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Truffles are ascomycetous ectomycorrhizal fungi: the accomplishment of their life cycle relies therefore on the establishment of symbiotic relationships with a narrow range of host plants. Three different phases have been identified in their life cycle: i) growth as filamentous vegetative mycelia, ii) organization of hypogeous fruitbodies with asci and ascospores, and iii) association of the fungal hyphae with the host root to form the fungal mantle and the Hartig net. The identification of truffles is mostly based on the morphological features of the fruitbodies (size and shape of asci, number and wall ornamentations of spores within the asci). However, many of the structures important for taxonomic identification are absent during the phase of filamentous growth and during the contact of hyphae with the host surface in the establishment of mycorrhizae.

The aim of this paper is to illustrate the strategies that were followed to develop molecular probes which would allow truffle identification during their different developmental phases.

DNA extracted from fruitbodies of 10 Tuber species, from mycorrhizae and from mycelia growing in vitro of T. magnatum and of T. borchii, was amplified in PCR experiments by using i) ITS and IGS primers designed for ribosomal gene spacers, ii) short arbitrary oligomers in RAPD experiments and iii) specific primers for T. magnatum designed on the sequence of a non-polymorphic RAPD band. Amplifications of DNA extracted from fruitbodies with ITS and IGS primers, followed by digestion with restriction enzymes Hinf I and Mbo I, led to genetic fingerprints which differentiated among the different species in the fruitbodies as well as in the mycorrhizae. RAPD fingerprints revealed a high degree of polymorphism among the different truffle species, but they showed a rather low polymorphism among fruitbodies of the same species, T. magnatum. A RAPD band of 1.5 kbp that was found to be specific for T. magnatum was cloned and partially sequenced. Two specific primers were designed and allowed us to distinguish unambiguously among the truffle fruitbodies, as they amplified DNA sequences only in T. magnatum samples.