Assessment of reproductive mode in Tuber melanosporum, the fungal species producing the most appreciated black truffles

Tuber melanosporum is an ectomycorrhizal ascomycete producing edible fruiting bodies, known as black truffles. For their distinctive taste and aroma the truffles produced by different Tu-ber spp. are appreciated worldwide. Therefore these fungi are of considerable economic importance and their market is steadily expanding. The demand for truffles greatly exceeds their natural production that has been largely dropped over the last dec-ades. Thus, the presence of these products in the market depends largely on their cultivation. How-ever, attempts of truffle cultivation by outplanting nursery-produced mycorrhizal plants have been not always, and not for all species, successful. Yet, most of the ecological and biological aspects that govern truffle life cycle such as their reproduction mode, are still under debate, since these fungi are neither amenable for in vitro cultivation nor for mating ex-periments under controlled conditions. Thus, many basic aspects concerning these fungal species have not been fully elucidated yet and truffle sex is a long lasting dilemma for mycologists.Mating in fungal species occurs either through a heterothallic (self-sterile) or homothallic (self-fertile) mechanism. In heterothallic species, mating takes places between two sexually compatible strains that are morphologically identical and are distinguished genetically by mating type. In homothallic species, each strain can mate itself.1 Mating type in the As-comycota is typically bipolar, determined by two possible DNA sequences at the mating-type locus (MAT). The alternative sequences at MAT locus are not alleles in the classic sense because they lack significant sequence similarity and encode different transcriptional regulators. The term idiomorph is used to describe this unusual genetic organization, which is common among all MAT loci from heter-othallic ascomycetes investigated to date. The two alternative MAT idiomorphs encode dif-ferent transcription factors that permit sexual com-patibility: the two key mating type genes conserved in all filamentous ascomycetes encode regulatory proteins with a high mobility group (HMG) or a ?-box DNA-binding domain in MAT1-2 and MAT1-1 idiomorphs, respectively. Until recently, the prevalent view was that truffles strictly self, since genetic analyses had never detect-ed heterozygotic profiles in their putatively diploid/dikaryiotic structures. By using highly informative molecular markers such as SSR and SNPs, we have recently provided evidence for the prevalence of the haploid phase in the truffle life cycle and for the occurrence of outcrossing in both T. melanosporumand Tuber magnatum.2-4Despite this new finding, however, the hypothesis of selfing is far from being settled.Here we took advantage of the recently pub-lished T. melanosporum genome,5 and by using a combination of bioinformatics and genome-based approach, we attempted to clone and characterized the second mating idiomorph in T. melano-sporum