LSU rDNA-based RFLP assays for discriminating species and strains of Alexandrium (Dinophyceae)

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and / or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these ''signature heterogeneities'' represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.