, 2001; Wang et al., 2006), but until now the molecular differences between these species as well as their potential capacity of inbreeding are largely unknown. Therefore, tools for Tuber species’ discrimination are still needed to avoid frauds in the truffle market. The intraspecies gSSH experiment in O. maius yielded, after subtraction of O. maius OmMa3 with O. maius OmMa2 genomic DNA and reverse C646 research buy dot blot analysis, 16 specific sequences: five were single independent sequences, whereas 11 formed three contigs (Table 3; accession numbers HN262662–HN262669). Of the singletons, one showed similarity to an l-galactonate dehydratase,
one to a short-chain dehydrogenase/reductase family protein and three found no similarity in databases. Of the contigs, one showed similarity to glutathione synthetase, one to acetoacetyl-coenzyme A synthetase and one found no similarity. OmMa3 and OmMa2 are two isolates derived from a serpentine soil, characterized by a high content in chromium and nickel (Vallino et al., 2011). These two isolates are genetically distinct, on the basis of genetic fingerprinting, and show different abilities to grow in the presence of heavy metals, OmMa3 growing considerably better than OmMa2 on Ni- and Cr-amended media (Vallino et al., 2011). Heavy metal tolerance is a trait of particular interest for documenting genetic changes during adaptation, as heavy metal toxicity
represents a strong directional selective pressure resulting in the substitution of tolerance alleles at some loci (Willems et al., 2007). The genetic basis of heavy metal tolerance is not fully understood, and the questions on how GPCR Compound Library clinical trial many genes are involved and on the dynamics of the alleles of these genes are still open. It is tempting to speculate that the sequences we have identified may represent genetic differences underlying different tolerance of the two isolates, but further investigations are needed. Interestingly, glutathione synthetase, the second enzyme in the glutathione
biosynthetic pathway, is known to be involved in metal tolerance (Pócsi et al., 2004; Reisinger et al., 2008). Glutathione plays a key role not only in metal detoxification but also in protecting cells from other environmental stresses, such as oxidative stress and xenobiotics (Memon & Schröder, 2009). Moreover, a recent study on Drosophila by Ortiz et al. (2009) nearly suggests that polymorphisms in GSH biosynthetic genes may be an important contributor to differential arsenic sensitivity. Therefore, this genomic region is a good candidate for further analyses on the genetic basis of metal tolerance in fungal isolates. In conclusion, our results show that gSSH is a quick and rather inexpensive approach that allows the identification of genomic differences both among (e.g. Tuber) and within (e.g. O. maius) fungal species. The sequences obtained by gSSH may be useful to identify species or strains as well as to investigate the genome plasticity, adaptation and evolution.