Development of a PCR strategy for thraustochytrid identification based on 18S rDNA sequence

The identification and characterization of the thraustochytrids, an emerging economically and biotechnologically important group of marine heterotrophic protists, is usually based on morphological characters. In this research we used molecular markers to identify thraustochytrids. We designed three sets of primers based on the 18S rDNA sequence alignment of known strains and employed a PCR (polymerase chain reaction) strategy to identify unknown thraustochytrid isolates. DNA from 26 thraustochytrids (three isolated from primary cell cultures of the tunicate Botryllus schlosseri and 23 from a coral holding aquarium) were amplified by these primers, revealing 21 isolates with three bands each, which were assigned to two groups according to PCR fragment sizes. Taxonomic characterizations were deduced by comparing with GenBank data. Four isolates were further studied by sequencing their 18S rDNA. Sequence alignments and phylogenetic analysis revealed that isolates from the coral aquarium (7-5 and 8-7) were highly similar to each other and 95.0-97.0% similar to Thraustochytrium multirudimentale and Schizochytrium minutum. Isolates from the tunicate primary cell Cultures (BS1 and BS2) were also closely related to each other and 84.3-86.0% similar to labyrinthulid quahog parasite and Thraustochyrium pachydermum. AFLP (amplified fragment-length polymorphism) analysis revealed 2.5-3.6% differences within the genomic DNA of each group, showing that each isolate is different, although isolates within each group may belong to the same species, in spite of differences found in the general morphology.