Testing A Selected Region of Tuber Mitochondrial Small Subunit rDNA as a Molecular Marker for Evolutionary and Bio-Diversity Studies

Summary

To set the basis for a molecular-evolutionary characterization of ascomycete ectomycorrhizal fungi belonging to the genus Tuber, we have worked out PCR conditions for the amplification of a selected region of the mitochondrial small subunit rDNA(mt-SrDNA). A couple of oligonucleotides (MS1, MS2) that had previously been reported to amplify a 700 bp mt-SrDNA fragment from a variety of fungi were initially utilized as PCR primers. Based on the partial sequence analysis of a Tuber magnatum derived PCR fragment and on comparison with all available ascomycete sequences, two nested PCR primers (MAS1-2) have been designed. DNA fragments (380 bp) corresponding in size to the homologous mt-SrDNA region of other ascomycetes were produced by amplification reactions programmed with template DNA derived from either T. magnatum or T. albidum, in the presence of primers MAS1-2. A preliminary sequence analysis has shown that both truffle-derived fragments have high similarity (75% to 79%) with Neurospora mt-SrDNA and exhibit a characteristic distribution of conserved (U) and highly divergent (V) regions. Sequence similarity values ranged from 96% in the case of the universal region U5 to 48% for the variable region V8. This indicates that, despite its rather narrow size, the mt-DNA region we have selected can be informative for both intermediate (up to the class level) as well as for very close (intraspecifíc) taxonomic comparisons.