Molecular Ecology (2003) 12, 1339–1348 doi: 10.1046/j.1365-294X.2003.01825.x © 2003 Blackwell Publishing Ltd Blackwell Publishing Ltd. COMMENTARY How much effort is required to isolate nuclear microsatellites from plants? J. SQUIRRELL,* P. M. HOLLINGSWORTH,* M. WOODHEAD,† J. RUSSELL,† A. J. LOWE ‡, M. GIBBY * and W. POWELL † *Royal Botanic Garden Edinburgh, 20 A Inverleith Row, Edinburgh, EH3 5LR, UK, †Scottish Crop Research Institute, Invergowrie, Dundee, Scotland, DD2 5DA, UK, ‡Centre for Ecology and Hydrology, Bush Estate, Penicuik, Midlothian, EH26 0QB, UK Abstract The attributes of codominance, reproducibility and high resolution have all contributed towards the current popularity of nuclear microsatellites as genetic markers in molecular ecological studies. One of their major drawbacks, however, is the development phase required to obtain working primers for a given study species. To facilitate project planning, we have reviewed the literature to quantify the workload involved in isolating nuclear microsatellites from plants. We highlight the attrition of loci at each stage in the process, and the average effort required to obtain 10 working microsatellite primer pairs. Keywords: enriched library, genotyping, microsatellites, molecular markers, SSRs Received 18 November 2002; revision received 23 January 2003; accepted 24 January 2003 Introduction Molecular ecologists embarking on a new project fre- quently face a dichotomous decision. Should one invest time and effort into the isolation of single locus codom- inant microsatellite markers, or instead utilize one of the off-the-shelf multilocus fingerprinting approaches, such as amplified fragment length polymorphisms (AFLPs), inter- simple sequence repeats (ISSRs) or random amplified polymorphic DNA markers (RAPDs)? The benefits of hypervariable codominant markers are well documented (Powell et al . 1996), but in many cases the perceived difficulties of microsatellite isolation act as a deterrent for the utilization of this class of markers. A review by Zane et al . (2002) describes some of the technical advances that have been made in recent years to facilitate microsatellite development. They cover a range of methods for obtaining sequences rich in microsatellite repeats (some of which can be undertaken in a matter of days), and also highlight the availability of companies who will undertake the construction of enriched microsatellite libraries as a commercial service ( c . 5000 –10 000 US dollars per library). Their review gives a useful overview of the workload, investment and methodologies for microsatellite library construction. The review is likely to persuade some researchers that microsatellite isolation is not too time-consuming or expensive to be undertaken in the course of a standard fixed-term project. However, the development of a library for microsatellite isolation is only the first stage in the process of developing a set of working microsatellite primers. The task of developing a working primer set from an enriched library can in itself represent a significant workload (Zane et al . 2002). In an attempt to provide an objective and comprehensive answer to the question ‘how much effort does it take to produce a set of working microsatellite loci?’, we have reviewed the literature to complement the Zane et al . (2002) review. Our goal has been to provide an empirical assessment of the workload once a library has been constructed. Our commentary deals exclusively with plants and is intended to capture the typical situation facing a researcher working on a previously unstudied organism, rather than focusing on model organisms. The information is intended to provide a realistic guide to the work involved in isolating plant microsatellites. The review was triggered by numerous enquiries into the workload involved, coupled with conflicting displays at conferences of bravado (‘it can be done in my laboratory in a week or two’) vs. doom and gloom (‘I spent my entire PhD isolating microsatellites, and not addressing biological questions’). Correspondence: Jane Squirrell. Fax: + 44 (0)131 2482901; E-mail: J.Squirrell@rbge.org.uk