Lenz Tobias L, Eizaguirre Christophe, Becker Sven, Reusch Thorsten B H
Department of Evolutionary Ecology, Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plön, Germany.
BMC Evol Biol. 2009 Mar 16;9:57. doi: 10.1186/1471-2148-9-57.
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.
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.
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.
在所有有颌脊椎动物中，主要组织相容性复合体（MHC）的高度多态性基因编码抗原呈递分子，这些分子在适应性免疫反应中起关键作用。其多态性由近期复制基因的多个拷贝组成，每个拷贝在种群内拥有许多等位基因，并且同一物种的等位基因之间存在高度核苷酸差异。越来越多的实验证据表明，MHC多态性是寄生虫和病原体平衡选择的结果。为了描述MHC多样性并分析维持其多样性的潜在机制，需要一种适用于此类高度可变基因的可靠基因分型技术。
我们提出了一种基因分型方案，该方案使用参考链介导的构象分析（RSCA），针对许多鱼类和鸟类物种（包括三刺鱼，Gasterosteus aculeatus）典型的近期复制的MHC IIB类基因进行了优化。此外，我们使用MHC IIB等位基因的综合质粒文库来确定由RSCA等位基因峰代表的等位基因的核苷酸序列。通过克隆和测序对RSCA分型进行验证，结果表明两种方法之间具有高度一致性，并为经典三刺鱼MHC基因的多态性提供了新的见解。对质粒文库的分析还揭示了RSCA技术的高分辨率和可重复性。
这种新的RSCA基因分型方案提供了一种快速、灵敏且可靠的方法来确定三刺鱼的MHC等位基因库。因此，它为在这种新兴模式生物的宿主 - 寄生虫共同进化和生态物种形成的未来高通量研究中使用这种高度多态性和适应性标记提供了一种有价值的工具。
