Earth and Life Institute - Research group Genetics, Reproduction, Populations, Université catholique de Louvain, Croix du Sud 2, Box L705 14, B-1348 Louvain-la-Neuve, Belgium.
Mol Ecol Resour. 2012 Jan;12(1):91-7. doi: 10.1111/j.1755-0998.2011.03072.x. Epub 2011 Sep 22.
Microsatellites represent one of the most commonly used genetic markers for population genetic studies. Traditionally, their development is quite time consuming, requiring construction of a genomic library enriched for repeated motifs. Using pyrosequencing, a fast and cost-effective new generation sequencing technique, we produced 24,340,862 bases in 63,860 short fragment reads, including 1170 dinucleotide motifs with a minimum of six repeats and 1383 trinucleotide motifs with a minimum of four repeats for the Marsh Cinquefoil, Comarum palustre L., an endangered marsh pioneer species. We selected 58 loci with SSR (Short Sequence Repeat) segments (at least 10 repeats) for a preliminary screening. Out of them, we screened 29 loci on a capillary sequencer after ligation in a vector and PCR using T7 forward primer labelled with FAM fluorescent dye and the specific unlabeled reverse primers. This procedure allowed us to screen large number of candidate loci with the same labelled primer and unlabelled specific primers. Finally, we characterized 20 polymorphic microsatellite markers, nine dinucleotides and 11 trinucleotides. We used these markers to assess genetic diversity and clonal structure in two Belgian populations. All loci showed a maximum of two alleles per individual, suggesting that they are from a diploid genome. One genet was detected in a newly extending population while 53 different genets in a long-term ecologically managed population. The number of alleles per locus ranged from 6 to 14 in this old population with an expected heterozygosity, ranging from 0.5964 to 0.8278. These preliminary results show a genet size up to 7.2 m.
微卫星是群体遗传学研究中最常用的遗传标记之一。传统上，它们的开发非常耗时，需要构建富含重复基序的基因组文库。我们使用焦磷酸测序技术，这是一种快速且具有成本效益的新一代测序技术，从 63860 个短片段读取中产生了 24340862 个碱基，其中包括 1170 个二核苷酸基序，其最小重复数为 6，1383 个三核苷酸基序，其最小重复数为 4，这些基序来自濒危沼泽先锋物种沼泽五福草（Comarum palustre L.）。我们选择了 58 个具有 SSR（短序列重复）片段（至少 10 个重复）的位点进行初步筛选。在其中，我们在载体中连接后，使用 T7 正向引物（用 FAM 荧光染料标记）和特定的未标记反向引物进行 PCR，在毛细管测序仪上筛选了 29 个位点。此过程允许我们使用相同标记引物和未标记特异性引物筛选大量候选位点。最后，我们对 20 个多态性微卫星标记进行了特征分析，这些标记包括 9 个二核苷酸和 11 个三核苷酸。我们使用这些标记来评估两个比利时种群的遗传多样性和克隆结构。所有位点在每个个体中最多显示两个等位基因，这表明它们来自二倍体基因组。在一个新扩展的种群中检测到一个单株，而在一个长期进行生态管理的种群中检测到 53 个不同的单株。在这个古老的种群中，每个位点的等位基因数从 6 到 14 不等，预期杂合度从 0.5964 到 0.8278。这些初步结果显示，一个单株的大小高达 7.2 米。
