Abstract
Single cell Chelex® DNA extraction and nested PCR amplification were used to examine partial gene sequences from natural diatom populations for taxonomic and phylogenetic studies at and above the level of species. DNA was extracted from cells that were either fresh collected or stored in RNAlater. Extractions from Lugol's fixation were also attempted with limited success. Three partial gene sequences (rbcL, 18S, and psbA) were recovered using existing and new primers with a nested or double nested PCR approach with amplification and success rates between 70 and 96%. An rbcL consensus tree grouped morphologically similar specimens and was consistent across the two primary sample treatments: fresh and RNAlater. This tool will greatly enhance the number of microscopic diatom taxa (and potentially other microbes) available for barcoding and phylogenetic studies. The near-term increase in sequence data for diatoms generated via routine single cell extractions and PCR will act as a multiproxy validation of longer-term next generation genomics.
Highlights
DNA barcoding has become common practice in animal and plant taxonomy (Hebert et al, 2003) with cytochrome c oxidase 1 (CO1), a mitochondrial gene, serving as the main animal barcoding gene (Hebert et al, 2004)
An additional 37 partial rbcL sequences were determined for a variety of diatom genera including Melosira C.Ag., Aulacoseira Thwaites, Synedra (Ulnaria) Ehrenberg, Eunotia Ehrenberg, Navicula Bory, Neidium Pfitzer, Placoneis C.Mereschkowsky, Frustulia C.Ag., Gyrosigma Hassall, Stauroneis Ehrenberg, Craticula Grunow, Sellaphora C.Mereschkowsky, Pinnularia Ehrenberg, Cymatopleura W.Sm., Encyonema Kütz., Gomphonema Ehrenberg, Nitzschia Hassall, Hantzschia Grunow, and Surirella Turpin
The development of novel DNA extraction protocols has accelerated the exploitation of microbial genetic studies in health (e.g., Richlen and Barber, 2005), environment (e.g., Neilan, 1995; Kermarrec et al, 2013), and even diatom taxonomic research (e.g., Evans et al, 2007; Poulicková et al, 2010)
Summary
DNA barcoding has become common practice in animal and plant taxonomy (Hebert et al, 2003) with cytochrome c oxidase 1 (CO1), a mitochondrial gene, serving as the main animal barcoding gene (Hebert et al, 2004). In plants the chloroplast genes ribulose 1,5-biphosphate (rbcL) and megakaryocyte-associated tyrosine kinase (MatK) serve as two of the preferred barcode genes for taxonomic identifications (CBOL Plant Work Group, 2009). This is in contrast with the situation faced in diatom barcoding where several regions are presently identified as prominent taxonomic markers (e.g., Yoon et al, 2002; Evans et al, 2007). Ribosomal complex genes ITS, 18S, and 28S were used both individually and in multi-gene studies to evaluate cryptic taxonomic variations at the genus and species levels (e.g., Amato et al, 2007; Vanormelingen et al, 2008; Poulicková et al, 2010; Kaczmarska et al, 2014). With little consensus as to which marker best delimits diatom groups, the ability to amplify several genes including new genes from a single cell is essential for diatom taxonomy using DNA barcodes
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