br Introduction Vitis vinifera L is
Introduction Vitis vinifera L. is one of the most worldwide-grown fruit crop and is mostly used in the wine industry (Bouquet, 2011). Domestication and history changed dramatically the biology of this species and today there are thousands of V. vinifera varieties, being many of them closely related (This et al., 2006). Olmo (1937) determined the number of chromosomes of 84 V. vinifera varieties by counting somatic plates of root-tips stained with Newton's gentian violet-iodine method. He concluded that the species is diploid and presents 38 small somatic chromosomes. These results were later confirmed by other authors in different varieties. Takusagawa (1951) reported the same number of chromosomes for 26 V. vinifera varieties, using meiotic plates of young flower buds stained with Eleidenhain’s iron haematoxylin. Raj and Seethaiah, 1969, Raj and Seethaiah, 1973 counted and measured mitotic chromosomes of 6 varieties using root-tips stained with aceto-orcein and shoot-tips stained with acetocarmine. Patil and Jadhav (1985) also counted and measured the mitotic chromosomes in root-tips of three V. vinifera varieties using a mixture of aceto-orcein and acetocarmine stains. Although these authors have reported a consensus number of chromosomes, they also described variations regarding the chromosomes length and XMD8-92 position among varieties. Other staining techniques have also been used to better visualize and study mitotic chromosomes. Pinto-Maglio et al. (2010) used Giemsa staining to measure mitotic chromosomes and fluorescent chromosome banding with the fluorochromes chromomycin A3 (CMA3) and DAPI, in order to characterize the constitutive heterochromatin in seven Vitis species, including V. vinifera variety Italia. This variety presented one pair plus one chromosome with one CMA3-positive terminal band and no DAPI contrastable bands. Giemsa staining was later used to construct a comprehensive ideogram of the same V. vinifera variety (Pierozzi, 2011; Pierozzi and Moura, 2016). Silver nitrate staining was previously used in 7 species of the genus Vitis (Haas and Alleweldt, 2000; Pierozzi, 2011) and in the hybrid Niagara (Vitis labrusca × V. vinifera) (Pierozzi and Moura, 2014) to visualize and score the nucleoli and NORs. This technique allows the detection of ribosomal DNA (rDNA) loci in metaphase that were transcriptionally active in the preceding interphase of the cell cycle (Jimenez et al., 1988). In V. vinifera a maximum of three nucleoli per cell and one chromosome pair with a terminal NOR band per metaphase were observed (Haas and Alleweldt, 2000; Pierozzi, 2011). Therefore, fluorescence in situ hybridisation (FISH) with 45S and 5S rDNA probes were performed in V. vinifera by different authors, indicating the existence of four 45S rDNA loci (Haas and Alleweldt, 2000; Houel et al., 2010; Pereira et al., 2005; Pereira et al., 2005, Pereira et al., 2014) and two 5S rDNA loci (Falistocco et al., 2007; Haas and Alleweldt, 2000; Pereira et al., 2014). FISH was also used in V. vinifera to localize the retrotransposon Gret1 (Pereira et al., 2005), several BAC clones from the Pinot Noir VvPN40024 library (Giannuzzi et al., 2011) and a telomeric sequence (Pereira et al., 2014). 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 cells of root-tips and leaves of seven V. vinifera varieties.