High Resolution Melting: un metodo innovativo per l’identificazione molecolare di diatomee potenzialmente tossiche appartenenti al genere Pseudo-nitzschia

The planktonic pennate diatom Pseudo-nitzschia (Bacillariophyceae) is a cosmopolitan genus commonly found in neritic and oceanic waters. The correct identification of Pseudo-nitzschia species has relevant implications for monitoring and management purposes, considering that toxigenic species may be morphologically similar or identical to non-toxigenic ones. Recent studies integrating molecular phylogenetic, morphological, ultrastructural and biological information have uncovered numerous cases of genetically distinct and at times reproductively isolated groups of strains that could not be distinguished easily or at all with microscopy. Molecular approaches constitute the most straightforward way to identify various taxa and elucidate their distribution over space and time. The high resolution melting (HRM) analysis can be an alternative technique to rapidly and accurately identity microalgal species and/or genotype cultured strains. Different melting profiles are obtained from the transition of double-strand (dsDNA) to single-strand DNA (ssDNA) as a result of a gradual temperature increase after PCR amplification. HRM has been used mainly for screening mutations linked to human diseases and for genotyping bacteria. To date, only one study applied HRM for microalgal species identification. The HRM analysis is faster, and less expensive than alternative approaches, such as microarray, or microsatellites. In the present study, we investigated the genetic diversity of several Pseudo-nitzschia species in monoclonal cultures obtained from environmental samples collected over one year in the Adriatic Sea using HRM based assay for species – specific identification. The HRM assay carried out on 21 strains of Pseudo-nitzschia spp. that were already identified, distinguished 3 different variants corresponding to the three different species of Pseudonitzschia, namely P. pungens, P. delicatissima and P. calliantha, as expected. The high resolution melting analysis is a powerful technique able to accurately identify Pseudo-nitzschia species that are difficult to be recognized by standard microscopy in environmental samples.