Oligonucleotide Probes for the Identification of Three Algal Groups by Dot Blot and Fluorescent Whole‐Cell Hybridization

Abstract

ABSTRACT. Photosynthetic pico-and nanoplankton dominate phytoplankton biomass and primary production in the oligotrophic open ocean. Species composition, community structure, and dynamics of the eukaryotic components of these size classes are poorly known primarily because of the difficulties associated with their preservation and identification. Molecular techniques utilizing 18S rRNA sequences offer a number of new and rapid means of identifying the picoplankton. From the available 18S rRNA sequence data for the algae, we designed new group-specific oligonucleotide probes for the division Chlorophyta, the division Haptophyta, and the class Pelagophyceae (division Heterokonta). Dot blot hybridization with polymerase chain reaction amplified target rDNA and whole-cell hybridization assays with fluorescence microscopy and flow cytometry were used to demonstrate probe specificity. Hybridization results with representatives from seven algal classes supported the phylogenetic affinities of the cells. Such group-or taxon-specific probes will be useful in examining community structure, for identifying new algal isolates, and for in situ detection of these three groups, which are thought to be the dominant algal taxa in the oligotrophic regions of the ocean.

An evaluation of the applicability of microarrays for monitoring toxic algae in Irish coastal waters

“…The only higher group probe for Prymnesiophyta (PrymS01_25_dT and PrymS02_25_dT; Simon et al, 2000;Table III-S3) to return a significant linear regression was PrymS01_25_dT normalised to POSITIVE_25_dT (R 2 = 0.98; P = 0.0400). However, both these higher group probes signal intensities were positively correlated except for PrymS02_25_dT normalised to DunGS02_25_dT_dT, which produced a negative linear regression (Table III- Table III-S3) for Prymnesiophyceae normalised signal intensity with both control probes had a positive linear relationship R 2 ≥ 0.84; but, only the normalised POSITIVE_25_dT curve was significant (P = 0.0276).…”

“…An increasing number of molecular probes targeting the 18S or 28S rRNA gene sequence are now available for many species of toxic algae (Groben et al 2004;Simon et al 2000). However, the adaption of existing FISH probes to the microarray format was not as straight forward because the sequence length, secondary structures and proteins can block accessibility of labelled targets to the corresponding binding sites of the 18S rRNA gene molecule ( Fig.…”

“…Cross reactivity testing was not tested on pure culture alone as it was not necessary due to previous studies using the same Prymnesium probes for FISH analysis and their specificity has already been verified by dot-blot hybridisation with PCR amplified with either 18S or 28S rDNA fragments of different target and non-target microalgae Simon et al 2000.…”

“…These probes can be applied to whole-cell or cell-homogenate based methods of detection. The whole-cell approach maintains the target species remains intact throughout the assay procedure; an example is Fluorescent in-situ Hybridisation (FISH) analysis that is used to quantify and identify target taxa in environmental samples Simon et al , 2000Touzet et al 2007;2010). Cell-homogenate probe techniques on the other hand require the lysis of cells.…”

Feeding selectivity of bivalve larvae on natural plankton assemblages in the Western English Channel

“…We therefore used primers to target the haptophyta, a group of nanoeukaryotes including the orders coccolithales, isochrysidales, phaeocystales and prymnesiales. Partial 18S rDNA solely found in haptophytes was selectively amplified using the forward primer Prym-429f: 5′-GCG CGT AAA TTG CCC GAA-3′ (Coolen et al 2004) and the reverse primer PRYM02: 5′-GGA ATA CGA GTG CCC CTG AC-3′ (Simon et al 2000). Two microlitres of extracted genomic DNA from experiments 1-7, in situ field animals and starved bivalves were used for PCR amplification.…”