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Diversidade Genómica E Genética Intra-Varietal Em Oliveira 'Galega Vulgar'

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Diversidade Genómica E Genética Intra-Varietal Em Oliveira 'Galega Vulgar' Diversidade genómica e genética intra-varietal em oliveira ‘Galega Vulgar’ Mónica Ramalhosa e Pereira Dissertação para a obtenção do Grau de Mestre em Engenharia Agronómica Orientadores: Mariana da Silva Gomes Mota Pedro Manuel Vieira Talhinhas Júri: Presidente: Doutor Joaquim Miguel Rangel da Cunha Costa, Professor auxiliar do Instituto Superior de Agronomia da Universidade de Lisboa. Vogais: Doutor António Manuel Cordeiro, Investigador auxiliar do Instituto Nacional de Investigação Agrária e Veterinária; Doutora Maria Teresa Victória Pires Antunes Barradas de Magalhães Carvalho, Investigadora auxiliar do Instituto Nacional de Investigação Agrária e Veterinária; Doutora Mariana da Silva Gomes Mota, Técnica superior do Instituto Superior de Agronomia da Universidade de Lisboa, orientadora. 2019 Agradecimentos Em primeiro lugar quero agradecer aos meus pais pois sem eles não estaria onde estou, nem seria quem sou. Aos meus avós, em especial à minha avó Almerinda, autora da pintura da capa. Aos meus primos, Diogo e Gabriel, e ao meu afilhado Tiago, que eu vi crescer e que me fez crescer. À minha família, pelo apoio que me deram. A todos os meus amigos que me acompanharam neste percurso e que contribuíram para que fosse a pessoa que sou hoje, desde amigas da primária até amigos de mestrado. À minha educadora de infância Sónia, que me acompanha desde pequena, e a todos os professores que contribuíram para a minha formação pessoal e académica. À professora Mariana Mota que não foi apenas minha orientadora, mas também amiga, pela atenção, preocupação e paciência. Ao professor orientador Pedro Talhinhas, sempre disponível e simpático, pronto a ajudar, obrigado por todo o tempo dedicado. À Engenheira Luísa Louro e à Dª Nídia pela companhia nas horas de laboratório, pelos almoços e conversas e pelas ajudas no trabalho. Às minhas companheiras de laboratório, à Nídia, à Zita, à Leonor, à Patrícia e à Filipa e aos companheiros Rodrigo e Martim, por tornarem o ambiente leve e divertido. Ao Engenheiro Cordeiro e à Engenheira Teresa pelo auxílio na colheita e por tornarem este trabalho possível, e também ao INIAV. Por fim ao Instituto Superior de Agronomia e a todas as escolas e instituições que tornaram possível o meu percurso académico. Este trabalho teve apoio financeiro, logístico e institucional do LEAF (Centro de Investigação em Agronomia, Alimentos, Ambiente e Paisagem), suportado pela Fundação para a Ciência e a Tecnologia (UID/AGR/04129/2013). i Resumo Em Portugal, a cultivar de oliveira Galega Vulgar é a mais cultivada e a mais apreciada pelas características sensoriais do azeite. Contudo, apresenta diversos obstáculos agronómicos, incluindo dificuldades na propagação vegetativa e difícil aptidão para a cultura em modo super-intensivo, sendo por isso muito importante em termos de programas de melhoramento de plantas. De modo a ultrapassar estas barreiras pretende-se avaliar a variabilidade genómica e genética de um banco de germoplasma constituído por 56 genótipos de ‘Galega Vulgar’ provenientes de diversas zonas do país, presentes no Pólo de Elvas do INIAV (Instituto Nacional de Investigação Agrária e Veterinária). A variabilidade genómica foi avaliada em todas as amostras através de citometria de fluxo sendo realizadas seis repetições para cada uma. Os valores médios de conteúdo nuclear obtidos para cada amostra variaram entre 3,20 pg/2C e 3,03 pg/2C representando uma variação de 5,31%. Estes resultados foram interpretados como representantes de uma variabilidade citogenómica entre as amostras, sendo que esta toma o seu valor máximo entre as amostras 1482 e 2092. A variabilidade genética foi avaliada através de marcadores moleculares após a aplicação de 10 “primers”, cinco ISSR (9MS, (GAC)5, (GTG)5, (GACA)4 e (GTGC)4) e cinco RAPD (OPC-04, OPC-07, OPC-08, OPC-10 e OPC-13) em 10 amostras pré-selecionadas com base nos resultados da avaliação genómica. A percentagem de polimorfismo foi semelhante para os dois tipos de “primers” sendo de 62,03% para os RAPD e 57,14% para os ISSR. O dendrograma gerado pelas matrizes resultantes da avaliação dos géis demonstrou que as amostra mais filogeneticamente diferentes são a 1053 e a 1482. Em conclusão a diversidade genómica e genética intra-varietal da ‘Galega Vulgar’ foi provada, sendo que estudos futuros poderão avaliar geneticamente as restantes amostras para descobrir novas dimensões de variabilidade. Palavras-chave: ‘Galega Vulgar’, RAPD, ISSR, citometria de fluxo, diversidade. ii Abstract In Portugal the olive cultivar Galega Vulgar is the most cultivated one and the most cherished for its sensory oil properties. However, it does present some agronomic barriers, including vegetative propagation problems and weak super-intensive performance, thus being of high relevance in plant breeding programs. In order to overcome these difficulties, the main goals are to analyse the genomic and genetic variability of a germplasm bank of 56 ‘Galega Vulgar’ genotypes from different areas of Portugal, currently at the INIAV (Instituto Nacional de Investigação Agrária e Veterinária) Elvas headquarters. Genomic variability was analysed in every accession through the means of flow cytometry, and for each genotype six samples were prepared and tested. The mean values of nuclear content obtained for each genotype varied between 3,20 pg/2C and 3,03 pg/2C, representing a variation of 5,31%. The interpretation of these results lead to the acknowledgement of genomic variability between the samples, peaking between the 1482 and the 2092 genotypes. The genetic variability was evaluated through molecular markers, using 10 primers, where five of them were ISSR (9MS, (GAC)5, (GTG)5, (GACA)4 e (GTGC)4) and the other five were RAPD (OPC-04, OPC-07, OPC-08, OPC-10 e OPC-13). These primers were applied in 10 genotypes which underwent a pre-selection based on the results of flow cytometry. The polymorphism percentage was similar for both types of primers, taking on the values of 62,03% for RAPD and 57,14% for ISSR. The dendrogram, which was the result of the binary band matrix from agarose electroforesis, revealed that the most distinct genotypes were 1053 and 1482. In conclusion, the genomic and genetic intra-cultivar diversity of ‘Galega Vulgar’ was proved, although future studies could lead on genetic evaluations of the remaining genotypes in order to unravel new variability dimensions. Keywords: ‘Galega Vulgar’, RAPD, ISSR, flow cytometry, diversity. iii Índice 1. Revisão Bibliográfica ................................................................................................................. 3 1.1. A oliveira .............................................................................................................................. 3 1.1.2. A cultivar Galega Vulgar ............................................................................................... 4 1.2. Método de avaliação de diversidade genética - marcadores moleculares ......................... 6 1.2.1. “Primers” mais utilizados em Olea europaea ................................................................ 6 1.2.2. RAPD ............................................................................................................................ 7 1.2.2.1. Vantagens e desvantagens dos RAPDs ................................................................. 7 1.2.3. ISSR .............................................................................................................................. 9 1.2.3.1. Vantagens e desvantagens dos ISSRs ................................................................ 11 1.3. Variabilidade inter-cultivar ................................................................................................. 11 1.4. Variabilidade intra-cultivar da Galega Vulgar .................................................................... 13 1.5. Método de avaliação de diversidade citogenómica - citometria de fluxo.......................... 14 1.5.1. Definição e funcionamento.......................................................................................... 14 1.5.2. A citometria de fluxo nas plantas ................................................................................ 17 1.5.3. Vantagens e desvantagens......................................................................................... 21 1.5.4. A citometria de fluxo e o método de Feulgen em Olea europaea .............................. 22 2. Material e métodos .................................................................................................................. 25 2.1. Material vegetal ................................................................................................................. 25 2.1.2. Conservação ............................................................................................................... 27 2.1.2.1. Citometria de Fluxo ............................................................................................... 27 2.1.2.2. Marcadores moleculares ....................................................................................... 27 2.2.1. Citometria de Fluxo ..................................................................................................... 28 2.2.1.1. Recolha de Dados ................................................................................................. 32 2.2.1.2. Tratamento estatístico ........................................................................................... 33 2.2.2. Marcadores moleculares ............................................................................................
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