A Comparison of Microsatellite Isolation Techniques Using Avian Genomes

Abstract

<p> In the past two decades or so. microsatellites have become a very widely used genetic tool in many disciplines of biology. Their major downfalL however. is that they often need to be isolated de novo before they can be applied to molecular studies. Traditional shotgun cloning can be successfuL but it is often overly costly and time consuming. Compounding this downfall, isolating microsatellites from some taxa has been shown to be difficult. For example. on average only 0.46% of all clones screened using avian genomes will yield positive clones. This is thought to be a result of a smaller avian genome, a requirement for flight. Several alternative methods have been developed for isolating microsatellites, but the choice as to which isolation method to use is often arbitrary. To address this. four species of birds. the smooth-billed ani (Crotophaga ani). herring gull (Larus argentatus), yellow-bellied elaenia (Elaenia flavogaster), and pukeko (Porphyria porphyria), representing four different orders were used to compare two alternative isolation methods. Enrichment via selective hybridization versus cloning with Lambda Zap phage vector were compared in terms of monetary requirements (total startup cost as well as per isolation attempt cost). and time requirements (total time from start to finish and hands-on experimentation time). No significant difference was detected in terms of number of polymorphic microsatellite loci isolated by each method (p = 0.57), with enrichment yielding more for the anis and elaenias, Lambda Zap yielding more for herring gulls. and both methods isolating equal numbers for pukekos. Nor was any difference found between the methods for dollars spent per sequence with repeat (SWR) using the startup cost (p = 0.30). Enrichment. however. proved to be significantly more effective in terms of dollars per SWR isolated using the per use cost (p = 0.004) as well as hands-on minutes per SWR (p = 0.01) and total minutes per SWR (p < 0.01 ). Based on these tindings. selective hybridization is the better choice for microsatellite isolation. </p>