A migração da citricultura paulista das regiões norte e central para a região sudoeste, tem submetido a cultura à clima mais chuvoso e, consequentemente, à maior ocorrência da podridão floral dos citros (PFC). A PFC, cujo agente causal é C. acutatum, induz a abscisão de frutos jovens e pode causar perdas de 100%. Alguns componentes do monociclo dessa doença ainda encontram-se indefinidos. Os processos de sobrevivência e colonização têm sido sustentados por evidências oriundas de experimentos não conclusivos. Até o momento, os métodos de detecção não têm sido eficazes para detectar o patógeno em folhas assintomáticas de citros, além de não serem capazes de quantificá-lo. Diante dessas lacunas, esse trabalho buscou avaliar o período de sobrevivência do patógeno na superfície de folhas assintomáticas entre as floradas; verificar se o patógeno coloniza os tecidos das folhas de citros; estabelecer um método sensível para detecção e quantificação de C. acutatum e C. gloeosporioides na superfície foliar e; monitorar quantitativamente os patógenos em campos de produção de citros no Estado de São Paulo. A sobrevivência do patógeno foi avaliada em condições controladas, casa de vegetação e condições ambientais. Foi notado que o patógeno sobreviveu até sete meses em condições controladas, até dez meses em casa de vegetação e até seis meses sob condições ambientais. Chuvas regulares associadas ao molhamento foliar prolongado, auxiliaram na manutenção e sobrevivência do inóculo na superfície foliar. O processo de colonização do patógeno foi investigado por microscopia de luz, transmissão e confocal. Não foi observada colonização do patógeno em tecidos foliares. O peg de penetração proveniente dos apressórios penetrou a cutícula e ficou restrito numa camada péctica, acima da parede periclinal da epiderme. Diferentes métodos foram avaliados para a detecção e quantificação de C. acutatum e C. gloeosporioides em folhas de citros. Foi desenvolvida uma PCR em tempo real (qPCR) e uma PCR multiplex em tempo real (qPCR multiplex), ambas específicas e altamente sensíveis à detecção dos patógenos. O DNA da planta não influenciou na amplificação da qPCR. As qPCR foram muito mais sensíveis que a PCR convencional e Nested-PCR. Distintos métodos para processamento das amostras e para obtenção do DNA foram avaliados. O método do congelamento e a maceração do tecido foliar, foram os mais eficazes para o processamento da amostras e, a extração do DNA (CTAB ou Qiagen) foi melhor método de obtenção do DNA para quantificação dos patógenos. O spot e extração do DNA foram validados para a detecção dos patógenos tanto para a qPCR quanto para a qPCR multiplex. O monitoramento dos patógenos foi realizado em áreas localizadas em duas distintas regiões do Estado de São Paulo. Foi notado considerável aumento na quantidade de inóculo quando ocorreram chuvas regulares, com muitos dias com chuvas. O inóculo se concentrou no interior da copa em épocas com baixo volume de chuvas. A quantidade de inóculo de C. acutatum na área localizada em Santa Cruz do Rio Pardo foi consideravelmente maior que na área de Barretos e ambas apresentaram quantidades similares de C. gloeosporioides. / The migration of the citrus culture in São Paulo State from the northern and central regions to the south-western region submitted the culture to a wetter climate and, consequently, a higher incidence of postbloom fruit drop (PFD). PFD, whose causal agents are C. acutatum and C. gloeosporioides, induces the abscission of young fruits and can causes loss of 100% of the culture. Some monocycle components of this disease are still undetermined. The survival and colonization processes are supported by evidences from inconclusive experiments. To date, the detection methods have not been effective in detecting and quantifying the pathogen in asymptomatic citrus leaves. This study aimed to assess the survival period of C. acutatum on the surface of asymptomatic leaves between the flowering seasons, to verify whether the pathogen colonizes the tissues of citrus leaves of citrus, to establish a sensitive method for detection and quantification of C. acutatum and C. gloeosporioides on leaves surface and to monitor quantitatively the pathogens in citrus orchards in the State of São Paulo in Brazil. The survival of the pathogen was evaluated under controlled conditions, in a greenhouse and in environmental conditions. The pathogen survived for seven months under controlled conditions, ten months in a greenhouse and six months under environmental conditions. Regular rainfall associated with the prolonged leaf wetness favored the maintenance and survival of the inoculum on the leaf surface. The colonization process of the pathogen was investigated by light, confocal and transmission microscopy. Pathogen colonization was not observed on leaf tissues. The penetration peg from appressoria penetrated the cuticle and was restricted in a pectic layer on the periclinal epidermal wall. Different methods were evaluated for the detection and quantification of C. acutatum and C. gloeosporioides on leaves of citrus plants. It was developed a realtime PCR (qPCR) and a real-time multiplex PCR (qPCR multiplex), both specific and highly sensitive in the detection of pathogens. The plant DNA did not influence the qPCR amplification. The qPCRs were much more sensitive than the conventional PCR and Nested-PCR. Different methods for sample processing and for DNA extraction were evaluated. The freezing and maceration methods of foliar tissue were the most effective for sample processing and the DNA extraction (CTAB or Qiagen) was best method for extracting the DNA for pathogen quantification. The Spot and DNA extraction were validated for the detection of pathogens for both qPCR and multiplex qPCR. The monitoring of pathogens was carried out in two separate regions of the State of São Paulo in Brazil. It was observed a considerable increase in the number of inoculum when regular rainfall occurred, with many rainy days. The inoculum was concentrated within the tree canopy in seasons with low volume of rainfall. The number of inoculum of C. acutatum the at region of Santa Cruz do Rio Pardo was considerably higher than in the region of Barretos and both showed similar amounts of C. gloeosporioides.

A genética molecular permite o entendimento dos mecanismos que regulam o desenvolvimento dos órgãos vegetais em resposta a fatores bióticos e abióticos. A regulação de vias gênicas é de fundamental importância no sucesso reprodutivo, evolutivo e econômico dos vegetais. Uma das modalidades de regulação é a pós transcricional através de microRNAs (miRNAs). Tratam-se de pequenos RNAs endógenos não codantes que possuem uma complementaridade quase perfeita em plantas. Em plantas, muitos genes-alvos de miRNA codificam-se para fatores de transcrição, como os genes SQUAMOSA Promoter-Binding Protein-Like (SPL/SBP). O microRNA156, conservado entre as Angiospermas, regula diversos fatores de transcrição da família SBP. O módulo miR156/SBP, denominado via da idade (AGE), atua ao longo do ciclo de vida dos vegetais regulando as transições de fase: juvenil-adulta e vegetativa-reprodutiva. O tomateiro, Solanum lycopersicum L., possui genes SlSBPs regulados pelo miR156 e com funcionalidade não esclarecida. Dentre aqueles, o gene SlSBP6c (Solyc12g038520) se destaca por possuir maior similaridade filogenética com SPL6s/SBP6s de solanáceas do que seus genes homólogos SlSBP6a e SlSBP6b o que sugere a hipótese de um possível ganho de função. Com o intuito de testar essa hipótese, o presente trabalho objetivou caracterizar funcionalmente o gene SlSBP6c. Para tanto, utilizou-se o genótipo 35S::rSlSBP6c, planta transformada de tomateiro cultivar Micro-Tom (MT) que super-expressa a versão resistente ao miR156 do gene SlSBP6, fusionada ao promotor viral 35S. Este material vegetal foi gerado pelo laboratório de Genética Molecular do Desenvolvimento Vegetal e cedido para execução desta pesquisa. O trabalho se desenvolveu em duas etapas: caracterização molecular e morfo-fisiológica. Primeiramente, os genes SlSBP6a, SlSBP6b e SlSBP6c de tomateiro foram analizados quanto a sua expressão ao longo do desenvolvimento em folhas e inflorescência. E, posteriormente, foram analisados a complexidade foliar, o tempo de florescimento, a transição do meristema vegetativo para o reprodutivo, o teor relativo de clorofila e a fotossíntese líquida. Os resultados obtidos demonstram que os genes SlSBP6a e SlSBP6c são semelhantes no padrão de expressão gênica na homologia das protéinas que o codificam, indicando similaridade funcional. A de-regulação do gene SlSBP6c leva ao aumento a complexidade foliar, o teor relativo de clorofila e a fotossíntese líquida. O atraso na transição do meristema vegetativo para o reprodutivo se evidencia por um florescimento tardio nas plantas que super-expressam o gene SlSBP6c. Os resultados demonstram que o gene SlSBP6c exerce funcionalidade no tomateiro atuando na transição de fase vegetativo-reprodutivo. / Molecular genetics allows the understanding of the mechanisms that regulate the development of plant organs in response to biotic and abiotic factors. The regulation of gene pathways is of fundamental importance in the reproductive, evolutionary and economic success of the plants. Research has been increasing the knowledge about post-transcriptional regulation through microRNAs (miRNAs). The miRNAs are small non-coding endogenous RNAs that have almost perfect complementarity in plants. Many miRNA target genes in plants are encoded by transcription factors, such as the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL / SBP) genes. MicroRNA156 is conserved among Angiosperms and regulates several transcription factors of the SBP family. The miR156 / SBP module, called the age pathway (AGE), acts throughout the life cycle of plants regulating phase transitions juvenile-adult and vegetative-reproductive. The tomato, Solanum lycopersicum L., has newly described and unintelligently regulated miR156 regulated SlSBPs. Among these, the gene SlSBP6c (Solyc12g038520) stands out for having greater phylogenetic similarity with solanaceous SBP6s than its homologous genes SlSBP6a and SlSBP6b, indicating a possible gain of function related to characteristics of this family of plants as fleshy fruits and composite leaves. In order to test this hypothesis the work aimed to characterize the SlSBP6c gene functionally. Using as a tool the 35S::rSlSBP6c genotype, transformed tomato plant micro-Tom (MT) that over-expresses the miR156 resistant version of the SlSBP6 gene, fused to the 35S viral promoter. The Laboratory of Molecular Genetics of Plant Development generated this plant material. The work was developed in two stages: molecular and morpho-physiological characterization. In the first the genes SlSBP6a, SlSBP6b and SlSBP6c of tomato were analyzed for their expression throughout the development in leaves and inflorescence. In the second, the leaf complexity, the flowering time, the transition from vegetative to the reproductive meristem, the relative chlorophyll content and the net photosynthesis were evaluated. The results obtained demonstrate that the SlSBP6a and SlSBP6c genes are very similar in the pattern of gene expression in the homology of the coding proteins, indicating a functional similarity. The SlSBP6c gene acts to increase leaf complexity, relative chlorophyll content and liquid photosynthesis. A late flowering in plants that overexpress the SlSBP6c gene evidences the delay in the transition from the vegetative to the reproductive meristem. The data set demonstrates that the SlSBP6c gene has important functionality in tomatoes acting on vegetative-reproductive phase transition.

Seleção natural divergente é a principal força que leva a diferenciação entre espécies. Nas Angiospermas, a preferencia do polinizador é considerada um dos agentes de seleção na diversificação de caracteres florais. Além disso, eles podem contribuir na delimitação de linhagens e diversificação de linhagens devido a possibilidade de reduzir fluxo gênico entre os fenótipos em simpatria, contribuindo para o isolamento reprodutivo. Nesse trabalho, nós avaliamos a interação entre padrões de variação geográfica floral e processos ecológicos associados aos polinizadores de Sinningia allagophylla. Nós amostramos traços florais e frequência de polinizadores ao longo da distribuição geográfica dessa espécie. Germinação de grãos de pólen e desenvolvimento da semente também foram amostradas para determinar a ocorrência de mecanismos de isolamento reprodutivos adicionais em ação. Nós mostramos que atributos florais de S. allagophylla apresentam um claro padrão de diferenciação geográfica. É possível classificar indivíduos dentro de três tipos florais, estando estes relacionados as vertentes de distribuição sul, noroeste e sudeste da espécie. Tipos florais intermediários ocorrem entre as regiões sudeste e o sul. Diversos grupos de polinizadores estão associados com o padrão geográficos dos atributos florais. Beija-flores estão presentes ao longo de toda a distribuição geográfica e visita tipos florais independentemente de seus atributos, mas abelhas são mais comuns nos extremos da distribuição e apresentam fidelidade a um dado tipo floral. Sobre condições de simpatria, pode haver fluxo gênico entre fenótipos distintos devido as visitas de beija-flores. No entanto, visitas seletivas de abelhas a um dos tipos florais e germinação do pólen e o desenvolvimento da semente reduzidos em indivíduos com traços florais intermediários resultam na ausência de coesão e perda dos fenótipos originais. Em conclusão, consideramos que mecanismos ecológicos associados em parte a interação planta-polinizador possam contribuir para a diferenciação das linhagens em Sinningia allagophylla. Tal informação adiciona a possibilidade de que mecanismos outros que os reconhecidamente vigentes possam contribuir para a especiação deste clado. / Divergent natural selection is a leading force of species differentiation. In flowering plants, pollinator\'s preference is considered to work as selective agent and can contribute to the diversification of floral traits. Because of pollinator role preventing gene flow under sympatric condition, they can also work as a component of reproductive isolation. Here we establish a link between geographic pattern and ecological process of differentiation in floral morphology and pollinator fidelity in Sinningia allagophylla. We sampled floral traits and pollinator frequency across the species geographic ranges. Germination of pollen grains and seed development were also sampled to determine the occurrence of additional reproductive isolation mechanisms in action. We show that floral traits of S. allagophylla present a clear pattern of geographic differentiation. It is possible to sort individuals within three floral types related to the south, southeast and northwest species range. Intermediate floral types occur between south-southeast distribution ranges. Diverse group of pollinators are associated with geographic pattern of floral traits. Hummingbirds are present across the entire geographic range and visit floral types independent of its traits, but bees are more frequent in the boundaries of distribution and show fidelity to one or other phenotype. Under sympatric condition, gene flow occurs between individuals with different floral traits due to hummingbird visits. However, bee preference of phenotypes, reduced pollen germination and seed development of individuals in the contact zones contributed to the maintenance of distinct phenotypes. In conclusion, we considered that ecological mechanisms associated with the plant-pollinator interaction are contributing partially to the differentiation of lineages in Sinningia allagophylla. This information sheds new light to the actual comprehension of speciation within this clade.

Submitted by Jadson Francisco de Jesus SILVA (jadson@uefs.br) on 2018-07-11T22:03:00Z No. of bitstreams: 1 MARIANA CHAVES - Dissertacao Mariana Chaves_ DEFINITIVA.pdf: 1571122 bytes, checksum: 655c891bef9d1aa8432a75b67d4a75e7 (MD5) / Made available in DSpace on 2018-07-11T22:03:00Z (GMT). No. of bitstreams: 1 MARIANA CHAVES - Dissertacao Mariana Chaves_ DEFINITIVA.pdf: 1571122 bytes, checksum: 655c891bef9d1aa8432a75b67d4a75e7 (MD5) Previous issue date: 2017-09-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Physalis angulata L. (Solanaceae) is a traditional medicinal species that can be found all over Brazil. The medicinal utility attributed derives from the production of complex substances of pharmacological interest as: vitaesteroids, fisalinas, flavonoids, steroids, fatty acids, carotenoids, ascorbic acid and alkaloids. For pharmaceutical production, the pharmaceutical industry requires a large amount of raw material, which would be very expensive and would generate significant impacts on the natural environment. In this way, it is necessary to develop breeding programs aimed at medicinal plants, in order to ensure the industrial production of special metabolites. In order to initiate the genetic improvement of a species it is necessary to obtain accurate information on several fundamental questions and one of them is the reproductive system. In this sense, the objective of this work was to carry out a study on the reproductive system of Physalis angulata, identifying the preferential crossing system through experiments of controlled pollinations, viability tests and pollen grain germination and floral visitors observations. All variables evaluated to estimate the preferential cross-breeding system of P. angulata indicated that this is a self-compatible species, and according to the egg pollen ratio it presents a facultative autogamous system. The dyes used to estimate pollen viability did not differ statistically. The pollen viability was high using the colorimetric method, however, there was no germination of the pollen tubes using the in vitro germination method, and further studies and adjustments of the culture medium were necessary. The hour factor influenced the viability of the pollen grains and reached 96% at 14 hours using the tetrazolium dye. The anthesis of the flowers of P. angulata lasted on average two days, they began the opening at 6 in the morning and the closure began at 14 o'clock in the field conditions; on the second day they reopen and close at the same time. In a greenhouse, the flowers opened at the same time, but the closure occurred around 5 pm and reopened the next day. The most frequent insects were of the order Hymenoptera (Apis mell?fera). / Physalis angulata L. (Solanaceae) ? uma esp?cie medicinal tradicional podendo ser encontrada em todo o territ?rio brasileiro. A utilidade medicinal atribu?da decorre da produ??o de subst?ncias complexas de interesse farmacol?gico como: vitaester?ides, fisalinas, flavon?ides, ester?ides, ?cidos graxos, caroten?ides, ?cido asc?rbico e alcal?ides. Para a produ??o de f?rmacos, a ind?stria farmac?utica requer uma grande quantidade de mat?ria-prima, o que seria muito dispendioso e geraria impactos expressivos no ambiente natural. Desse modo, faz-se necess?rio o desenvolvimento de programas de melhoramento voltados para plantas medicinais, visando assegurar a produ??o industrial de metab?licos especiais. Para iniciar o melhoramento gen?tico de uma esp?cie ? necess?rio obter informa??es precisas sobre v?rias quest?es fundamentais e uma delas ? o sistema reprodutivo. Nesse sentido, o objetivo desse trabalho foi realizar um estudo sobre o sistema reprodutivo de Physalis angulata, identificando o sistema de cruzamento preferencial por meio de experimentos de poliniza??es controladas, testes de viabilidade e germinabilidade dos gr?os de p?len e observa??es dos visitantes florais. Todas as vari?veis avaliadas para estimar o sistema preferencial de cruzamento de P. angulata indicaram que se trata de uma esp?cie auto-compat?vel, e segundo a rela??o p?len ?vulo apresenta um sistema aut?gamo facultativo. Os corantes utilizados para estimar a viabilidade pol?nica n?o diferiram estatisticamente. A viabilidade pol?nica foi alta utilizando o m?todo colorim?trico, entretanto n?o houve germina??o dos tubos pol?nicos utilizando o m?todo de germina??o in vitro, sendo necess?rios mais estudos e ajustes dos meios de cultura. O fator hora influenciou a viabilidade dos gr?os de p?len e alcan?ou 96% ?s 14 horas utilizando o corante tetraz?lio. A antese das flores de P. angulata durou em m?dia dois dias, iniciaram a abertura ?s 6 da manh? e o encerramento iniciou ?s 14 horas em condi??es de campo; no segundo dia reabrem e fecham no mesmo hor?rio. Em casa de vegeta??o, as flores abriram no mesmo hor?rio, mas o fechamento ocorreu por volta das 17 horas e reabriram no dia seguinte. Os insetos visitantes mais frequentes foram da ordem Hymenoptera (Apis mell?fera).

A genética molecular permite o entendimento dos mecanismos que regulam o desenvolvimento dos órgãos vegetais em resposta a fatores bióticos e abióticos. A regulação de vias gênicas é de fundamental importância no sucesso reprodutivo, evolutivo e econômico dos vegetais. Uma das modalidades de regulação é a pós transcricional através de microRNAs (miRNAs). Tratam-se de pequenos RNAs endógenos não codantes que possuem uma complementaridade quase perfeita em plantas. Em plantas, muitos genes-alvos de miRNA codificam-se para fatores de transcrição, como os genes SQUAMOSA Promoter-Binding Protein-Like (SPL/SBP). O microRNA156, conservado entre as Angiospermas, regula diversos fatores de transcrição da família SBP. O módulo miR156/SBP, denominado via da idade (AGE), atua ao longo do ciclo de vida dos vegetais regulando as transições de fase: juvenil-adulta e vegetativa-reprodutiva. O tomateiro, Solanum lycopersicum L., possui genes SlSBPs regulados pelo miR156 e com funcionalidade não esclarecida. Dentre aqueles, o gene SlSBP6c (Solyc12g038520) se destaca por possuir maior similaridade filogenética com SPL6s/SBP6s de solanáceas do que seus genes homólogos SlSBP6a e SlSBP6b o que sugere a hipótese de um possível ganho de função. Com o intuito de testar essa hipótese, o presente trabalho objetivou caracterizar funcionalmente o gene SlSBP6c. Para tanto, utilizou-se o genótipo 35S::rSlSBP6c, planta transformada de tomateiro cultivar Micro-Tom (MT) que super-expressa a versão resistente ao miR156 do gene SlSBP6, fusionada ao promotor viral 35S. Este material vegetal foi gerado pelo laboratório de Genética Molecular do Desenvolvimento Vegetal e cedido para execução desta pesquisa. O trabalho se desenvolveu em duas etapas: caracterização molecular e morfo-fisiológica. Primeiramente, os genes SlSBP6a, SlSBP6b e SlSBP6c de tomateiro foram analizados quanto a sua expressão ao longo do desenvolvimento em folhas e inflorescência. E, posteriormente, foram analisados a complexidade foliar, o tempo de florescimento, a transição do meristema vegetativo para o reprodutivo, o teor relativo de clorofila e a fotossíntese líquida. Os resultados obtidos demonstram que os genes SlSBP6a e SlSBP6c são semelhantes no padrão de expressão gênica na homologia das protéinas que o codificam, indicando similaridade funcional. A de-regulação do gene SlSBP6c leva ao aumento a complexidade foliar, o teor relativo de clorofila e a fotossíntese líquida. O atraso na transição do meristema vegetativo para o reprodutivo se evidencia por um florescimento tardio nas plantas que super-expressam o gene SlSBP6c. Os resultados demonstram que o gene SlSBP6c exerce funcionalidade no tomateiro atuando na transição de fase vegetativo-reprodutivo. / Molecular genetics allows the understanding of the mechanisms that regulate the development of plant organs in response to biotic and abiotic factors. The regulation of gene pathways is of fundamental importance in the reproductive, evolutionary and economic success of the plants. Research has been increasing the knowledge about post-transcriptional regulation through microRNAs (miRNAs). The miRNAs are small non-coding endogenous RNAs that have almost perfect complementarity in plants. Many miRNA target genes in plants are encoded by transcription factors, such as the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL / SBP) genes. MicroRNA156 is conserved among Angiosperms and regulates several transcription factors of the SBP family. The miR156 / SBP module, called the age pathway (AGE), acts throughout the life cycle of plants regulating phase transitions juvenile-adult and vegetative-reproductive. The tomato, Solanum lycopersicum L., has newly described and unintelligently regulated miR156 regulated SlSBPs. Among these, the gene SlSBP6c (Solyc12g038520) stands out for having greater phylogenetic similarity with solanaceous SBP6s than its homologous genes SlSBP6a and SlSBP6b, indicating a possible gain of function related to characteristics of this family of plants as fleshy fruits and composite leaves. In order to test this hypothesis the work aimed to characterize the SlSBP6c gene functionally. Using as a tool the 35S::rSlSBP6c genotype, transformed tomato plant micro-Tom (MT) that over-expresses the miR156 resistant version of the SlSBP6 gene, fused to the 35S viral promoter. The Laboratory of Molecular Genetics of Plant Development generated this plant material. The work was developed in two stages: molecular and morpho-physiological characterization. In the first the genes SlSBP6a, SlSBP6b and SlSBP6c of tomato were analyzed for their expression throughout the development in leaves and inflorescence. In the second, the leaf complexity, the flowering time, the transition from vegetative to the reproductive meristem, the relative chlorophyll content and the net photosynthesis were evaluated. The results obtained demonstrate that the SlSBP6a and SlSBP6c genes are very similar in the pattern of gene expression in the homology of the coding proteins, indicating a functional similarity. The SlSBP6c gene acts to increase leaf complexity, relative chlorophyll content and liquid photosynthesis. A late flowering in plants that overexpress the SlSBP6c gene evidences the delay in the transition from the vegetative to the reproductive meristem. The data set demonstrates that the SlSBP6c gene has important functionality in tomatoes acting on vegetative-reproductive phase transition.

Titre du projet: Recherche de marqueurs moléculaires précoces de l’anomalie florale mantled du palmier à huile Objectifs : - identifier des marqueurs d’expression de la variation somaclonale mantled par comparaison entre les transcriptomes conformes et variants.- valider la capacité de discrimination des marqueurs sélectionnés lors des stades précoces du processus in vitro. Stratégie et Méthode: Analyse transcriptomique de l’inflorescence normale de palmier à huile et construction d’un transcriptome de référence. Technique : RNAseq, séquençage Illumina.Identification des séquences et voies de régulation d’intérêt. Technique: analyse bioinformatique des données de séquençage.Comparaison entre les trancriptomes issus d’inflorescences normales vs. mantled par re-séquençage de banques obtenues ) partir de différents génotypes clonaux. Technique : Illumina.Identification des séquences présentant de manière cohérente des profils d’expression dépendant du phénotype. Technique : analyse bioinformatique des données de séquençage, analyse statistique des profils d’expression. Validation des marqueurs candidats sur des paires de régénérants normal/mantled issus de lignées clonales variées, ainsi que sur des cultures in vitro à différents stades du processus de régénération. Technique : PCR quantitative (q-PCR). / Project title : Search for early molecular markers of the mantled floral variation of oil palmObjectives : - identifying expression markers of the mantled somaclonal variation through the comparison between the true-to-type and the variant transcriptome. - assessing the discriminating power of the selected markers at early stages of the in vitro process.Strategy and Methods : Transcriptomic analysis of the normal oil palm inflorescence, construction of a reference transcriptome. Technique : RNAseq, Illumina sequencing.Identification of sequences and pathways of interest. Technique : bioinformatic analysis of sequencing data.Comparison between the normal and the mantled inflorescence transcriptome through the re-sequencing of libraries generated from several different clonal lines. Technique : Illumina. Identification of sequences displaying consistently a phenotype-dependent differential expression pattern. Technique : bioinformatic analysis of sequencing data, statistical analysis of expression patterns. Validation of candidate markers on normal/mantled regenerant palm pairs from different clonal lines and on normal-/mantled-derived in vitro cultures at various stages of the industrial regeneration process. Technique : quantitative PCR (q-PCR).

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-03-05T22:11:04Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EcologiaPolinizacaoTapereba.pdf: 1407104 bytes, checksum: 9e24bf0fcc0c51b02eecbe91c0390bfa (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-03-08T13:16:28Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EcologiaPolinizacaoTapereba.pdf: 1407104 bytes, checksum: 9e24bf0fcc0c51b02eecbe91c0390bfa (MD5) / Made available in DSpace on 2013-03-08T13:16:28Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EcologiaPolinizacaoTapereba.pdf: 1407104 bytes, checksum: 9e24bf0fcc0c51b02eecbe91c0390bfa (MD5) Previous issue date: 2009 / Este trabalho teve como objetivo caracterizar o sistema de polinização de Spondias mombin L., obtendo dados sobre a, sistema reprodutivo e os visitantes florais. Spondias mombin floresceu durante a estação seca e produziu frutos maduros na estação chuvosa. É uma espécie andromonóica, produzindo flores hermafroditas e masculinas, com números e viabilidades polínicas semelhantes. A análise da razão pólen/óvulo revelou que é uma espécie que se enquadra no tipo de sistema reprodutivo xenogâmico, necessitando da polinização cruzada realizada, neste caso, pelos insetos. Spondias mombin foi visitada por uma variedade de insetos de pequeno porte e generalistas como abelhas, moscas, vespas e besouros. As abelhas Apis mellifera Linneu, 1758, Tetragona goettei (Friese, 1900) e Trigona hyalinata (Lepeletier, 1836) apresentaram maior freqüência e, seu comportamento, tocando as partes reprodutivas das flores, sugere que são os principais polinizadores desta espécie vegetal. Além disso, a análise do pólen coletado pelos principais polinizadores demonstrou a fidelidade destes ao taperebá e o grande poder de recrutamento desta espécie. / The objective of this study was to characterize the pollination system of Spondias mombin L., through its phenology, floral biology, reproductive system and floral visitors. Spondias mombin blossomed during the dry station and produced mature fruits in the rainy station. It is an andromonoic species, that produces hermaphrodites and masculine flowers similars numbers and pollen viability. The analysis of pollen/ovule ratio showed that this species fits in the xenogamic reproductive system, and it needs the cross-pollination realized by insects. Spondias mombin was visited by a variety of small and generalistic insects as bees, flies, wasps and beetles. The bees Apis mellifera Linneu, 1758, Tetragona goettei (Friese, 1900) and Trigona hyalinata (Lepeletier, 1836) showed higher frequency and, their behavior to touch the reproductive parts of the flowers, suggest that they are the main pollinators of this plant species. In addition, the analysis of the collected pollen by the main pollinators demonstrated the fidelity of them to the taperebá and the great power of recruitment of this species.

De nombreuses populations voient leurs conditions de vie modifiées par les changements globaux. Au-delà de leurs conséquences écologiques, les changements globaux peuvent également modifier les régimes de sélection des populations. Le déclin récent des pollinisateurs pourrait altérer fortement le succès reproducteur de nombreuses populations de plantes à fleurs. Cependant, ses conséquences évolutives n'ont pas été étudiées jusqu'ici. Cette thèse traite de la possibilité d'adaptation des stratégies de reproduction des plantes face aux changements globaux et plus spécifiquement face au déclin des pollinisateurs. Cette question a été abordée en deux temps. Premièrement, l'analyse de l'abondante littérature théorique et empirique sur les systèmes de reproduction des plantes et dans un moindre mesure la construction d'un modèle d'évolution des traits floraux d'attraction ont permis de clarifier des scénarios d'évolution à court terme. Deuxièmement, une approche empirique originale a été menée, consistant à comparer directement les traits de populations ancestrales et descendantes de trois espèces annuelles, à partir de la culture en jardin commun de graines anciennes et récentes en provenance de régions où des indices de déclin des pollinisateurs existent. Cette approche permet de mettre clairement en évidence l'évolution génétique. Les données existantes indiquent que le déclin des pollinisateurs peut accentuer la limitation pollinique et par conséquent augmenter la sélection sur les traits floraux. Par ailleurs, la variation génétique substantielle dans les populations suggère que l'évolution rapide des populations de plantes est possible. L'analyse de la littérature et notre étude théorique suggèrent que l'accroissement de la capacité d'autofécondation autonome ou l'accroissement de l'attraction des pollinisateurs sont deux scénarios d'évolution possibles. Deux types de réponses évolutives ressortent de nos travaux expérimentaux. D'abord, une avancée du calendrier de floraison a été retrouvée chez les trois espèces étudiées. Ce résultat souligne le rôle de l'évolution génétique, et pas seulement de la plasticité phénotypique, dans les avancées de phénologies de printemps que l'on retrouve chez de très nombreux organismes. Ensuite, contrairement aux traits de phénologie, les traits floraux ont évolué dans des directions opposées selon les espèces. Ainsi, des traits floraux a priori plus attractifs ont évolué chez une des espèces tandis que pour une autre, des traits floraux a priori moins attractifs ont évolué, mais s'accompagnent d'une meilleure capacité d'autofécondation autonome. Cette étude confirme que les traits de reproduction des plantes peuvent évoluer en quelques décennies seulement. La possibilité du sauvetage évolutif des populations, par l'évolution rapide des traits et des stratégies de reproduction des plantes, est une perspective de recherche qui découle de ces résultats. / Global change alters life conditions of numerous populations. Beyond ecological consequences, global change can also modify selection regimes in populations. While the recent pollinator decline may specifically affect the reproductive success of flowering plants, its evolutionary consequences have not been studied yet. This thesis deals with the possibility of adaptation of plant reproductive strategies under global change and more specifically under pollinator decline. This question was addressed in two steps. First, the analysis of the extensive literature on plant mating systems, and, to a lesser extent, the construction of a model for the evolution of attractive floral traits, allowed us to clarify evolutionary scenarios at short-time scales. Second, we conducted an original empirical approach, consisting in the direct comparison of ancestral and descendant populations by re-growing old and recent seeds under identical conditions. This approach allowed us to test whether genetic evolution of reproductive traits occurred in the context of pollinator decline for three annual plant species. Data from the literature indicates that pollinator decline likely increases pollen limitation and thus selection on floral traits. Moreover, the substantial genetic variation within populations suggests that rapid evolution is possible. Increased autonomous selfing or increased pollinator attraction are two possible routes of plant adaptation to pollinator decline emerging from the analysis of the literature and from our theoretical study. Our empirical work brings out two types of evolutionary trends. Firstly, earlier flowering phenology was found in all three studied species. This result shows that genetic evolution, not only phenotypic plasticity; certainly contribute to the spring phenological advancements reported for numerous species. Secondly, unlike phenological traits, floral traits evolved in opposite directions depending on the species. Showy floral traits evolved in a species while joint evolution of autonomous selfing with a reduction of floral attractiveness seemed to evolve in another species. This study shows that plant reproductive traits can evolve in a few decades. Whether or not rapid evolution of plant reproductive traits can act as an evolutionary rescue for threatened populations is a research question that arises from these results.

<p>Natural selection mediated by pollinators has influenced the evolution of floral diversity of the flowering plants (angiosperms). The scope of this thesis was to study: 1) phenotypic selection, 2) mating systems, and 3) floral shifts involved in plant speciation. Model plant species were <i>Platanthera bifolia</i> and <i>P. chlorantha</i> (Orchidaceae). These orchids are moth-pollinated, strictly co-sexual (bisexual flowers), and produce a spike that displays 10-20 white flowers.</p><p>I explored the influence of characters on plant fitness by using multiple linear regressions. Pollen removal (male fitness) and fruit set (female fitness) increased with more flowers per plant in three <i>P. bifolia</i> populations. There was selection towards longer spurs in a dry year when average spur length was shorter than in normal-wet years. Female function was sensitive to drought, which enabled an application of the male function hypothesis of floral evolution (Bateman's principle). The results show that selection may vary between populations, years, and sex-functions.</p><p>I examined inbreeding by estimating levels of geitonogamy (self-pollination between flowers of an individual) with an emasculation method in two <i>P. bifolia</i> populations. Geitonogamy did not vary with inflorescence size. Levels of geitonogamy was 20-40% in the smaller, but non-significant in the larger population. This may relate to lower number of possible mates and pollinator activity in the smaller population.</p><p><i>Platanthera bifolia</i> exhibits the ancestral character state of tongue-attachment of pollinia on the pollinator. Its close relative <i>P. chlorantha</i> attaches its pollinia onto the pollinator's eyes. To explore the mechanism of a floral shift, pollination efficiency and speed was compared between the two species. The results showed no differences in pollination efficiency, but <i>P. chlorantha</i> had faster pollen export and import. Efficiency of pollination in terms of speed may cause floral shifts, and thus speciation.</p>

<p><i>Echiochilon</i>, <i>Ogastemma</i> and <i>Sericostoma</i> are revised resulting in the recognition of 15 species of <i>Echiochilon</i> and one <i>Ogastemma</i> species. Several species are placed in synonymy and three new species are described, <i>E. baricum</i>, <i>E. callianthum</i> and <i>E. cyananthum</i>. The single species of <i>Sericostoma</i> is shown to be nested within <i>Echiochilon</i>.</p><p>The plastid <i>atpB</i> gene was sequenced for <i>Echiochilon</i> and <i>Ogastemma</i> from the Old World and <i>Antiphytum</i> from the New World, plus for a selection of 33 other Boraginaceae taxa. They were analysed together with selected outgroup taxa to give a framework of the tribes of Boraginoideae. The analysis gave support for establishing the new tribe Echiochileae for <i>Antiphytum</i>, <i>Echiochilon</i> and <i>Ogastemma</i>, and for merging the traditionally accepted tribe Eritrichieae with Cynoglosseae. The ITS region was sequenced for all but one species of <i>Echiochilon</i> and for representatives of <i>Antiphytum</i> and <i>Ogastemma</i>. Phylogenetic analysis of Echiochilon revealed that the strongly zygomorphic-flowered species form a paraphyletic group. The morphological data gave results fairly congruent with the ITS phylogeny.</p><p>Biogeographic interpretations of the ITS and <i>atpB</i> phylogenies indicated a trans-Atlantic dispersal of <i>Antiphytum</i> as the most plausible explanation to the Old/New World disjunction. Analyses using DIVA (Dispersal Vicariance Analysis) of the distributions of the <i>Echiochilon</i> species indicated an ancestor to <i>Echiochilon</i> with a wide distribution over northern Africa and Arabia to India.</p>