Abstract
BackgroundHigh-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms.Methodology/Principal FindingsThis study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples.Conclusions/SignificanceThese results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography.
Highlights
The development of innovative methods for detecting genetic variation has progressively enhanced the study of evolutionary relationships and processes [1]
At least 1 mg of total genomic DNA was extracted from silica gel desiccated (Asplenium) or air dried (Garovaglia) leaf material using a modification of the standard CTAB procedure [15] as specified in Trewick et al [16] except that extractions were incubated in 500 mL CTAB buffer, 50 ml sarkosyl and 10 ml proteinase-K and purified by phenol-chloroform extraction
The frequencies of polymorphic markers detected on the Asplenium and Garovaglia discovery arrays were 6% and 15% respectively, resulting in 444 and 905 polymorphic markers recovered respectively (Table 5)
Summary
The development of innovative methods for detecting genetic variation has progressively enhanced the study of evolutionary relationships and processes [1]. High-throughput genomic tools such as the DNA microarray platform have sparked a remarkable increase in data production, leading to new evolutionary insights [2]. Their application is nearly exclusively restricted to model and near-model organisms [2,3]. The detection of genetic variation in non-model plants and animal—the majority of life on earth—is largely restricted to direct sequencing of previously-identified variable loci or arbitrarily amplified dominant (AAD) markers, especially RAPDs, ISSRs and AFLPs [4]. High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, is predominantly restricted to model or nearmodel organisms
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