Molecular typing of the red-tide dinoflagellate Gonyaulax polyedra in phytoplankton suspensions

Rollo, Franco; Sassaroli, Stefano; Boni, Laurita; Marota, Isolina

doi:10.3354/ame009055

Cited by 12 publications

(5 citation statements)

References 10 publications

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“…The distinctive morphology and relative ease of culturing L. polyedrum have led to numerous studies, and the species has become one of the best described dinoflagellates in terms of its cell chemistry, life cycle, physiology, behavior, ecology, and fossil record (reviewed in Lewis and Hallett 1997). Contrastingly, only one study by Rollo et al. (1995) described a genetic marker for the molecular typing of L. polyedrum, and, to date, no detailed population genetic study has been conducted on this species.…”

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confidence: 99%

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES¹

Frommlet

Iglesias‐Rodríguez

2008

Journal of Phycology

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In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high-resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two-step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1-5.8S-ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species.

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confidence: 99%

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES¹

Frommlet

Iglesias‐Rodríguez

2008

Journal of Phycology

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“…Because of their rapidity, PCR-based methods are used more and more. The PCR amplification technique of targeting ribosomal DNA (rDNA) regions has been successfully employed for the detection of various toxic dinoflagellates in seawater samples (8,11,19,44,48). The rDNA genes have been the regions of choice for the development of molecular assays because these sequences are repeated in tandem in high copy numbers and are highly conserved (24,36,37,50,55).…”

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confidence: 99%

Development of a Real-Time PCR Assay for Rapid Detection and Quantification ofAlexandrium minutum(a Dinoflagellate)

Galluzzi

Penna

Bertozzini

et al. 2004

Appl Environ Microbiol

260

214

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The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.

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“…4). Assuming that K. mikimotoi has a minimum cell DNA quota similar as the one reported for K. brevis (~50 pg/cell) and the number of rRNA gene copies per dinoflagellate cell is ~230 copies (Kim and Martin 1974; Walker 1982; Rollo et al 1995; Kamykowski et al 1998; Yoon et al 2005), 10 fg of genomic DNA corresponded to 0.05 copies of rRNA gene. Following the above calculation, 1 pg and 10 pg of genomic DNA correspond to 4.6 and 46 gene copies, respectively.…”

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confidence: 71%

“…In contrast, inferior levels of detection (~5 ng) were documented for both sets of probes when challenged with enzymatic end labeled amplicons. After correcting for PCR product length and rRNA gene copies, the detection limits of 500 pg and 5 ng correspond to 5.5 × 10 6 and 5.5 × 10 7 copies, respectively (Rollo et al 1995). …”

Section: Discussion and Commentsmentioning

confidence: 99%

Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology

Diaz

Jacobson²,

Goodwin

et al. 2010

Limnology & Ocean Methods

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Harmful algal bloom (HABs) events are a threat to public health, marine life, commercial fisheries, tourism, recreation, and the environment. These outbreaks are known to decrease water quality by increasing the biological O 2 demand, which can cause die-offs of marine life (Figley et al. 1979). Expanded spatial distribution of HABs can cover over a thousand kilometers and are triggered by the effects of winds, currents, storms, and ship ballast activities (Tester and Steidinger 1997;Doblin et al. 2004;Bolch and Salas 2007). Increases in HAB frequency and duration are probably fueled by changes in sea surface temperature, salinity, grazing, turbulence, irradiance, or increases in nutrient loads derived from run off from agricultural and urban areas (Reynolds 1989;Sterner 1989;Thomas and Gibson 1990; Anderson et al. 2002;Xiaonan and Wei 2004).HABs are formed by species of phytoplankton or cyanobacteria, which can produce toxins that can be transferred through marine food webs, affecting consumers at higher trophic levels including fish, shellfish, marine mammals, and humans (Sellner et al. 2003;Smayda 1997 AbstractHarmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT ® nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5¢ terminal labeling technique, the chemicallabeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

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Molecular typing of the red-tide dinoflagellate Gonyaulax polyedra in phytoplankton suspensions

Cited by 12 publications

References 10 publications

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES¹

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES¹

Development of a Real-Time PCR Assay for Rapid Detection and Quantification ofAlexandrium minutum(a Dinoflagellate)

Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology

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Molecular typing of the red-tide dinoflagellate Gonyaulax polyedra in phytoplankton suspensions

Cited by 12 publications

References 10 publications

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES1

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES1

Development of a Real-Time PCR Assay for Rapid Detection and Quantification ofAlexandrium minutum(a Dinoflagellate)

Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology

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Product

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MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES¹

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES¹