Design and testing of ITS probes for distinguishing Phaeocystis species

Oligonucleotide probes provide rapid, easy identification of species difficult to identify by more traditional means, e.g. light microscopy. Phaeocystis is a difficult genus to identify as a unicell and thus presents a good candidate for probe development. Hybridisation using PCR products or cell lysates rather than whole cells as targets provides access to more highly variable regions of the genome, such as non-coding spacer regions of the ribosomal operon for detection of fine scale molecular variation not available in the coding regions. The ITS region is an excellent region of the genome to discriminate between Phaeocystis species. PCR amplified ITS-1 sequence from one Phaeocystis antarctica strain was labelled with digoxigenin and hybridised to total nucleic acids from 35 Phaeocystis strains, the prymnesiophyte Emiliania huxleyi, the diatom Cylindrotheca closterium, the rhodophyte Phycodrys austrogeorgia, the phaeophyte Desmarestia aculeata and the chlorophyte Acrosiphonia arcta. Strong signals were observed in all cold water species, i.e. Phaeocystis antarctica and Phaeocystis pouchetii, whereas other warm-water Phaeocystis spp. were not labelled or only weakly labelled with the ITS-1 probe. No hybridisations were observed in all other genera. A short oligonucleotide probe for all cold-water Phaeocystis spp. and for Phaeocystis pouchetii was designed from the ITS-1. Both probes were labelled with digoxigenin and tested in a dot blot analysis against PCR products from 23 Phaeocystis species. Only the two cold water species or Phaeocystis pouchetii were, respectively, labelled by their specific probe.