A Survey of Polymerase Chain Reaction (PCR) Amplification Studies of Unicellular Protists Using Single‐Cell PCR

Pinheiro, Marcel D.O.

doi:10.1111/j.1550-7408.2009.00439.x

Cited by 21 publications

(17 citation statements)

References 51 publications

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“…). Such high genome copy numbers in ciliates make DNA extractions un‐needed as they are ideally suited for single‐cell PCR's (Lynn & Pinheiro ) (see Fig. S1, Supporting information).…”

Section: Resultsmentioning

confidence: 99%

TheD1‐D2 region of the large subunit ribosomalDNAas barcode for ciliates

Stoeck

Przyboś

Dunthorn

2013

Molecular Ecology Resources

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Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1-D2 region of the ribosomal DNA large subunit (LSU-rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1-D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1-D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1-D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour-joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1-D2 region as an excellent candidate for an official CBOL barcode for ciliated protists.

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“…). Such high genome copy numbers in ciliates make DNA extractions un‐needed as they are ideally suited for single‐cell PCR's (Lynn & Pinheiro ) (see Fig. S1, Supporting information).…”

Section: Resultsmentioning

confidence: 99%

TheD1‐D2 region of the large subunit ribosomalDNAas barcode for ciliates

Stoeck

Przyboś

Dunthorn

2013

Molecular Ecology Resources

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“…Formalin-preserved specimens are not, however, suitable candidates for PCR amplification of material for molecular genetics (Lynn and Pinheiro, 2009). A combination of Lugol's solution for fixation and alcohol for long-term preservation may suit the needs of both the microscopist (morphology) and the geneticist (PCR).…”

Section: B Preparationmentioning

confidence: 99%

Chrysophyceae and Phaeothamniophyceae

Nicholls¹,

Wujek

2015

Freshwater Algae of North America

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“…Linking morphological and molecular analysis of protists in environmental samples is a methodological challenge. Single‐cell PCR has been successfully demonstrated for a range of organisms (Lynn and Pinheiro 2009 and references therein). Even preserved plankton samples, with a variety of preservatives, were used for single‐cell PCR (Marin et al.…”

mentioning

confidence: 99%

“…2005). Furthermore, most of the studies were based on live material (Lynn and Pinheiro 2009). The method introduced by Auinger et al.…”

mentioning

confidence: 99%

Cultivation-Independent Species Identification of Dinobryon Species (Chrysophyceae) by Means of Multiplex Single-Cell Pcr1

Jost

Medinger

Boenigk

2010

Journal of Phycology

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With the discovery of a high molecular diversity of protists, a discrepancy between morphological and molecular species richness estimates became apparent. Solving the current concerns requires a comparative analysis of different sequences combined with morphological analyses of single cells originating from preserved field samples. We refined a single‐cell PCR (SC‐PCR) protocol for analyzing cells from field samples preserved with Lugol’s iodine solution. We linked microscopic screening with multiplex PCR targeting the SSU rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and the mitochondrial cytochrome oxidase 1 (CO1) in a single PCR reaction. Using this method, we investigated the intraspecific molecular variation in Dinobryon populations originating from two lakes in the Salzkammergut area of Austria. All investigated genetic markers showed two separated clusters within the investigated populations of Dinobryon divergens O. E. Imhof, indicating a reproductive isolation of the two coexisting populations. Based on these findings, we describe a lineage, which is morphologically similar to D. divergens but, based on the molecular data, is reproductively isolated.

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A Survey of Polymerase Chain Reaction (PCR) Amplification Studies of Unicellular Protists Using Single‐Cell PCR

Cited by 21 publications

References 51 publications

TheD1‐D2 region of the large subunit ribosomalDNAas barcode for ciliates

TheD1‐D2 region of the large subunit ribosomalDNAas barcode for ciliates

Chrysophyceae and Phaeothamniophyceae

Cultivation-Independent Species Identification of Dinobryon Species (Chrysophyceae) by Means of Multiplex Single-Cell Pcr1

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