Quantificação do conteúdo de DNA nuclear de Eucalyptus spp. por citometrias de fluxo e de imagem

O gênero Eucalyptus, da família Myrtaceae, apresenta cerca de 800 espécies e vários híbridos naturais. Além da importância biológica, é de múltiplo interesse comercial, destacando-se na indústria farmacêutica e na produção de biomassa lenhosa. A quantificação do conteúdo de DNA nuclear é útil em est...

Nível de Acesso:openAccess
Publication Date:2005
Main Author: Boaventura, Carolina Ribeiro Diniz lattes
Orientador/a: Carvalho, Carlos Roberto de
Format: Dissertação
Language:por
Published: Universidade Federal de Viçosa
Áreas de Conhecimento:
Online Access:http://www.locus.ufv.br/handle/123456789/10541
Citação:BOAVENTURA, Carolina Ribeiro Diniz. Quantificação do conteúdo de DNA nuclear de Eucalyptus spp. por citometrias de fluxo e de imagem. 2006. 61 f. Dissertação (Mestrado em Genética e Melhoramento) - Universidade Federal de Viçosa, Viçosa. 2005.
Resumo inglês:The Eucalyptus genus of Myrtaceae family has about 800 species and some natural hybrids. Beyond the biological importance, it is of multiple commercial interests, being distinguished in the pharmaceutical industry and the production of pulp biomass. The quantification of the nuclear DNA content is useful in studies of phylogenetics. Also it is important information for genome mapping. For nuclear DNA content determination, flow cytometry is considered the most appropriate methodology. However, Eucalyptus studies present differences between values. Another technique whose results allow the determination of the nuclear DNA amount is the image cytometry. The objective of this work was to reevaluate the divergences in the measures of nuclear DNA in Eucalyptus, for the application of flow and image cytometries. The measures had been carried through with the use of flow cytometer and stain with propidium iodide, of the isolated nuclei. The reference standards were leaves of Raphanus sativus cv. Saxa (1.11 pg), Lycopersicon esculentum cv. Stupicke (1.96 pg) and Gallus gallus female Leghorn erythrocytes (2.33 pg). Histograms of relative fluorescence intensity have generated after analysis of the isolated nuclei of combined internal standards with E. globulus, E. grandis and E. urophylla. The values in pg of nuclear DNA found for E. globulus and E. grandis xhad been of 1.39 and 1.41 (standard R. sativus), 1.20 and 1.22 (standard L. esculentum), and 1,05 and 1,09 (standard G. gallus). The quantification for E. urophylla pg was of 1.01 (standard G. gallus). The Eucalyptus species analyzed did not present differences among themselves greater than 5.4% in genomic DNA content in pg. However, intraspecific variations in only one plant, resultant of the different standards, presented divergences of up to 32%. The image cytometry analyses, with Feulgen reaction stain nuclei, were carried out to analyze the E. globulus DNA content, whose values were of 1.16 (standard R. sativus) and 1.02 pg (standard L. esculentum). The fluorochrome use (DNA- intercalating or base specific) and the secondary compounds interference in the staining were considered critical points in the analysis for the flow cytometry; besides the standards of reference and the coefficients of variation for both the cytometries. In all the analyses the bigger variation coefficients were 3.80 (flow cytometry) and 4.83 (image cytometry). For the comparison between the applied methodologies, the increase of the estimate of the nuclear DNA content was confirmed in E. globulus by flow cytometry, about 16% greater as compared to image cytometry. The ratios between plant standards remained the same ones in both techniques. Although the most precise nuclear DNA quantification in picogramas for E. globulus was proceeding from flow cytometry of leaves, by CV value, the most accurate was considered the average of meristem image cytometry data, whose value was 1.09 pg DNA/2C.