( 2008), to substantially increase efficiency while reducing costs during primer development by combining custom-labeling of primers by the investigator during the PCR reaction while also multiplexing multiple primer sets together for each sample. Here we expand upon a method, first mentioned in Culley et al. Costs further increase both in terms of necessary consumables and labor if PCR and fragment analysis are conducted separately for each individual primer and for every single sample.
One of the most costly steps in this process is the fluorescent labeling of individual microsatellite primers, with costs varying depending upon the dye used (e.g., 6-FAM, VIC, NED) and the manufacturer. Regardless of the initial approach, putative primers must eventually be designed and tested ( Zane et al., 2002). There are several techniques for developing these markers, including the traditional approach using bacterial colonies containing microsatellite inserts or newer methods involving next-generation sequencing. Within the past decade, microsatellites have often been the marker of choice for many of these types of studies however, in most cases, development of these markers is typically time-consuming and expensive (e.g., Squirrell et al., 2003). Molecular markers developed for plant and animal species have been instrumental in answering many biological questions about the natural world, including, for example, those focusing on mating systems, effective population sizes, speciation, and taxonomy.
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