genetic diversity in the Major Histocompatibility Complex (MHC) to measure the ability of the black grouse to respond to environmental changes.

I partly characterized MHC class II in black grouse and found striking similarities with chicken MHC class II. I demonstrated that black grouse possess a similar compact MHC as chicken with few MHC class II B (BLB) and Y (YLB) loci. I did not find evidence of balancing selection in YLB so I concentrated further studies on BLB.

I developed a PCR-based screening method for amplifying and separating expressed BLB alleles in European black grouse populations. Small fragmented populations had lost neutral genetic diversity (based on microsatellites and SNPs) compared to samples from the historical distribution and contemporary large populations. There was also a trend, albeit less pronounced, for reduced MHC diversity in these populations. Neutral markers in small isolated populations were affected by increased levels of genetic drift and were therefore genetically differentiated compared to other populations. MHC markers on the other hand, were not subjected to genetic drift to the same extent probably due a long historic process of balancing selection.

Inferences of heterozygosity and evolutionary patterns as well as detailed correlations to reproductive success and diseases cannot be performed until MHC can be amplified in a locus-specific manner. Therefore, I developed a single locus sequence-based typing method for independently amplifying MHC class II B loci (BLB1 and BLB2). I found that BLB1 and BLB2 were duplicated in a common ancestor to chickens and black grouse and that these loci are subjected to homogenizing concerted evolution due to inter-genetic exchange between loci after species divergence.  I could also verify that both BLB1 and BLB2 were transcribed in black grouse and under balancing selection.

This collection of work has significance for future conservation of black grouse as well as research and management of zoonotic diseases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 59 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 869
Major Histocompatibility Complex, BLB, balancing selection, concerted evolution, bottleneck, fragmentation, 3'UTR
National Category
Evolutionary Biology Ecology Genetics
Research subject
Biology with specialization in Population Biology
urn:nbn:se:uu:diva-160042 (URN)978-91-554-8188-9 (ISBN)
Public defence
2011-11-25, Zootissalen, Evolutionary Biology Centre, Villavägen 9, Uppsala, 09:15 (English)
Available from: 2011-11-04 Created: 2011-10-13 Last updated: 2011-11-10Bibliographically approved

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