Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti Nucleolar activity and physical location of ribosomal DNA loci in Vitis vinifera L. by silver staining and sequential FISH Cláudia Castro a , Ana Carvalho a , Ivo Pavia b , Fernanda Leal a,c , José Moutinho-Pereira b , José Lima-Brito a,c, ⁎ a Biosystems and Integrative Sciences Institute, University of Tras-os-Montes and Alto Douro (BioISI-UTAD), 5001-801 Vila Real, Portugal b Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Tras-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal c Department of Genetics and Biotechnology, University of Tras-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal ARTICLE INFO Keywords: Fluorescent in situ hybridisation (FISH) Grapevine Nucleolar activity Salt-nylon silver staining ABSTRACT 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. 1. 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 re- lated (This et al., 2006). Olmo (1937) determined the number of chromosomes of 84 V. vi- nifera 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 Elei- denhain’s iron haematoxylin. Raj and Seethaiah (1969, 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 chro- mosomes in root-tips of three V. vinifera varieties using a mixture of aceto-orcein and acetocarmine stains. Although these authors have re- ported a consensus number of chromosomes, they also described var- iations regarding the chromosomes length and centromere 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 A 3 (CMA 3 ) 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 CMA 3 -positive terminal band and no DAPI contrastable bands. Giemsa staining was later used to construct a comprehensive ideogram of the same V. vini- fera 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 vi- sualize and score the nucleoli and NORs. This technique allows the detection of ribosomal DNA (rDNA) loci in metaphase that were https://doi.org/10.1016/j.scienta.2017.12.064 Received 20 October 2017; Received in revised form 27 December 2017; Accepted 30 December 2017 ⁎ Corresponding author at: Departamento de Genética e Biotecnologia, Ed. Blocos Laboratoriais, A0.04, Universidade de Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal. E-mail address: jbrito@utad.pt (J. Lima-Brito). Scientia Horticulturae 232 (2018) 57–62 0304-4238/ © 2017 Elsevier B.V. All rights reserved. T