Identification of ectomycorrhizal fungi of the genus Tuber by RFLP analysis and species specific primers of the ITS region

AMICUCCI, ANTONELLA¹, LUANA BERTINI¹, LUCIA POTENZA¹, ISMAELA ROSSI¹, SABRINA ZEPPA¹, GIOVANNA GIOMARO² & VILBERTO STOCCHI¹.

¹Istituto di Chimica Biologica "Giorgio Fornarini", Università di Urbino, Via Saffi, 2 - 61029 Urbino (PS) - ITALY. ²Istituto Orto Botanico, Via Bramante, 28 - 61029 Urbino (PS) - ITALY.

PCR-based techniques for a reliable molecular identification of five species of white truffles are reported. The sequences of the ITS region of Tuber magnatum Pico, T. borchii Vittad., T. maculatum Vittad., T. dryophilum Berk. & Br. and T. puberulum Berk. & Br. were analysed. From the analysis of the sequences species-specific restriction fragment length polymorphisms patterns were determined for all five species. The alignment of the ITS sequences allowed us to design a pair of primers for each species under study. Furthermore the avaiability of species-specific primers led us to set up a multiplex PCR. The molecular methods described provide efficient identifications tools: the RFLP analysis is useful to distinguish different species and to evaluate the presence of contaminants; the multiplex PCR can be used for rapid and unambiguous identification in a single step reaction, showing the probable presence of several species in the same root system.

Key words: identification, ITS, RFLP, SCAR marker, multiplex PCR, Tuber.