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Localização Da Heterocromatina Em Vernonia Scorpioides E Rolandra Fruticosa (VERNONIEAE:ASTERACEAE) Por Meio Dos Fluorocromos CMA E DAPI

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Localização Da Heterocromatina Em Vernonia Scorpioides E Rolandra Fruticosa (VERNONIEAE:ASTERACEAE) Por Meio Dos Fluorocromos CMA E DAPI XIII JORNADA DE ENSINO, PESQUISA E EXTENSÃO – JEPEX 2013 – UFRPE: Recife, 09 a 13 de dezembro. Localização da heterocromatina em Vernonia scorpioides e Rolandra fruticosa (VERNONIEAE:ASTERACEAE) por meio dos fluorocromos CMA e DAPI Maria Angélica Oliveira Marinho1, Ana Raquel Oliveira Marinho4, Maria Rita Cabral Sales-Melo2, Maria Betânia Melo de Oliveira3 e Reginaldo de Carvalho1 Introdução A tribo Vernonieae é uma das mais importantes da família Asteraceae, sendo relativamente bem estudada citogeneticamente quanto ao número cromossômico e morfometria cariotípica, entretanto, pouco se sabe sobre as características especificas da cromatina de seus representantes. Na tribo as descrições estão restritas a pequenos grupos como é o caso das espécies de Vernonia, V. condensata (Sale-de-Melo, 2010), V. adamantium, V. fruticulosa e V. germinata (Oliveira, 2008). Os fluorocromos são substâncias fluorescentes muito utilizados em estudos citogenéticos, já que apresentam especificidade de ligação com bases específicas de DNA. O 4’-6- diamidino-2, fenilindol (DAPI), marca regiões ricas em bandas Adenina (A) e Timina (T) e a cromomicina A3 (CMA) que marca regiões de DNA ricas em Guanina (G) e Citosina (C) são os mais utilizados (Sumner, 1990). Guerra (1988) utilizou estes fluorocromos em um estudo com cinco populações de Eleutherine bulbosa (2n=12), família Iridaceae, verificando eventos citogenéticos evolutivos complexos como a inversão pericêntrica e duplicação em tandem. Silva (2010) estudou 23 acessos de 13 espécies de Asteraceae com o intuito de analisar as suas relações evolutivas e percebeu variação na quantidade, localização e tamanho de sítios de DNAr, identificando vários tipos de rearranjos como inversões, transposição e translocações. Além da típica caracterização cariotípica, estes corantes podem ser empregados na citotaxonomia que é um ramo da citogenética que estuda eventos de natureza citogenética associados a evolução cariotípica e citotaxonomia, podendo auxiliar na solução de questões taxonômicas pertinentes (Stace, 1989). Sendo assim, este trabalho teve como objetivo localizar a porção heterocromática de duas espécies da tribo Vernonieae, mas de gêneros diferentes, através da técnica de coloração diferencial CMA/DAPI buscando obter novos dados para a caracterização do cariótipo. Material e métodos A. Coloração diferencial CMA/DAPI Foram estudadas duas espécies Vernonia scorpioides (Lam.) Pers e Rolandra fruticosa Rottb coletadas nas cidades de Recife e Buíque. A preparação cromossômica com a técnica de coloração diferencial foi realizada segundo o protocolo de Schweizer e Ambros (1994). Pontas de raízes foram pré-tratadas com 8- hidroxiquinoleina (8HQ) 2mM durante 24 h e fixadas em Carnoy (etanol: ácido acético, 3:1). Logo após, as raízes foram digeridas com por 2 horas em uma solução de celulase (2%) e pectinase (20%) a 37ºC e esmagadas em ácido acético a 45%. As lâminas foram envelhecidas por três dias à temperatura ambiente e, posteriormente, coradas por 60 minutos com CMA a 0,5 mg/mL, e contracoradas com DAPI 2 g/ml por 30 minutos e montadas em tampão McIlvaine-glicerol (1:1). As imagens de fluorescência foram capturadas em microscópio de epifluorescência Leica DM 2500 equipado com câmera digital DFC 345FX. Os ideogramas foram desenhados usando o programa Power Point da Microsoft baseados na localização das bandas heterocromáticas. Resultados e Discussão O número cromossômico encontrado para as espécies foi 2n= 52 para Rolandra fruticosa Rottb e 2n= 60 para Vernonia scorpioides (Lam.) Pers (Figura 1). Em Vernonieae, de maneira geral, os estudos citogenéticos têm sido limitados à utilização da coloração convencional (Jones, 1979; Dematteis, 1996, 2002; Dematteis & Fernandez, 2000; Oliveira et al., 2007). Dados da literatura fazem referência ao uso da técnica CMA/DAPI revelando que no genoma destas espécies, há uma predominância de heterocromatina rica em guaninas e citosinas para o corante cromomicina e nenhuma banda positiva para o corante DAPI. Oliveira (2008), por exemplo, relatou para algumas espécies do gênero 1 Universidade Federal Rural de Pernambuco; Departamento de Biologia, Genética; Programa de Pós-graduação em Botânica; Laboratório de Citogenética Vegetal, Recife, Pernambuco, Brasil. 2 Universidade Federal Rural de Pernambuco; Departamento de Biologia, Botânica; Recife, Pernambuco, Brasil. 3 Universidade Federal de Pernambuco; Departamento de Bioquímica, Recife, Pernambuco, Brasil. e-mail para correspondência: [email protected] 4 Universidade Federal Rural de Pernambuco; Unidade Acadêmica de Serra Talhada, Departamento de Genética; Serra Talhada, Pernambuco, Brasil. XIII JORNADA DE ENSINO, PESQUISA E EXTENSÃO – JEPEX 2013 – UFRPE: Recife, 09 a 13 de dezembro. Vernonia um número máximo de seis bandas CMA+. Todos os registros descritos até o momento para Vernonia apontam para uma composição da heterocromatina do tipo CMA+/DAPI-. Na família Asteraceae há registro de espécies apresentando regiões heterocromáticas ricas em adenina e timina (AT), como é o caso de representantes dos gêneros Cichorium e Artemisia (Silva, 2010). O uso de fluorocromos base-específicos CMA e DAPI revelou três padrões diferentes de bandeamento cromossômico nas espécies estudadas: DAPI+/CMA0, e DAPI+/CMA- (Figura 1). Apesar Rolandra fruticosa e Vernonia scorpioides apresentarem bandas heterocromáticas ricas em AT, foram observadas algumas diferenças na quantidade e localização entre as duas espécies. Para Rolandra fruticosa, foram encontradas quatro bandas CMA+/DAPI- terminais e um par DAPI+/CMA0 distais. Já o padrão encontrado em Vernonia scorpioides revelou oito bandas CMA+/DAPI- distais com variação no tamanho, dois blocos grandes e seis muito pequenos todos e localizados terminalmente, além de cinquenta e oito bandas DAPI+/CMA- teloméricas distribuídas ao longo do conjunto diploide cromossômico. Para estas espécies foram localizadas apenas duas regiões organizadoras de nucléolos (RONs) que corresponderam ÀS bandas CMA positivas. Oliveira (2008) relatou apenas seis bandas CMA+ e nenhuma DAPI+ e um número cromossômico diploide 2n=56 para Vernonia scorpioides. Rolandra fruticosa não apresenta registros anteriores para este tipo de técnica Para Schwarzacher et al. (1980), as mudanças na quantidade de heterocromatina são um parâmetro expressivo no entendimento de questões genéticas e estudos de filogenia. As duas espécies analisadas pertencem ao Novo Mundo e podem se tratar de uma tendência evolutiva no grupo. As análises destas duas espécies permitiram a diferenciação citogenética e a construção de ideogramas espécie- espécíficos, evidenciando suas diferenças interespecíficas. Segundo Silva (2010) mapas citogenéticos são de grande importância para compreender a organização de genomas e identificação de processos divergentes na evolução. A variação do padrão de bandas dessas espécies mostra uma grande variabilidade da heterocromatina na tribo Vernonieae, mostrando a importante contribuição desse tipo de investigação no estudo da caracterização cariotípica. Agradecimentos À Universidade Federal Rural de Pernambuco - UFRPE, ao Programa de Pós-graduação em Botânica – PPGB e ao Conselho de Desenvolvimento Científico e Tecnológico - CNPq pela concessão da bolsa. Referências Dematteis, M. Cytotaxonomic analysis of South American species of Vernonia (Vernonieae: Asteraceae). Bot. Jour. Linn. Soc., v.139, p.401-408, 2002. Dematteis, M. Estudios cromosomicos en especies Argentinas de Vernonia (Asteraceae). Bonplandia, v.9, n.1-2, p.103-110, 1996. Dematteis, M.; Fernández, A. Chromosomes studies on nine South American species of Vernonia (Vernonieae, Asteraceae). Caryologia, v.53, n.1, p.61, 2000. Guerra, M. Mitotic and meiotic analysis of a pericentric inversion associated with a duplication in Eleutherine bulbosa (Iridaceae). Chromosoma, Berlin, v. 97, p.80-87, 1988. Jones, S. B. Chromosome numbers of Vernonieae (Compositae). Bull. Torrey Bot. Club. 1979a. 106(2): 79-84. Oliveira, V.M.; Forni-Martins, E.R.; Semir, J. Cytotaxonomic studies in six species of Vernonia (Asteraceae: Vernonieae). Caryologia, v.60, p.37–47, 2007. Oliveira, V.M. Caracterização cariotípica de espécies de Vernonia Schreb. (Asteraceae: Vernonieae) com técnicas de diferencial longitudinal de cromossomos (bandeamentos e hibridação de DNA in situ). Instituto de biologia. Campinas, SP. 2008. Tese Doutorado. Salles-de-Melo, M.R.; Lucena, R.M.; Semir, J.O.; Carvalho, R.; Pereira, C.A. E Benko-Iseppon, A.M. Karyological features and cytotaxonomy of the tribe Vernonieae (Asteraceae). Springer, v.285, n.3-4, p.189-199, 2010. Silva, E.B.C. Citogenética evolutiva na família Asteraceae usando fluorocromos. CMA/DAPI e FISH com sondas de DNAr 45S e 5S. Universidade Federal de Pernambuco. Recife. 2010. Tese Doutorado. Schweizer, D.E Ambros, P.F. Chromosome banding: stain combinations for specific regions. In: GOSDEN, J. R. (Ed.). Methods in molecular biology: Chromosome analysis protocols. Humana Press, v.29, p97-112, 1994. Schwarzacher, T.; Ambros, P.; Schweizer, D. Application of Giemsa banding to orchid karyotype analysis. Plant Systematic and Evolution, v.134, n.3-4, p.293-297, 1980. Stace, C.A. Plant Taxonomy and biossistematics. 2 edition. New York, Cambridge University Press. 1989. Sumner, A.T. Chromosome banding. Cambridge: Cambridge University Press. 1990. XIII JORNADA DE ENSINO, PESQUISA E EXTENSÃO – JEPEX 2013 – UFRPE: Recife, 09 a 13 de dezembro. Figura 1. Ideogramas de coloração diferencial CMA/DAPI de espécies de Vernonieae: A- Rolandra fruticosa Rottb e B- Vernonia scorpioides (Lam.) Pers. .
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