Abstract
Microsatellites are the markers of choice for a variety of population genetic studies. The recent advent of next-generation pyrosequencing has drastically accelerated microsatellite locus discovery by providing a greater amount of DNA sequencing reads at lower costs compared to other techniques. However, laboratory testing of PCR primers targeting potential microsatellite markers remains time consuming and costly. Here we show how to reduce this workload by screening microsatellite loci via bioinformatic analyses prior to primer design. Our method emphasizes the importance of sequence quality, and we avoid loci associated with repetitive elements by screening with repetitive sequence databases available for a growing number of taxa. Testing with the Yellowstripe Goatfish Mulloidichthys flavolineatus and the marine planktonic copepod Pleuromamma xiphias we show higher success rate of primers selected by our pipeline in comparison to previous in silico microsatellite detection methodologies. Following the same pipeline, we discover and select microsatellite loci in nine additional species including fishes, sea stars, copepods and octopuses.
Highlights
Microsatellite loci remain one of the most popular choices for population genetic studies
Can we increase the primer-to-marker conversion rate through selection of microsatellite loci via bioinformatics analysis? Focusing on three reef fishes, two sea stars, two copepods and two octopuses as case studies, we explore bioinformatic analyses to reduce the likelihood of the two most common pitfalls researchers encounter in the development of microsatellite markers: failed PCR amplification and unspecific amplification of multiple loci
Genomic DNA was extracted from a fish fin clip (Mulloidichthys vanicolensis and M. flavolineatus), octopus muscle tissue (Octopus cyanea and O. oliveri) or whole copepods (Haloptilus longicornis and Pleuromamma xiphias) by one of two methods: (1) Qiagen DNeasy kits or (2) incubation with proteinase K at 56uC for 1 h followed by two extractions with phenol:chloroform:isoamyl alcohol (25:24:1), one extraction with chloroform:isoamyl alcohol (24:1) and one ethanol precipitation
Summary
Microsatellite loci remain one of the most popular choices for population genetic studies This success may be credited to several attributes including their ability to provide contemporary estimates of migration, distinguish relatively high rates of migration from panmixia, and resolving pedigrees [1,2,3,4,5]. In spite of their potential to address a myriad of issues in molecular ecology, evolution, and conservation, until recently the expertise, time and costs of initially developing microsatellite markers remained deterrents for many. The longer read lengths of the 454 pyrosequencing platform (Roche 454 Life Science, Bradford CT, USA) have made it the preferred approach for microsatellites to date (but see [12]), and the continued rapid advances of NGS technology will make microsatellites even cheaper and easier to develop in the future
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