Distribution and localization of microsatellites in the Perigord black truffle genome and identification of new molecular markers.

The level of genetic diversity and genetic structure in the Perigord black truffle (Tuber melanosporum Vittad.) has been debated for several years, mainly due to the lack of appropriate genetic markers. Microsatellites or simple sequence repeats (SSRs) are important for the genome organisation, phenotypic diversity and are one of the most popular molecular markers. In this study, we surveyed the T. melanosporum genome (1) to characterise its SSR pattern; (2) to compare it with SSR patterns found in 48 other fungal and three oomycetes genomes and (3) to identify new polymorphic SSR markers for population genetics. The T. melanosporum genome is rich in SSRs with 22,425 SSRs with mono-nucleotides being the most frequent motifs. SSRs were found in all genomic regions although they are more frequent in non-coding regions (introns and intergenic regions). Sixty out of 135 PCR-amplified mono-, di-, tri-, tetra, penta, and hexa-nucleotides were polymorphic (44%) within black truffle populations and 27 were randomly selected and analysed on 139 T. melanosporum isolates from France, Italy and Spain. The number of alleles varied from 2 to 18 and the expected heterozygosity from 0.124 to 0.815. One hundred and thirty-two different multilocus genotypes out of the 139 T. melanosporum isolates were identified and the genotypic diversity was high (0.999). Polymorphic SSRs were found in UTR regulatory regions of fruiting bodies and ectomycorrhiza regulated genes, suggesting that they may play a role in phenotypic variation. In conclusion, SSRs developed in this study were highly polymorphic and our results showed that T. melanosporum is a species with an important genetic diversity, which is in agreement with its recently uncovered heterothallic mating system.

Expression patterns of endothelial and inducible nitric oxide synthase isoforms in corpora lutea of pseudopregnant rabbits at different luteal stages.

Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P

Cloning and characterization of two repeated sequences in the symbiotic fungus Tuber melanosporum Vitt.

Two repeated DNA sequences of European strains of the symbiotic fungus Tuber melanosporum were isolated and characterized. One of these, SS14, representing about 0.05% of the fungal genome, was shown to be a T. melanosporum-specific sequence by Southern and dot-blot hybridization. The second one, named SS15, is about 0.0025% of the entire genome, and it is specific not only to T. melanosporum but also to the Asian black truffle Tuber indicum. Neither of these two fragments hybridizes with any of the other European truffle species tested. By sequence analysis of these two fragments, PCR primers were designed and used to selectively amplify DNA from T. melanosporum ascocarps and ectomycorrhizae by simple and multiplex PCR. No amplification products were obtained with DNA from either mycorrhizal roots or fruit bodies of other ectosymbiotic fungi. The two identified genomic traits also provided useful information for a better understanding of the phylogenetic relationships among black truffle species and for testing T. melanosporum intraspecific variability.

The volatile organic compounds from the mycelium of Tuber borchii Vitt.

The mycelium of T. borchii (characterized by DNA analysis) grown in sterile liquid medium produced some VOCs. The VOCs were retained on carbographs by passing a flow of helium, isolated and characterized in a GC-MS equipment after a thermal desorption. The compounds present in the VOCs from the mycelium cultures, but not in the VOCs from the control cultures, contained 29 compounds. The main compounds were 1,3-ditertbutylbenzene (16.1 ng/l), 3-methylheptane (9.2 ng/l), butan-2-one (8.8 ng/l), ethynylbenzene (5.6 ng/l), and octan-3-one (4.9 ng/l).

A maize anthocyanin transactivator induces pigmentation in hairy roots of dicotyledonous species.

Several dicotyledonous species were infected with an Agrobacterium rhizogenes binary vector harbouring the plasmid 121.Sn which contains the maize gene Sn under the constitutive promoter CaMV35S. In maize, Sn transactivates the anthocyanin pathway in different tissues. The aim of this work was to test the efficiency of this gene to regulate the anthocyanin pathway in heterologous systems and verify its suitability as a reporter gene. The pigmentation of the hairy roots was compared with hairy roots stained for ß-glucuronidase activity, which were used as a control. In two polymorphic genotypes of Lotus angustissimus, DNA integration and expression were assayed. The maize gene is competent to induce anthocyanin pigmentation in different species, but the complexity of the regulatory mechanisms of anthocyanin synthesis restricts the use of Sn as a reporter gene.

Typing Tuber melanosporum and Chinese black truffle species by molecular markers.

Morphologically similar species such as Chinese black truffles and Tuber melanosporum were typed by restriction length polymorphism of internal transcribed spacers. This analysis together with sequence comparison revealed the presence of high genetic variability among fruit bodies collected in China. Selection of primer pairs allowed the internal transcribed spacer region of both Chinese truffles and T. melanosporum to be specifically amplified.

Expression of the neuron-specific FE65 gene marks the development of embryo ganglionic derivatives.

The major transcript of the FE65 gene is a neuron-specific mRNA that encodes a nuclear protein whose aminoterminal domain strongly activates the transcription of a reporter gene when fused to a heterologous DNA-binding domain. FE65 gene expression is regulated during neuronal differentiation of the NTERA-2 cell line, and it is temporally and spatially restricted during mouse embryo development. It is first detected around day 10 of gestation in the basal plate of the neural tube, and then, at the subsequent stages of development and in the newborn animals, it is found solely in neural structures. Its expression is most abundant in the neural crest derivatives (e.g. spinal and encephalic ganglia), ganglionic structures of sense organs (ganglionic layer of the retina and olfactory epithelium), as well as the ganglionic structures of the autonomic nervous system. Thus FE65 gene expression can be considered a marker of the development of embryo ganglionic derivatives.