Quantification of diatom and dinoflagellate biomasses in coastal marine seawater samples by real-time PCR

Abstract

Two real-time PCR assays targeting the small-subunit (SSU) ribosomal DNA (rDNA) were designed to assess the proportional biomass of diatoms and dinoflagellates in marine coastal water. The reverse primer for the diatom assay was designed to be class specific, and the dinoflagellate-specific reverse primer was obtained from the literature. For both targets, we used universal eukaryotic SSU rDNA forward primers. Specificity was confirmed by using a BLAST search and by amplification of cultures of various phytoplankton taxa. Reaction conditions were optimized for each primer set with linearized plasmids from cloned SSU rDNA fragments. The number of SSU rDNA copies per cell was estimated for six species of diatoms and nine species of dinoflagellates; these were significantly correlated to the biovolumes of the cells. Nineteen field samples were collected along the Swedish west coast and subjected to the two real-time PCR assays. The linear regression of the proportion of SSU rDNA copies of dinoflagellate and diatom origin versus the proportion of dinoflagellate and diatom biovolumes or biomass per liter was significant. For diatoms, linear regression of the number of SSU rDNA copies versus biovolume or biomass per liter was significant, but no such significant correlation was detected in the field samples for dinoflagellates. The method described will be useful for estimating the proportion of dinoflagellate versus diatom biovolume or biomass and the absolute diatom biovolume or biomass in various aquatic disciplines.

FIG. 1.

Correlation between SSU rDNA copy number estimated by real-time PCR and cell biovolume of cultured algal strains. Symbols: ▪, dinoflagellates; ▴, diatoms.

FIG. 2.

(A) Cell abundances (10³ liter⁻¹) of each target class, diatoms and dinoflagellates, in field samples collected during May and June 2007. (B) Biovolumes (cubic millimeters per liter). (C) rDNA copies (10⁹ liter⁻¹). Error bars represent standard deviations.

FIG. 3.

Correlation between the ratio of dinoflagellate and diatom SSU rDNA copies and biovolumes per liter of seawater in field samples collected along the Swedish west coast. The linear regression was significant (P < 0.01). y is the ratio of dinoflagellate to diatom biovolumes, and x is the ratio of dinoflagellate to diatom SSU rDNA copies obtained by the two real-time PCR assays.

FIG. 4.

Correlation between the number of diatom SSU rDNA copies and diatom biovolume per liter of seawater in field samples collected along the Swedish west coast. The linear regression was significant (P < 0.001). y is the log of the diatom biovolumes (cubic micrometers), and x is the log of the number of diatom SSU rDNA copies obtained by the real-time PCR assay.

FIG. 5.

Correlation between SSU rDNA copy number estimated by real-time PCR and cell length of cultured algal strains from this study and from that of Zhu et al. (50). Species analyzed in this study are in bold type.