The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today's truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a 'symbiosis toolbox'. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at approximately 125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for approximately 58% of the genome. In contrast, this genome only contains approximately 7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis-'the symbiosis toolbox'-evolved along different ways in ascomycetes and basidiomycetes.

Summary• In light of the recent finding that Tuber melanosporum, the ectomycorrhizal ascomycete that produces the most highly prized black truffles, is a heterothallic species, we monitored the spatial distribution of strains with opposite mating types (MAT) in a natural truffle ground and followed strain dynamics in artificially inoculated host plants grown under controlled conditions.• In a natural truffle ground, ectomycorrhizas (ECMs), soil samples and fruit bodies were sampled and genotyped to determine mating types. Simple sequence repeat (SSR) markers were also used to fingerprint ECMs and fruit bodies. The ECMs from nursery-inoculated host plants were analysed for mating type at 6 months and 19 months post-inoculation.• In open-field conditions, all ECMs from the same sampling site showed an identical mating type and an identical haploid genotype, based on SSR analysis. Interestingly, the gleba of fruit bodies always demonstrated the same genotype as the surrounding ECMs. Although root tips from nursery-grown plants initially developed ECMs of both mating types, a dominance of ECMs of the same MAT were found after several months.• The present study deepens our understanding of the vegetative and sexual propagation modes of T. melanosporum. These results are highly relevant for truffle cultivation.

Tuber spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of Tuber biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single Tuber magnatum truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that T. magnatum outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the T. magnatum life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other Tuber spp.Tuber spp. are Ascomycetes fungi that establish an ectomycorrhizal symbiosis with trees and shrubs. As a result of this mutualistic symbiosis, ascocarps known as truffles are produced. Some Tuber spp. produce edible truffles that, given their distinctive taste and aroma, are highly valued by gourmets. Research on these fungi has focused on promoting the cultivation of these fungi to meet increasing worldwide demand and to provide replacements for the catastrophic decline in their natural production (10). Truffle cultivation is no longer an agronomic practice confined to Europe, where the most profitable species are endemic. Truffle plantations have been established in various countries worldwide, including New Zealand and Israel. Nevertheless, the understanding of many basic aspects of truffle biology is still in its infancy, and the ecological requirements for some of these species are still not known. One of the most elusive goals has been discerning the reproductive system of Tuber spp. The reproductive structures of these species in pure cultures have not been reported, and axenic spore germination remains an unresolved problem (26). Furthermore, the mating-type genes have never been characterized in truffles.Molecular markers are being developed to type each truffle species to overcome the difficulty of identifying these species solely on their morphological traits (1,8,11,17,18,19,22,23). By combining molecular markers with an appropriate sampling strategy, we may be able to critically evaluate the truffle reproductive system and life cycle even without reproducing the entire life cycle in the lab. To date, Tuber melanosporum Vittad. and Tuber magnatum Pico, the finest black and white truffle species, respectively, have been regarded as selfing species. When codominant markers were evaluated, heterozygous ascocarps were not detected (2,3,7,15,16). These studies proceed from the assumption that the ascocarps are diploid (dikaryotic) structures.We recently used simple sequence repeat (SSR) markers and a large survey of...

Summary The genome of Tuber melanosporum has recently been sequenced. Here, we used this information to identify genes involved in the reproductive processes of this edible fungus. The sequenced strain (Mel28) possesses only one of the two master genes required for mating, that is, the gene that codes for the high mobility group (HMG) transcription factor (MAT1‐2‐1), whereas it lacks the gene that codes for the protein containing the α‐box‐ domain (MAT1‐1‐1), suggesting that this fungus is heterothallic. A PCR‐based approach was initially employed to screen truffles for the presence of the MAT1‐2‐1 gene and amplify the conserved regions flanking the mating type (MAT) locus. The MAT1‐1‐1 gene was finally identified using primers designed from the conserved regions of strains that lack the MAT1‐2‐1 gene. Mating type‐specific primer pairs were developed to screen asci and gleba from truffles of different origins and to genotype single ascospores within the asci. These analyses provided definitive evidence that T. melanosporum is a heterothallic species with a MAT locus that is organized similarly to those of ancient fungal lineages. A greater understanding of the reproductive mechanisms that exist in Tuber spp. allows for optimization of truffle plantation management strategies.

Summary• Tuber melanosporum is an ectomycorrhizal ascomycete producing edible ascocarps. The prevalent view is that this species strictly selfs, since genetic analyses have never detected heterozygotic profiles in its putatively diploid/dikaryotic gleba. The selfing model has also forged the experimental approaches to assess the population genetic variability. Here, the hypothesis that T. melanosporum outcrosses was tested.• To this end, SSR (simple sequence repeats) and ITS (internal transcribed spacer) markers were employed to fingerprint asci and the surrounding gleba within single ascocarps. The distribution of genetic variability was also investigated at different geographical levels using single (SSR and ITS) and multilocus (AFLP, amplified fragment length polymorphism) markers.• It is shown that T. melanosporum outcrosses since asci display additional alleles besides those present in the surrounding, uniparental, gleba. Furthermore, SSR and AFLP data reveal a high rate of intrapopulation diversity within samples from the same ground and root apparatus and the highest rate of genetic variability within the southernmost populations of the distributional range.• These data call for a profound re-examination of T. melanosporum mating system, life cycle and strategies for managing man-made plantations. They also strongly support the idea that the last glaciation restricted the species distribution to the Italian and Spanish peninsulas.