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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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Suitability of reference alleles (FLR). Distribution of genetic distances between alleles in the collected library and a) a suitable FLR with a broad distribution of genetic distances, b) a less suitable FLR with a narrow distribution. Distance is measured in pair-wise nucleotide p-distance. Note the different scale of the y-axis between panel a) and b).
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Figure 3: Suitability of reference alleles (FLR). Distribution of genetic distances between alleles in the collected library and a) a suitable FLR with a broad distribution of genetic distances, b) a less suitable FLR with a narrow distribution. Distance is measured in pair-wise nucleotide p-distance. Note the different scale of the y-axis between panel a) and b).

Mentions: The selection of suitable reference alleles is a crucial step in the optimization of RSCA. Selection criteria were: First, the reference alleles should represent sequence variants that do not occur in the screened populations. Second, they should not be too genetically distant to assure reliable hybridisation. And third, to increase resolution, the individual reference sequences should be as dissimilar from each other as possible. Due to the trans-species polymorphism of the MHC [57] it is even possible to use reference sequences from closely related species [25]. However, this increases the risk that certain alleles do not hybridize, which again increases with the complexity of the template (number of alleles) because of competition between alleles during the hybridisation reaction. Generally, when no sequence information is available, the resolution of the reference alleles can be tested by screening a subset of individuals, followed by a comparison of the peak distribution between references to choose the ones that show the broadest distribution and the highest number of peaks. For our study we tested nine cloned MHC IIB sequence variants of three-spined sticklebacks from a West Canadian population (TBH Reusch & T Reimchen, unpublished data), which due to long divergence time [58] are unlikely to carry any European alleles. Due to the existence of a database of more than 120 sequence variants that have been sequenced over the years [15,53,54] [TBH Reusch, KM Wegner, C Eizaguirre & TL Lenz, unpublished data], we were able to compare the genetic distance between the Canadian alleles and our local ones, to be able to choose candidate alleles that differed from each other more, but from the local alleles on average less than the population average. Additionally we estimated the distribution of the genetic distance between each reference and all the known alleles from our local populations to choose the flattest and broadest distribution (see Fig 3 for examples). A broad distribution promises the highest resolution of alleles, because genetic distance and molecule mobility in the gel matrix are highly correlated due to the three-dimensional structure of the heteroduplex, which increases in complexity with more mismatches between the reference and the target strand. A flat and broad distribution of the genetic distances between reference allele and the allele test pool therefore leads to a broader distribution of allele peaks and avoids overlap between them.


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)

Suitability of reference alleles (FLR). Distribution of genetic distances between alleles in the collected library and a) a suitable FLR with a broad distribution of genetic distances, b) a less suitable FLR with a narrow distribution. Distance is measured in pair-wise nucleotide p-distance. Note the different scale of the y-axis between panel a) and b).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Suitability of reference alleles (FLR). Distribution of genetic distances between alleles in the collected library and a) a suitable FLR with a broad distribution of genetic distances, b) a less suitable FLR with a narrow distribution. Distance is measured in pair-wise nucleotide p-distance. Note the different scale of the y-axis between panel a) and b).
Mentions: The selection of suitable reference alleles is a crucial step in the optimization of RSCA. Selection criteria were: First, the reference alleles should represent sequence variants that do not occur in the screened populations. Second, they should not be too genetically distant to assure reliable hybridisation. And third, to increase resolution, the individual reference sequences should be as dissimilar from each other as possible. Due to the trans-species polymorphism of the MHC [57] it is even possible to use reference sequences from closely related species [25]. However, this increases the risk that certain alleles do not hybridize, which again increases with the complexity of the template (number of alleles) because of competition between alleles during the hybridisation reaction. Generally, when no sequence information is available, the resolution of the reference alleles can be tested by screening a subset of individuals, followed by a comparison of the peak distribution between references to choose the ones that show the broadest distribution and the highest number of peaks. For our study we tested nine cloned MHC IIB sequence variants of three-spined sticklebacks from a West Canadian population (TBH Reusch & T Reimchen, unpublished data), which due to long divergence time [58] are unlikely to carry any European alleles. Due to the existence of a database of more than 120 sequence variants that have been sequenced over the years [15,53,54] [TBH Reusch, KM Wegner, C Eizaguirre & TL Lenz, unpublished data], we were able to compare the genetic distance between the Canadian alleles and our local ones, to be able to choose candidate alleles that differed from each other more, but from the local alleles on average less than the population average. Additionally we estimated the distribution of the genetic distance between each reference and all the known alleles from our local populations to choose the flattest and broadest distribution (see Fig 3 for examples). A broad distribution promises the highest resolution of alleles, because genetic distance and molecule mobility in the gel matrix are highly correlated due to the three-dimensional structure of the heteroduplex, which increases in complexity with more mismatches between the reference and the target strand. A flat and broad distribution of the genetic distances between reference allele and the allele test pool therefore leads to a broader distribution of allele peaks and avoids overlap between them.

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