Testing A Selected Region of Tuber Mitochondrial Small Subunit rDNA as a Molecular Marker for Evolutionary and Bio-Diversity Studies
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.
KeywordsSmall Subunit rRNA Gene Universal Region Partial Sequence Analysis Truffle Species White Truffle
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