Molecular Ecology and Evolution Programme, Department of Genetics, University of Pretoria, Pretoria 0002, South Africa.
Mol Ecol Resour. 2012 Jul;12(4):740-52. doi: 10.1111/j.1755-0998.2012.03138.x. Epub 2012 Apr 18.
Recently, 454 sequencing has emerged as a popular method for isolating microsatellites owing to cost-effectiveness and time saving. In this study, repeat-enriched libraries from two southern African endemic sparids (Pachymetopon blochii and Lithognathus lithognathus) were 454 GS-FLX sequenced. From these, 7370 sequences containing repeats (SCRs) were identified. A brief survey of 23 studies showed a significant difference between the number of SCRs when enrichment was performed first before 454 sequencing. We designed primers for 302 unique fragments containing more than five repeat units and suitable flanking regions. A fraction (<11%) of these loci were characterized with 18 polymorphic microsatellite loci (nine in each of the focal species) being described. Sanger sequencing of alleles confirmed that size variation was because of differences in the number of tandem repeats. However, a case of homoplasy and sequencing errors in the 454 sequencing were identified. These newly developed and four previously isolated loci were successfully used to identify polymorphic markers in nine other economically important species, representative of sparid diversity. The combination of newly developed markers with data from previous sparid cross-species studies showed a significant negative correlation between genetic divergence to focal species and microsatellite transferability. The high level of transferability we described (48% amplification success and 32% polymorphism) suggests that the 302 microsatellite loci identified represent an excellent resource for future studies on sparids. Microsatellite marker development should commonly include tests of transferability to reduce costs and increase feasibility of population genetics studies in nonmodel organisms.
最近，454 测序技术因其成本效益和节省时间而成为分离微卫星的流行方法。在这项研究中，对来自两个南部非洲特有 Sparidae 鱼类（Pachymetopon blochii 和 Lithognathus lithognathus）的重复富集文库进行了 454 GS-FLX 测序。从中鉴定出 7370 个含有重复序列（SCR）的序列。对 23 项研究的简要调查显示，在进行 454 测序之前先进行富集时，SCR 的数量存在显著差异。我们为包含超过五个重复单元和合适侧翼区域的 302 个独特片段设计了引物。这些位点中只有一小部分（<11%）具有 18 个多态性微卫星位点（每个焦点物种 9 个）被描述。等位基因的 Sanger 测序证实，大小变异是由于串联重复数的差异所致。然而，在 454 测序中发现了同形现象和测序错误的情况。这些新开发的和四个以前分离的位点成功地用于鉴定 9 个其他经济上重要的 Sparid 物种中的多态性标记，代表了 Sparid 的多样性。新开发的标记与以前 Sparid 跨物种研究的数据相结合，显示出遗传分化与微卫星转移之间存在显著的负相关。我们描述的高转移能力（48%的扩增成功率和 32%的多态性）表明，鉴定出的 302 个微卫星位点代表了 Sparids 未来研究的极好资源。微卫星标记的开发通常应包括转移能力的测试，以降低成本并增加非模型生物种群遗传学研究的可行性。
