Sequential silver nitrate staining and FISH has

Sequential silver nitrate staining and FISH has been used to study rDNA in several plant species (Hasterok and Maluszynska, 2000; Idziak and Hasterok, 2008). The conjugation of these two techniques for rDNA studies has the advantage of establishing the activity, location and number of rDNA loci. As far as we know, silver nitrate staining and sequential FISH was never performed in V. vinifera. Hence, in this work, we aimed to obtain high-quality chromosome spreads for silver nitrate staining and sequential FISH performed with the 45S rDNA probe pTa71, to study the nucleolar activity and to physically map the rDNA loci in interphase and metaphase BSI-201 synthesis of root-tips and leaves of seven V. vinifera varieties.
Materials and methods
Results In the leaves of the studied varieties, one to three nucleoli per nucleus were observed (Fig. 1a), except for the variety Bastardo were a maximum of two nucleoli per nucleus were detected. In the root-tip cells, most of the varieties presented one to three nucleoli per interphase nucleus apart from the variety Pinot Noir that showed one to four nucleoli per nucleus (Fig. 1b). The average frequencies of the nucleoli scored in the leaves and root-tips of different V. vinifera varieties are presented in Table 1. In all varieties, independently of the tissue, one single nucleolus per nucleus appeared more frequently, justifying the fact of all average values of nucleoli per cell were lower than 2 (Table 1). For the average number of nucleoli per cell, the Tukey test distributed the 7 varieties into 5 groups with statistically significant differences (p < 0.001) among them. When comparing the different varieties, independently of the tissue, there are statistically significant differences (p < 0.001) for the nucleoli average frequencies. The interaction leaf × variety showed statistically significant differences (p < 0.001) among the studied varieties for all the parameters, with the obvious exception of the average frequency of four nucleoli. Once again, in the average number of nucleoli per cell data, the Tukey test distributed the 7 varieties into 5 groups. This did not happen for the interaction root × variety. Here, only Pinot Noir stands out because it is the only variety that presented four nucleoli. Due to the small size of the chromosomes that hampers the accurate identification of Ag-NORs and to the reduced mitotic index, particularly in the leaf tissue, it was not possible to score the number of Ag-NORs in a high number of metaphase cells of the 7 varieties to achieve valuable statistical data. Nevertheless, in the root-tip cells of most of the varieties, one to three Ag-NORs per metaphase were observed (Fig. 2a to c; Table 1). In fact, the Ag-NORs identified after silver nitrate staining were further confirmed by sequential FISH performed with the 45S rDNA probe, pTa71 (Fig. 2c and d; Table 1). Except for Tinto Cão that showed three rDNA loci after FISH (Fig. 2d), the remaining varieties presented four rDNA loci independently of the maximum number of Ag-NORs previously detected (Fig. 2e; Table 1).