Oligonucleotide primers for the universal amplification of β-tubulin genes facilitate phylogenetic analyses in the regnum Fungi

Among genes coding for proteins with basic structural functions in all eukaryotes, the highly conserved and functionally essential gene for beta-tubulin is receiving increasing attention in the reconstruction of phylogenies within a broad organismic range. We therefore constructed a set of twelve universally applicable primers that allow reliable amplification of beta-tubulin genes among all major eukaryotic kingdoms including fungi (Fungi), animals (Animalia) and green plants (Planta). For primer design, the amino acid sequences of 35 beta-tubulin genes from Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Animalia, Oophyta and Planta were aligned and used for the definition of four well-conserved regions. These are suitable priming sites in PCR amplification experiments. Out of these amino acid regions twelve primers were designed which initiate especially the amplification of fungal beta-tubulin genes. In four pairwise primer applications gene fragments of up to 1500 bp in size could be isolated, which comprise nearly complete beta-tubulin genes from twelve species representative of the Fungi. The sequences of seven beta-tubulin fragments were obtained from Allomyces moniliformis, A. neomoniliformis, Blastocladiella britannica, Chytridium confervae, Mortierella isabellina and Trametes versicolor, respectively. Reliable amplification of beta-tubulin over a broad spectrum of organisms provides a strong basis for the establishment of both deep-level phylogenies and studies of complex species groups based on beta-tubulin gene trees.