Nucleolar activity and physical location of ribosomal DNA loci in Vitis vinifera L. by silver staining and sequential FISH

Vitis vinifera L. is one of the most important fruit crops in the world, having thousands of different varieties. This diploid species has 38 small chromosomes that have been characterised with Giemsa staining, fluorochrome banding, silver staining, and fluorescence in situ hybridisation (FISH). Previous works reported that the number of ribosomal DNA (rDNA) loci in V. vinifera is four. In this work, we intend to study the nucleolar activity and to physically map the 45S rDNA loci in seven varieties of V. vinifera using an optimized protocol of silver nitrate staining and sequential FISH, respectively. Nucleoli and mitotic chromosomes of root-tips and leaves of the seven V. vinifera varieties were stained with silver nitrate. The number of nucleoli per interphase cell was scored and statistically analysed. Most of the varieties showed a maximum of three nucleoli per nucleus and three Ag-NORs per metaphase. The variety Bastardo showed a maximum of two nucleoli per nucleus in leaf tissue whereas Pinot Noir presented a maximum of four nucleoli per interphase in roots. However, in both tissues of all varieties, 1 nucleolus per nucleus was most frequent. High-quality chromosome spreads were selected for sequential FISH with the 45S rDNA probe pTa71 which allowed the physical location of four 45S rDNA loci in all varieties.