CORESTA Congress, Kyoto, 2004, AP 15

Genetic fingerprinting of Nicotiana species by ISSR markers

Istituto Sperimentale per il Tabacco, Scafati, Italy

The application of DNA technology, for assessing the genetic diversity in plants, has progressed rapidly in the last years, expecially with the development of PCR techniques. In the Inter Simple Sequence Repeat (ISSR) technique DNA fragments located between adiacent, oppositely oriented microsatellites (Simple Sequence Repeats, SSR), are amplified by Polymerase Chain Reaction (PCR) using primers that are anchored at 5' or 3' end of a repeat region and extend into the flanking region. The aim of this paper was to value the potential of ISSR technique for the characterization and the identification of Nicotiana species. Genomic DNA of 30 Nicotiana species, belonging to almost all the sections of the genus, was extracted and amplified utilizing 10 different primers (14-22 bp). PCR products were resolved on agarose gel, stained with ethidium bromide and visualized by UV transilluminator. The electrophoretic banding patterns were recordered and analzed by utilizing an image analysis system. Amplification profiles of Nicotiana species examined revealed a high degree of polymorphism, as generally different band patterns were observed. The number of markers generated per primer ranged from 15 to 30, and the dimensions from 200 and 2500 bp. On the bases of these results, it may be expected that ISSR technique is a useful tool to individuate molecular markers able to characterize all Nicotiana species. To complete the analysis of the Nicotiana genus, further experiments of amplification with other species are in progress. The availability of Nicotiana species fingerprints, by means of ISSR technique, may contribute to a better characterization of this genus and may be a powerful monitoring tool in maintaining botanic collection.