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RSCA genotyping of MHC for high-throughput evolutionary studies in the model organism three-spined stickleback Gasterosteus aculeatus.

Lenz TL, Eizaguirre C, Becker S, Reusch TB - BMC Evol. Biol. (2009)

Bottom Line: Experimental evidence is accumulating that MHC polymorphism is a result of balancing selection by parasites and pathogens.Analysis of the plasmid library additionally reveals the high resolution and reproducibility of the RSCA technique.It therefore provides a valuable tool to employ this highly polymorphic and adaptive marker in future high-throughput studies of host-parasite co-evolution and ecological speciation in this emerging model organism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Evolutionary Ecology, Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plön, Germany. Lenz@evolbio.mpg.de

ABSTRACT

Background: In all jawed vertebrates, highly polymorphic genes of the major histocompatibility complex (MHC) encode antigen presenting molecules that play a key role in the adaptive immune response. Their polymorphism is composed of multiple copies of recently duplicated genes, each possessing many alleles within populations, as well as high nucleotide divergence between alleles of the same species. Experimental evidence is accumulating that MHC polymorphism is a result of balancing selection by parasites and pathogens. In order to describe MHC diversity and analyse the underlying mechanisms that maintain it, a reliable genotyping technique is required that is suitable for such highly variable genes.

Results: We present a genotyping protocol that uses Reference Strand-mediated Conformation Analysis (RSCA), optimised for recently duplicated MHC class IIB genes that are typical for many fish and bird species, including the three-spined stickleback, Gasterosteus aculeatus. In addition we use a comprehensive plasmid library of MHC class IIB alleles to determine the nucleotide sequence of alleles represented by RSCA allele peaks. Verification of the RSCA typing by cloning and sequencing demonstrates high congruency between both methods and provides new insight into the polymorphism of classical stickleback MHC genes. Analysis of the plasmid library additionally reveals the high resolution and reproducibility of the RSCA technique.

Conclusion: This new RSCA genotyping protocol offers a fast, but sensitive and reliable way to determine the MHC allele repertoire of three-spined sticklebacks. It therefore provides a valuable tool to employ this highly polymorphic and adaptive marker in future high-throughput studies of host-parasite co-evolution and ecological speciation in this emerging model organism.

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Mobility values of 83 alleles with the selected three reference alleles (FLR). Mean and SD of each allele from four independent runs are shown. Arrows indicate the allele pairs that can not be differentiated with the three FLRs. Alleles are presented in the order of mobility with the reference FLR3_1_4 (top panel).
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Figure 4: Mobility values of 83 alleles with the selected three reference alleles (FLR). Mean and SD of each allele from four independent runs are shown. Arrows indicate the allele pairs that can not be differentiated with the three FLRs. Alleles are presented in the order of mobility with the reference FLR3_1_4 (top panel).

Mentions: The three mean mobility values for each of the 83 allele variants averaged over four independent runs are shown in Fig 4. To estimate the resolution of the RSCA typing method, we compared all 83 alleles pair-wise. The mean mobility difference between alleles was 46.5, 42.3 and 27.3 units for the three FLRs. In comparison, the average standard deviation of the independently obtained mobility values of an allele was 1.2, 1.0 and 1.4 units. In seven (0.2%) of 3,403 possible allele pairs, the two alleles were not distinguishable according to our definition outlined above. Six of them differed by 1 bp and one by 2 bp. The other 11 pairs with 1 bp and seven with 2 bp difference were distinguished unambiguously by using the three different FLRs (Fig 4).


RSCA genotyping of MHC for high-throughput evolutionary studies in the model organism three-spined stickleback Gasterosteus aculeatus.

Lenz TL, Eizaguirre C, Becker S, Reusch TB - BMC Evol. Biol. (2009)

Mobility values of 83 alleles with the selected three reference alleles (FLR). Mean and SD of each allele from four independent runs are shown. Arrows indicate the allele pairs that can not be differentiated with the three FLRs. Alleles are presented in the order of mobility with the reference FLR3_1_4 (top panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662802&req=5

Figure 4: Mobility values of 83 alleles with the selected three reference alleles (FLR). Mean and SD of each allele from four independent runs are shown. Arrows indicate the allele pairs that can not be differentiated with the three FLRs. Alleles are presented in the order of mobility with the reference FLR3_1_4 (top panel).
Mentions: The three mean mobility values for each of the 83 allele variants averaged over four independent runs are shown in Fig 4. To estimate the resolution of the RSCA typing method, we compared all 83 alleles pair-wise. The mean mobility difference between alleles was 46.5, 42.3 and 27.3 units for the three FLRs. In comparison, the average standard deviation of the independently obtained mobility values of an allele was 1.2, 1.0 and 1.4 units. In seven (0.2%) of 3,403 possible allele pairs, the two alleles were not distinguishable according to our definition outlined above. Six of them differed by 1 bp and one by 2 bp. The other 11 pairs with 1 bp and seven with 2 bp difference were distinguished unambiguously by using the three different FLRs (Fig 4).

Bottom Line: Experimental evidence is accumulating that MHC polymorphism is a result of balancing selection by parasites and pathogens.Analysis of the plasmid library additionally reveals the high resolution and reproducibility of the RSCA technique.It therefore provides a valuable tool to employ this highly polymorphic and adaptive marker in future high-throughput studies of host-parasite co-evolution and ecological speciation in this emerging model organism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Evolutionary Ecology, Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plön, Germany. Lenz@evolbio.mpg.de

ABSTRACT

Background: In all jawed vertebrates, highly polymorphic genes of the major histocompatibility complex (MHC) encode antigen presenting molecules that play a key role in the adaptive immune response. Their polymorphism is composed of multiple copies of recently duplicated genes, each possessing many alleles within populations, as well as high nucleotide divergence between alleles of the same species. Experimental evidence is accumulating that MHC polymorphism is a result of balancing selection by parasites and pathogens. In order to describe MHC diversity and analyse the underlying mechanisms that maintain it, a reliable genotyping technique is required that is suitable for such highly variable genes.

Results: We present a genotyping protocol that uses Reference Strand-mediated Conformation Analysis (RSCA), optimised for recently duplicated MHC class IIB genes that are typical for many fish and bird species, including the three-spined stickleback, Gasterosteus aculeatus. In addition we use a comprehensive plasmid library of MHC class IIB alleles to determine the nucleotide sequence of alleles represented by RSCA allele peaks. Verification of the RSCA typing by cloning and sequencing demonstrates high congruency between both methods and provides new insight into the polymorphism of classical stickleback MHC genes. Analysis of the plasmid library additionally reveals the high resolution and reproducibility of the RSCA technique.

Conclusion: This new RSCA genotyping protocol offers a fast, but sensitive and reliable way to determine the MHC allele repertoire of three-spined sticklebacks. It therefore provides a valuable tool to employ this highly polymorphic and adaptive marker in future high-throughput studies of host-parasite co-evolution and ecological speciation in this emerging model organism.

Show MeSH