Species-specific probe, based on 18S rDNA sequence, could be used for identification of the mucilage producer microalga Gonyaulax fragilis (Dinophyta)

Abstract

In the Adriatic Sea, the correlation between mucilage phenomena and the presence of Gonyaulax fragilis (Schutt) Kofoid (Dinophyta) has been recently demonstrated. The application of PCR-based methods and the development of species-specific molecular probes might represent powerful technologies for rapid and specific monitoring of microalgal species in seawater samples. Here, we report sequencing of the small subunit (SSU) ribosomal RNA gene (18S rDNA) of G. fragilis and its comparative analysis within the Dinophyta. Total DNAs were extracted and amplified from cultured cells of G. fragilis, which were isolated from natural phytoplanktonic association in the northern Adriatic Sea. Total 18S rDNA gene was amplified using 16S1N and 16S2N primers and sequenced using ad hoc designed internal primers. The primers amplified a product of expected size (length 1700/1800 bp). The phylogenetic analysis carried out by comparing G. fragilis sequence to homologous sequences of Lingulodinium polyedrum (Stein) Dodge, Gonyaulax spinifera (Claparede et Lachmann) Diesing, Protoceratium reticulatum (Claparede et Lachmann) Butschli revealed a great nucleotide divergence of G. fragilis SSU sequence. Therefore, the SSU sequence could be used as species-specific marker for the identification of this mucilage producer microalga. In addition, such sequence could be used as target to design oligonucleotide probes for the construction of DNA microchips as diagnostic tool for the routine monitoring of harmful algae in seawater.