Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556-5645 USA.
BMC Genomics. 2009 Dec 9;10:590. doi: 10.1186/1471-2164-10-590.
Microsatellite markers have proven useful in genetic studies in many organisms, yet microsatellite-based studies of the dengue and yellow fever vector mosquito Aedes aegypti have been limited by the number of assayable and polymorphic loci available, despite multiple independent efforts to identify them. Here we present strategies for efficient identification and development of useful microsatellites with broad coverage across the Aedes aegypti genome, development of multiplex-ready PCR groups of microsatellite loci, and validation of their utility for population analysis with field collections from Haiti.
From 79 putative microsatellite loci representing 31 motifs identified in 42 whole genome sequence supercontig assemblies in the Aedes aegypti genome, 33 microsatellites providing genome-wide coverage amplified as single copy sequences in four lab strains, with a range of 2-6 alleles per locus. The tri-nucleotide motifs represented the majority (51%) of the polymorphic single copy loci, and none of these was located within a putative open reading frame. Seven groups of 4-5 microsatellite loci each were developed for multiplex-ready PCR. Four multiplex-ready groups were used to investigate population genetics of Aedes aegypti populations sampled in Haiti. Of the 23 loci represented in these groups, 20 were polymorphic with a range of 3-24 alleles per locus (mean = 8.75). Allelic polymorphic information content varied from 0.171 to 0.867 (mean = 0.545). Most loci met Hardy-Weinberg expectations across populations and pairwise FST comparisons identified significant genetic differentiation between some populations. No evidence for genetic isolation by distance was observed.
Despite limited success in previous reports, we demonstrate that the Aedes aegypti genome is well-populated with single copy, polymorphic microsatellite loci that can be uncovered using the strategy developed here for rapid and efficient screening of genome supercontig assemblies. These loci are suitable for genetic and population studies using multiplex-PCR.
微卫星标记已被证明在许多生物体的遗传研究中非常有用，但由于可用的可检测和多态性基因座数量有限，基于微卫星的登革热和黄热病媒介蚊子埃及伊蚊的研究受到了限制，尽管已经进行了多项独立的努力来鉴定它们。在这里，我们提出了在埃及伊蚊基因组中进行广泛覆盖的有效鉴定和开发有用微卫星的策略，开发了微卫星基因座的多重准备 PCR 组，并验证了它们在海地实地采集的种群分析中的实用性。
从 79 个假定的微卫星基因座中，代表 42 个埃及伊蚊基因组全基因组序列超级基因座组装中的 31 个基序，33 个微卫星在四个实验室品系中作为单拷贝序列扩增，每个基因座有 2-6 个等位基因。三核苷酸基序代表了大多数（51%）多态性单拷贝基因座，并且这些基序都不在假定的开放阅读框内。开发了 7 组每组 4-5 个微卫星基因座，用于多重准备 PCR。使用 7 个多重准备基因座组来研究在海地采集的埃及伊蚊种群的群体遗传学。在这些基因座组中代表的 23 个基因座中，有 20 个具有多态性，每个基因座有 3-24 个等位基因（平均值=8.75）。等位基因多态性信息含量从 0.171 到 0.867 不等（平均值=0.545）。大多数基因座在整个种群中都符合 Hardy-Weinberg 预期，并且成对的 FST 比较确定了一些种群之间存在显著的遗传分化。没有观察到遗传隔离与距离有关的证据。
尽管以前的报告中取得的成功有限，但我们证明，埃及伊蚊基因组中存在大量的单拷贝、多态性微卫星基因座，可以使用这里开发的策略快速有效地筛选基因组超级基因座组装来揭示这些基因座。这些基因座适合使用多重 PCR 进行遗传和种群研究。
