Identification of the dinoflagellate community during Cochlodinium polykrikoides (Dinophyceae) blooms using amplified rDNA melting curve analysis and real-time PCR probes

Abstract

The dinoflagellate community present during blooms of the fish killing dinoflagellate Cochlodinium polykrikoides was characterized by DNA melting curve analysis and direct sequencing of the SSU rDNA amplified from environmental sample extracts. PCR amplification of genomic DNA from Gaedo water samples using dinoflagellate-specific SSU rDNA primers yielded 280 clones, which were screened by closed tube PCR-melting curve analysis targeting a region of the SSU rDNA, enabling high throughput analysis. Twenty-eight clones producing distinct melting curve patterns were sequenced, and their phylogenetic information revealed that C. polykrikoides co-occurred with morphologically similar species including Gymnodinium impudicum and Gymnodinium catenatum. Temporal variations of C. polykrikoides and G. impudicum abundances in South Sea were also examined by species-specific real-time TaqMan-based PCR probes developed in this study. C. polykrikoides- and G. impudicum-specific real-time PCR probes were designed targeting the internal transcribed spacer 2 ribosomal DNA region. The probe specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR products from environmental samples. The real-time PCR assays showed that C. polykrikoides cell densities peaked in August at 16,928cellsmL−1, while G. impudicum was present at low abundances (below 25cellsmL−1). Our amplified rDNA melting curve protocol provides a facile method for the characterization of the dinoflagellate community, and the real-time PCR assay could be an alternative method for rapid and sensitive enumeration of harmful dinoflagellates in the marine environment.