Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica

• Ano de defesa: 2008
• Autor(a) principal: Clarindo, Wellington Ronildo
• Orientador(a): Carvalho, Carlos Roberto de
• Banca de defesa: Sakiyama, Ney Sussumu , Picoli, Edgard Augusto de Toledo
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Genética e Melhoramento
• Departamento: Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me
• País: BR
• Palavras-chave em Português: Café; Citogenética; Citometrias de fluxo e imagem; Cultura de tecidos vegetais
• Palavras-chave em Inglês: Coffee; Cytogenetics; flow and image cytometric techniques; Plant tissue culture
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL
• Link de acesso: http://locus.ufv.br/handle/123456789/1273

The application of cytogenetic, flow and image cytometric techniques has been enabling the genome characterization of Coffea arabica L. and Coffea canephora Pierre ex Froehner. These approaches have been providing relevant information for evolutionary studies and breeding programs. Considering the need of increasing the information available on the genomes of C. arabica and C. canephora, the present study improved cytogenetic and cytometric strategies in order to provide new data about the genomes of these species. Initially, cytogenetic methodologies applied to suspensions of cell aggregates of C. canephora yielded chromosomes with adequate structure for morphometric characterization and assembly of the karyogram for this species. Application of the banding techniques Ag-NOR and Hsc-FA allowed the identification of the active NOR in the short arm of chromosome 6. After employing the same procedures used in C. canephora to cell aggregates of C. arabica, metaphasic and prometaphasic chromosomes were obtained presenting satisfactory resolution for the accomplishment of the cytogenetic analyses and assembly of the karyogram of this species. The morphometric analyses evidenced both morphologically identical and distinct chromosomes, which suggests that C. arabica is a allotetraploid, non-segmental, originated from the crossing between two species with similar genome. Flow cytometry was used to quantify the nuclear DNA and determine the base composition of C. canephora and C. arabica. Preliminary analyses showed that the procedures involving use of the OTTO buffers and antioxidant substances resulted in histograms showing peaks of G0/G1 nuclei, with coefficients of variation considered adequate for cytometric analyses. Moreover, it was demonstrated that, in comparison to Pisum sativum and Raphanus sativus, Solanum lycopersicum was the plant used as standard of known DNA that offered the most accurate estimation of the nuclear DNA contents of C. arabica and C. canephora. In a second stage, it was verified that the mean size of the nuclear genome of C. arabica (2.62 picograms) and C. canephora (1.41 picograms), obtained through use of the fluorochrome propidium iodide, was identical to that measured in nuclei stained with the dye SYBR Green I. However, SYBR Green I rendered more precise cytometrical analyses, seeing that the peaks resulting from G0/G1 nuclei presented coefficients of variation under those obtained with propidium iodide. Apart from the absolute DNA content, the base composition was also determined for both species by use of nuclei stained with 4',6 -diamidino-2- phenylindole (DAPI) and chromomycin A3 (CMA3). C. arabica presented a percentage of AT bases equivalent to 63.04%, and 36.96% GC, while C. canephora presented 65.27% AT and 34.73% GC. The association of cytogenetic methodologies with flow and image cytometries allowed estimation of the DNA content of each chromosome of C. arabica and C. canephora. The results obtained also showed that C. arabica is a true allotetraploid, and additionally provided evidence that C. canephora is a possible progenitor of that species. The presented data supplied new information on the characteristics, composition and genome organization of C. arabica and C. canephora, and represented an advance in the genome research of the coffee tree.