Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica
Autor(a) principal: | |
---|---|
Data de Publicação: | 2008 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | http://locus.ufv.br/handle/123456789/1273 |
Resumo: | 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. |
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Clarindo, Wellington Ronildohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4702819H9Otoni, Wagner Camposhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786133Y6Caixeta, Eveline Teixeirahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728636Z7Carvalho, Carlos Roberto dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780878T0Sakiyama, Ney Sussumuhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781483H8Picoli, Edgard Augusto de Toledohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4768537Z52015-03-26T12:45:16Z2008-04-022015-03-26T12:45:16Z2008-02-26CLARINDO, Wellington Ronildo. Utilization of cytogenetic, flow and image cytometric techniques for genome characterization of Coffea canephora and C. arabica. 2008. 113 f. Tese (Doutorado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2008.http://locus.ufv.br/handle/123456789/1273The 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.A aplicação de técnicas citogenéticas, de citometria de fluxo e de imagem tem possibilitado a caracterização do genoma de Coffea arabica L. e Coffea canephora Pierre ex Froehner. Esses trabalhos vêm provendo relevantes informações a estudos evolutivos e para o melhoramento da cultura. Levando em conta a necessidade de ampliação das informações acerca do genoma de C. arabica e C. canephora, esse estudo aprimorou estratégias citogenéticas e citométricas para gerar novos dados acerca do genoma dessas espécies. Inicialmente, metodologias citogenéticas aplicadas em suspensões de agregados celulares de C. canephora geraram cromossomos com morfologia adequada para caracterização morfométrica e montagem do cariograma da espécie. A aplicação das técnicas de bandeamento Ag- NOR e Hsc-FA possibilitaram a identificação da NOR ativa no braço curto do cromossomo 6. Após empregar os mesmos procedimentos utilizados para C. canephora em agregados celulares de C. arabica, foram obtidos cromossomos metafásicos e prometafásicos apresentando resolução suficiente para realização das análises citogenéticas e para montagem do cariograma da espécie. As análises morfométricas evidenciaram tanto cromossomos morfologicamente idênticos quanto diferentes, sugerindo que C. arabica é um alotetraplóide, não segmental, formado a partir do cruzamento de duas espécies com genoma similar. A citometria de fluxo foi empregada para mensurar o conteúdo de DNA nuclear e determinar a composição de bases de C. canephora e C. arabica. Análises preliminares mostraram que os procedimentos envolvendo o uso dos tampões OTTO e de compostos antioxidantes resultaram histogramas apresentando picos de núcleos em G0/G1 com coeficientes de variação considerados adequados para análises citométricas. Além disso, foi demonstrado que, em comparação com Pisum sativum e Raphanus sativus, Solanum lycopersicum foi a planta utilizada como padrão de DNA conhecido que gerou as estimativas mais acuradas acerca do conteúdo de DNA nuclear de C. arabica e C. canephora. Numa segunda etapa foi verificado que o tamanho médio do genoma nuclear de C. arabica (2,62 picogramas) e C. canephora (1,41 picogramas) obtido por meio do fluorocromo iodeto de propídeo foi idêntico ao mensurado com núcleos corados com SYBR Green I. Entretanto, o corante SYBR Green I proporcionou análises citométricas mais precisas, visto que os picos gerados por núcleos na fase G0/G1 apresentaram coeficientes de variação abaixo dos obtidos com iodeto. Além do conteúdo absoluto de DNA, a composição de bases também foi mensurada para as duas espécies utilizando núcleos corados com 4 ,6 -diamidino- 2-fenilindole (DAPI) e cromomicina A3 (CMA3). C. arabica apresentou um percentual de bases AT equivalente a 63,04% e GC 36,96%, enquanto o percentual de base AT de C. canephora foi 65,27% e GC 34,73%. A associação de metodologias citogenéticas e citométricas de fluxo e imagem permitiu estimar o conteúdo de DNA de cada cromossomo de C. arabica e C. canephora. Os resultados obtidos também mostraram que C. arabica é um verdadeiro allotetraplóide e geraram evidências que C. canephora é um possível progenitor dessa espécie. Os dados apresentados forneceram novas informações acerca das características, composição e organização do genoma de C. arabica e C. canephora e representam um avanço na prospecção do genoma do cafeeiro.Universidade Federal do Espírito Santoapplication/pdfporUniversidade Federal de ViçosaDoutorado em Genética e MelhoramentoUFVBRGenética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; MeCaféCitogenéticaCitometrias de fluxo e imagemCultura de tecidos vegetaisCoffeeCytogeneticsflow and image cytometric techniquesPlant tissue cultureCNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETALUtilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabicaUtilization of cytogenetic, flow and image cytometric techniques for genome characterization of Coffea canephora and C. arabicainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf1266042https://locus.ufv.br//bitstream/123456789/1273/1/texto%20completo.pdf769860186de8c70d271a11b7c76033c9MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain199447https://locus.ufv.br//bitstream/123456789/1273/2/texto%20completo.pdf.txt3ebe0b8d8da5514998081e84d53d54ebMD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3819https://locus.ufv.br//bitstream/123456789/1273/3/texto%20completo.pdf.jpg384978d293318d383153a6cb4048580aMD53123456789/12732016-04-06 23:10:08.342oai:locus.ufv.br:123456789/1273Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-07T02:10:08LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica |
dc.title.alternative.eng.fl_str_mv |
Utilization of cytogenetic, flow and image cytometric techniques for genome characterization of Coffea canephora and C. arabica |
title |
Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica |
spellingShingle |
Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica Clarindo, Wellington Ronildo Café Citogenética Citometrias de fluxo e imagem Cultura de tecidos vegetais Coffee Cytogenetics flow and image cytometric techniques Plant tissue culture CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL |
title_short |
Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica |
title_full |
Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica |
title_fullStr |
Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica |
title_full_unstemmed |
Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica |
title_sort |
Utilização de técnicas citogenéticas, de citometria de fluxo e de imagem para caracterização do genoma de Coffea canephora e C. arabica |
author |
Clarindo, Wellington Ronildo |
author_facet |
Clarindo, Wellington Ronildo |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4702819H9 |
dc.contributor.author.fl_str_mv |
Clarindo, Wellington Ronildo |
dc.contributor.advisor-co1.fl_str_mv |
Otoni, Wagner Campos |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786133Y6 |
dc.contributor.advisor-co2.fl_str_mv |
Caixeta, Eveline Teixeira |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728636Z7 |
dc.contributor.advisor1.fl_str_mv |
Carvalho, Carlos Roberto de |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780878T0 |
dc.contributor.referee1.fl_str_mv |
Sakiyama, Ney Sussumu |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781483H8 |
dc.contributor.referee2.fl_str_mv |
Picoli, Edgard Augusto de Toledo |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4768537Z5 |
contributor_str_mv |
Otoni, Wagner Campos Caixeta, Eveline Teixeira Carvalho, Carlos Roberto de Sakiyama, Ney Sussumu Picoli, Edgard Augusto de Toledo |
dc.subject.por.fl_str_mv |
Café Citogenética Citometrias de fluxo e imagem Cultura de tecidos vegetais |
topic |
Café Citogenética Citometrias de fluxo e imagem Cultura de tecidos vegetais Coffee Cytogenetics flow and image cytometric techniques Plant tissue culture CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL |
dc.subject.eng.fl_str_mv |
Coffee Cytogenetics flow and image cytometric techniques Plant tissue culture |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL |
description |
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. |
publishDate |
2008 |
dc.date.available.fl_str_mv |
2008-04-02 2015-03-26T12:45:16Z |
dc.date.issued.fl_str_mv |
2008-02-26 |
dc.date.accessioned.fl_str_mv |
2015-03-26T12:45:16Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
CLARINDO, Wellington Ronildo. Utilization of cytogenetic, flow and image cytometric techniques for genome characterization of Coffea canephora and C. arabica. 2008. 113 f. Tese (Doutorado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2008. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/1273 |
identifier_str_mv |
CLARINDO, Wellington Ronildo. Utilization of cytogenetic, flow and image cytometric techniques for genome characterization of Coffea canephora and C. arabica. 2008. 113 f. Tese (Doutorado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2008. |
url |
http://locus.ufv.br/handle/123456789/1273 |
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por |
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por |
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Universidade Federal de Viçosa |
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Doutorado em Genética e Melhoramento |
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UFV |
dc.publisher.country.fl_str_mv |
BR |
dc.publisher.department.fl_str_mv |
Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me |
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Universidade Federal de Viçosa |
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