Summary• Glaciations and postglacial migrations are major factors responsible for the present patterns of genetic variation we see in natural populations in Europe. For ectomycorrhizal fungi, escape from refugia can only follow range expansion by their specific hosts.• To infer phylogeographic relationships within Tuber melanosporum , sequences of internal transcribed spacers (ITS) and the 5.8S coding region of the ribosomal DNA repeat were obtained for 188 individuals sampled over the entire distribution of this species in France, and in north-western Italy and north-eastern Spain.• Ten distinct ITS haplotypes were distinguished, mapped and treated using F -and N ST -statistics and nested clade (NCA) analyses. They showed a significant genetic differentiation between regional populations. NCA revealed a geographical association of ITS haplotypes, an old fragmentation into two major groups of populations, which likely colonized regions on different sides of the French Central Massif.• This re-colonization pattern is reminiscent of the one observed for host trees of the Perigord truffle, such as oaks and hazelnut trees. This suggests that host postglacial expansion was one of the major factors that shaped the mycobiont population structure.

Mycorrhizal fungi of the genus Tuber are classified by morphological characters that allow differentiation of most species. However, some economically important species are difficult to differentiate on morphological grounds. When morphological traits are not sufficient to discriminate between taxa, other markers are needed. Genetic variation of fruit bodies of 12 Tuber taxa was studied by the random amplified polymorphic DNA (RAPD) technique. High interspecific variability was observed between most species. Moreover, important infraspecific variation occurred in all species, except Tuber brumale s.L, Tuber melanosporum, and Tuber magnatum. Southern hybridization patterns of RAPD products of the various species were used to confirm the data. Relationships among Tuber species were determined by cluster analyses. UPGMA analyses revealed several main clusters and a low genetic similarity between taxa. These results indicate that RAPD and polymerase chain reaction are useful for analysing genetic variation within Tuber species. Most species can be identified by differences in their amplified DNA profiles. However, the two pairs of closely related taxa Tuber aestivum – Tuber uncinatum and Tuber brumale var. brumale – Tuber brumale var. moschatum did not appear to differ genotypically. Key words: Tuber, RAPD, Southern, UPGMA, inter- and infra-specific variability.

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Identification of some economically important Tuber species using classical morphological characteristics is sometimes difficult. We report here the molecular characterization of a species coming from China, Tuber indicum, mistaken with Tuber melanosporum species. Using restriction analysis of the amplified internal transcribed spacer (ITS) of rDNA, ITS sequence analysis, and sequence characterized amplified region markers, with DNA from fruit bodies or mycorrhizae, genetic variation was found between these two species, allowing to differentiate and characterize them.

We have developed new DNA extraction and purification procedures for investigation of mycorrhized seedlings and canned truffles. Use of these procedures on approximately 100 mg initial material enabled good sample representation. For mycorrhized seedlings, Taq polymerase inhibitors were discarded irrespective of tree species. In routine analysis we systematically used consensus primers ITS1/ITS4 to check the absence of Taq polymerase inhibitors and the presence of fungus DNA. Positive response with ITS validates other positive or negative PCR results. Absence of amplification with ITS prevents validation of other results. For canned truffles, DNA harvested from ascocarps sterilized for one and a half hours at 115 degrees C was amplified with specific primers. We have developed consensus primers, named R12/F12, to check for the presence of amplifiable fungus DNA and the absence of Taq polymerase inhibitors. Here also, positive response with consensus R12/F12 validates other positive or negative PCR results. We have developed one primer pair specific for T. brumale and another specific for T. melanosporum. We can then characterize these two taxa, which enables the use of "truffle or truffled" French designations. We can also characterize T. indicum, the Asiatic black truffle that might fraudulently be sold as T. melanosporum and T. brumale. These three specific primer pairs were used independently of DNA extraction from tree seedlings or canned truffles. Our process is specific, sensitive, convenient, and quick.