Evaluation of copper-induced DNA damage in Vitis vinifera L. using Comet-FISH
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RESEARCH ARTICLE
Evaluation of copper-induced DNA damage in Vitis vinifera L. using Comet-FISH Cláudia Castro 1 & Ana Carvalho 1,2,3
&
Isabel Gaivão 3,4 & José Lima-Brito 1,2,3
Received: 5 March 2020 / Accepted: 25 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract The contamination of soils and water with copper (Cu) can compromise the crops production and quality. Fungicides containing Cu are widely and intensively used in viticulture contributing to environmental contamination and genotoxicity in Vitis vinifera L. Despite the difficulty in reproducing field conditions in the laboratory, hydroponic solutions enriched with Cu (1, 10, 25 and 50 μM) were used in forced V. vinifera cuttings to evaluate the DNA damage in leaves of four wine-producing varieties (‘Tinta Barroca’, ‘Tinto Cão’, ‘Malvasia Fina’ and ‘Viosinho’). Alkaline comet assay followed by fluorescence in situ hybridisation (Comet-FISH) was performed with the 45S ribosomal DNA (rDNA) and telomeric [(TTTAGGG)n] sequences as probes. This study aimed to evaluate the tolerance of the four varieties to different concentrations of Cu and to determine which genomic regions were more prone to DNA damage. The comet assay revealed comets of categories 0 to 4 in all varieties. The DNA damage increased significantly (p < 0.001) with the Cu concentration. ‘Tinto Cão’ appeared to be the most sensitive variety because it had the highest DNA damage increase in 50 μM Cu relative to the control. Comet-FISH was only performed on slides of the control and 50 μM Cu treatments. Comets of all varieties treated with 50 μM Cu showed rDNA hybridisation on the head, ‘halo’ and tail (category III), and their frequency was significantly higher than that of control. The frequency of category III comets hybridised with the telomeric probe was only significantly different from the control in ‘Malvasia Fina’ and ‘Tinta Barroca’. Comet-FISH revealed partial damage on rDNA and telomeric DNA in response to Cu but also in control, confirming the high sensitivity of these genomic regions to DNA fragmentation. Keywords Comet assay . Comet-FISH . Copper . Genotoxicity . Grapevine . Ribosomal DNA . Telomeric sequence
Introduction Responsible Editor: Gangrong Shi * Ana Carvalho [email protected] 1
Biosystems & Integrative Sciences Institute-University of Tras-os-Montes and Alto Douro (BioISI-UTAD), Quinta de Prados, 5000-801 Vila Real, Portugal
2
Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Tras-os-Montes and Alto Douro, Quinta de Prados, 5000-801 Vila Real, Portugal
3
Department of Genetics and Biotechnology, Blocos Laboratoriais, A1.09, University of Tras-os-Montes and Alto Douro, Quinta de Prados, 5000-801 Vila Real, Portugal
4
Animal and Veterinary Research Centre (CECAV), University of Tras-os-Montes and Alto Douro, Quinta de Prados, 5000-801 Vila Real, Portugal
Copper (Cu) is an essential micronutrient for higher plants, acting as a critical cofactor for proteins, and it is involve
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