Molecular Ecology Group, Institute of Ecology, University of Innsbruck, 6020 Innsbruck, Austria.
Mol Genet Genomics. 2011 Oct;286(3-4):225-35. doi: 10.1007/s00438-011-0641-0. Epub 2011 Aug 17.
This study describes a new method for identifying microsatellite loci that will reliably amplify and show high degree of polymorphism in a given species. Microsatellites are the most powerful codominant markers available today, but the development of novel loci remains a labour-intensive and expensive process. In de novo isolation, approaches using next generation sequencing (NGS) are gradually replacing ones using Escherichia coli libraries, resulting in unparalleled numbers of candidate loci available. We present a systematic review of published microsatellite primer notes and show that, on average, about half of all candidate loci are lost due to insufficient PCR amplification, monomorphism or multicopy status in the genome, no matter what isolation strategy is used. Thus, the screening of candidate loci remains a major step in marker development. Re-assessing capillary-electrophoresis genotyped loci via high-resolution melting analysis (HRM), we evaluate the usefulness of HRM for this step. We demonstrate its applicability in a genotyping case study and introduce a fast, HRM-based workflow for the screening of microsatellite loci. This workflow may readily be applied to NGS-based marker development and has the potential to cut the costs of traditional testing by half to three quarters.
本研究描述了一种新的方法，用于鉴定微卫星基因座，该方法在给定物种中可靠扩增并显示高度多态性。微卫星是目前最强大的共显性标记，但新基因座的开发仍然是一个劳动密集型且昂贵的过程。在从头开始的分离中，使用下一代测序（NGS）的方法逐渐取代了使用大肠杆菌文库的方法，导致可用的候选基因座数量空前。我们对已发表的微卫星引物注释进行了系统回顾，结果表明，无论使用何种分离策略，平均约有一半的候选基因座由于 PCR 扩增不足、基因组中的单态性或多拷贝状态以及没有多态性而丢失。因此，候选基因座的筛选仍然是标记开发的主要步骤。我们通过高分辨率熔解分析（HRM）重新评估毛细管电泳基因分型基因座，以评估 HRM 在该步骤中的有用性。我们在基因分型案例研究中证明了其适用性，并引入了一种基于 HRM 的快速微卫星基因座筛选工作流程。该工作流程可以很容易地应用于基于 NGS 的标记开发，并有可能将传统测试的成本降低一半至四分之三。
