Microrganismos endofíticos são aqueles, cultiváveis ou não, que habitam o interior da planta hospedeira sem causar danos aparentes, podendo ou não apresentar estruturas externas visíveis. Esta interação endófito-planta é intrínseca a determinadas espécies de plantas e/ou bactérias. Embora bactérias do gênero Curtobacterium sejam normalmente estudadas como fitopatógenas, este grupo vem sendo isolado como endófito de diferentes espécies vegetais, tais como citros, trevo, arroz, batata, milho, olmeiro, café e álamo. Tais bactérias vem sendo estudada no controle de doenças em pepino, batata e fumo, na promoção de crescimento, interagindo com bactérias promotoras de crescimento (PGPR) e fitopatógenas. Neste contexto, o objetivo do presente estudo foi estudar a variabilidade genética e fisiológica de Curtobacterium sp. isolados endofiticamente de citros, por meio das técnicas moleculares ARDRA, RAPD, AFLP e sequenciamento do gene 16S rRNA, além de características fisiológicas (coloração da colônia, fitopatogenicidade e perfil enzimático). Além dos isolados endofíticos de citros, foram utilizados isolados endofíticos e fitopatogênicos de diversas culturas proveniente de diferentes regiões do Brasil e outros países. Embora a coloração das colônias seja uma característica altamente variável, foi observada correlação, onde os isolados endofíticos de citros apresentaram coloração variando de alaranjado a róseo, enquanto os outros isolados analisados apresentaram coloração amarelada e creme. A análise por ARDRA possibilitou a formação de 4 ribotipos (A, B, C e D), sendo que todos os fitopatógenos foram agrupados no ribotipo D. As análises de variabilidade por meio do RAPD e AFLP permitiram a separação dos isolados bacterianos em 2 grupos principais, endófitos e fitopatógenos, além de mostrar grande variabilidade dentro do grupo de isolados endofíticos de citros. De acordo com os resultados do sequenciamento do gene 16S rRNA, os isolados endofíticos de citros foram agrupados separadamente das demais espécies analisadas. Os resultados do presente estudo demonstram que os isolados endofíticos de citros apresentam genótipo divergente, sugerindo que estes isolados possam pertencer a uma nova espécie ou subespécie de Curtobacterium. / The endophytic microorganisms are those, cultivated or not, that inhabit the interior of the plant host without causing apparent damages, presenting or not visible external structures. This interaction microorganisms-plant is specific to certain species of plants and/or bacteria. Although bacteria from genus Curtobacterium genus are normally studied as phytopathogens, this group has been isolated as endophytes from several plants, such as citrus, potato, rice, maize, coffee, elm trees, red clover and poplar. Also, this bacterium has been associated to disease biocontrol in tobacco, cucumber and potato; influencing host growth or interacting with plant growth-promoting rhizobacteria and other phytopathogenic bacteria. Therefore, the aim of this study was to evaluate the genetic and physiological variability of citrus endophytic strains of Curtobacterium by physiological traits (colony color, pathogenicity and enzymatic profile), as well as molecular techniques, such as ARDRA, RAPD, AFLP and 16S rRNA gene sequencing. In the present study endophytic and phytopathogenic strains from many different plants and regions around the world were used. Although the colony color is a high variable character, correlation was observed, since phytopathogenic strains presented yellow and ivory colors, while endophytes from citrus presented orange and pink colors. Four ARDRA profile (A, B, C, D) were observed in the evaluated population, being all phytopathogenic strains include in the ribotype D. The RAPD and AFLP analysis allocated the endophytes and phytopathogens in two different major groups, besides a high variability inside citrus endophytes cluster. According to 16S rRNA gene sequence analysis, citrus endophytic were clustered in a different group of others Curtobacterium strains. The results of the present study demonstrated that the citrus endophytic strains is a divergent genotype, suggesting that citrus endophytic strains could belong to a new Curtobacterium species or subspecies.

As espécies selvagens relacionadas ao tomateiro desenvolveram-se em uma ampla gama de latitudes que compreende o sul do Equador ao norte do Chile, ocupando diferentes habitats e constituindo uma fonte de diversidade genética natural. Essa diversidade tem sido utilizada no melhoramento de tomateiro, sobretudo para introgressão de genes de resistência a patógenos e mais recentemente de genes afetando a qualidade dos frutos. Embora a variação genética natural tenha a relevância de algo que foi selecionado na natureza e que pode ter implicações evolutivas, ela ainda é pouco explorada em estudos básicos. Para o uso intensivo desse recurso é necessário lançar mão de cruzamentos e retrocruzamentos em larga escala entre as espécies selvagens e o tomateiro cultivado (Solanum lycopersicum). Para tanto, dispomos de uma cultivar miniatura de tomateiro, a cv Micro-Tom (MT), a qual pode crescer nas mesmas condições requeridas para a planta modelo Arabidopsis thaliana. Com o uso da cv MT, podemos explorar de forma adequada a variação genética natural das espécies selvagens de tomateiro, sendo que a identificação de alterações fenotípicas é muito mais evidente quando podemos visualizar a planta como um todo. Dessa forma, vários alelos ainda não conhecidos foram resgatados dessas espécies, os quais levam a fenótipos distintos daqueles encontrados nos parentais, tais como frutos cor rosa-salmão vindo de S. neorickii, frutos de superfície opaca vindo de S. chmielewskii e frutos de coloração verde escuro vindo de S. galapagense. Esse modelo parece ser de especial importância para isolar genes de efeito maior. Assim, isolamos um locus vindo de S. pimpinellifolium que aumenta o número de flores por inflorescência da cv MT de 7 para 11. Sendo o tomateiro uma planta cultivada, os alelos oriundos das espécies selvagens, além de úteis para estudos genéticos e fisiológicos, podem ser aproveitados em programas de pré-melhoramento. / Tomato wild species evolved in a wide range of latitudes, from the south of Ecuador to the north of Chile, which comprise different habitats and are a source of natural genetic variation. This diversity is commonly used in tomato breeding, especially for the introgression of genes for resistance to pathogens and, recently, for fruit quality. Although natural genetic variation usually has an adaptive significance, which means that it may have evolutionary implications, it is still poorly explored in basic research. For the intensive utilization of this resource, it is necessary to perform large scale crosses and backcrosses between wild species and cultivated tomato. Aiming to this purpose, we used the miniature tomato cultivar, cv Micro-Tom (MT), which can growth in the same minimum requirements of the Arabidopsis thaliana model. Using MT one can adequately explore the natural genetic variation of wild species with less time and space requirements. In addiction, phenotypical alterations are more evident in MT, since one can easily visualize the whole plant. Thus, alleles hitherto unknown were isolated from tomato wild related species, leading to distinct phenotypes, such as pink-salmon fruits from S. neorickii, opaque fruit surface from S. chmielewskii and dark green fruits from S. galapagense. The MT model seems to be particularly adequate for the isolation of major genes and QTLs of great effects. Thereby, we had isolated a S. pimpinellifolium locus that increases the number of flowers per inflorescence, from 7 to 11, in MT. Being the tomato a cultivated plant, the alleles obtained in the wild species, besides to be useful for genetic and physiological studies, can be used in pre-breeding programs.

Orientadores: Anete Pereira de Souza, Ronan Xavier Corrêa / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:19:58Z (GMT). No. of bitstreams: 1 CerqueiraSilva_CarlosBernardMoreno_D.pdf: 123175108 bytes, checksum: 668158b424ffa91922546922a5837ce2 (MD5) Previous issue date: 2014 / Resumo: O gênero Passiflora, cujas espécies são popularmente conhecidas como maracujazeiros, destaca-se na família Passifloraceae tanto pelo número de espécies (aproximadamente 520) quanto pela importância econômica associada à parte destas espécies. Os maracujazeiros ocorrem em diferentes países, sendo sua diversidade amplamente representada nas Américas, onde a Colômbia e o Brasil se destacam com aproximadamente 170 e 150 espécies de Passiflora, respectivamente. Economicamente os maracujazeiros despertam interesse pela beleza de suas flores, presença de princípios ativos medicinais, extração de óleos essenciais para indústria de cosméticos, produção de frutos para consumo in natura ou produção de derivados. O Brasil se destaca como maior produtor de maracujá, embora a produtividade nacional seja baixa (média de 14 T/ha-1 ano-1), quando comparada ao potencial da passicultura (± 50 T/ha -1 ano-1). Em parte essa baixa produtividade é ocasionada pela ausência de cultivares adaptadas às diferentes regiões produtoras e pela suscetibilidade das cultivares às principais enfermidades que acometem a cultura. Embora crescente, os programas de melhoramento genético do maracujazeiro apresentam resultados modestos frente às demandas existentes. Entre os obstáculos enfrentados pelos melhoristas, está a reduzida representatividade do gênero Passiflora em bancos de germoplasma, bem como a escassez de informações biológicas e agronômicas para maioria dos acessos. Assim, foi objetivo desta tese gerar informações genéticas e moleculares que contribuam para o melhoramento do maracujazeiro. Inicialmente foram construídas revisões críticas relacionadas aos avanços obtidos no melhoramento e na conservação das Passiflora com o uso de marcadores moleculares, bem como a cerca da principal doença que acomete a passicultura (virose do endurecimento dos frutos). Posteriormente foram obtidos, a partir de bibliotecas genômicas enriquecidas de microssatélites, 25, 17 e 52 novos pares de primers para P. cincinnata, P. edulis e P. setacea respectivamente, sendo observado um percentual de locos polimórficos inferior a 30% e um número médio de cinco alelos por loco. Testes de amplificação cruzada foram realizados para 14 espécies de maracujazeiros, sendo observada uma média de 70% de amplificação cruzada. De posse destes marcadores, foi estimada a distância e quantificada a estrutura genética entre 116 acessos (representados por 364 plantas distribuídas em três espécies). A partir dos dados de genotipagem e das análises estatísticas (descritivas, frequentistas e Bayesianas) foi observado baixa diversidade entre os acessos, e níveis moderado a alto de estruturação (com 0,08 ? Gst ? 0,38) e percentuais de alelos privados variando entre 20 e 40% entre os grupos sugeridos pelas estimativas Bayesianas (K=2 para P. cincinnata; K=3 ou 5, para P. edulis, e; K=2 ou 3 para P. setacea). Coleções nucleares representativas para até 100% da diversidade alélica amostrada foram sugeridas. Com base na caracterização de locos microssatélites e na avaliação de sintomas associadas à virose do endurecimento, verrugose e antracnose, observados em 36 acessos de P. edulis (amarelo e roxo), foi possível identificar grupos de acessos a serem priorizados em programas de (pré) melhoramento dedicados ao incremento de resistência em variedades cultivadas. Os resultados obtidos contribuem para o desenvolvimento dos programas de pré-melhoramento e melhoramento genético do maracujazeiro, além de auxiliarem no manejo e na conservação da variabilidade genética do gênero / Abstract: The genus Passiflora, whose species are popularly known as passion fruits, stands out in the family Passifloraceae both by the number of species (about 520) as well as the associated economic importance of some of these species. Passion fruits occur in different countries, with their diversity widely represented in the Americas, where Colombia and Brazil stand out with approximately 170 and 150 species of Passiflora, respectively. Economic interest in passion fruit emerged from the beauty of their flowers, presence of active medicinal principles, extraction of essential oils for cosmetics industry, fruits production for fresh consumption or derivatives production. Brazil is considered the largest producer of passion fruit, although national productivity is low (average 14 T/ha-1 year-1) when compared to passion fruit culture potential (± 50 T/ha -1 year-1). This low productivity is caused in part by the lack of adapted cultivars to the different production regions and due cultivars susceptibility to passion fruit major diseases. Despite an increasing rate, passion fruit breeding programs shows modest results versus existing demands. Among the obstacles faced by breeders, stand out the genus Passiflora reduced representation in germplasm banks, as well as the scarcity of biological and agronomic information for most accessions. Thus, the aim of this thesis was to generate information genetics and molecular that contributes to the passion fruit genetic breeding. Initially, critical reviews related to advances in Passiflora breeding and conservation using molecular markers, as along with information about the main disease affecting the passiculture (passion fruit woodiness disease) were presented. Novel primer pairs for P. cincinnata, P. edulis and P. setacea, in number of 25, 17 and 52 respectively, were subsequently obtained from microsatellite enriched genomic libraries, being observed a less than 30% polymorphic loci and an average number of five alleles per locus. Cross-amplification tests were performed for 14 species of passion fruit and an average of 70% cross-amplification was observed. Using these markers, genetic distance and structure were estimated among 116 accessions (represented by 364 plants distributed among three species). From genotyping and statistical analyzes data (descriptive, frequentist and Bayesian), low genetic diversity among accessions was observed. Also, Bayesian estimated suggested groups (K=2 for P. cincinnata, K=3 or 5 for P. edulis, and, K=2 or 3 for P. setacea) showed moderate to high levels of structuring (0.08 ? Gst ? 0.38) along with private alleles percentage ranging from 20 to 40%. Representative core collections ensuring up to 100% of the sampled allelic diversity were suggested. Based on microsatellite loci characterization and symptoms evaluation of associated woodiness virus, scab and anthracnose, observed in 36 accessions of P. edulis (yellow and purple), it was possible to identify groups of accessions to be prioritized in (pre) breeding programs dedicated to resistance improving in cultivars. These results contribute to the passion fruit pre-breeding and breeding programs development, and assist the genus genetic variability management and conservation / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Orientadores: Carmen Silvia Passos Lima, Maria de Lourdes Setsuko Ayrizono / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T01:40:41Z (GMT). No. of bitstreams: 1 Guimaraes_JoseLuizMiranda_D.pdf: 4302732 bytes, checksum: 2d6604590176d72460b4a606d6ba2dd6 (MD5) Previous issue date: 2012 / Resumo: O desenvolvimento de câncer colorretal (CCR) é resultado de uma complexa interação de variáveis, incluindo elementos externos, como a exposição a agentes ambientais e dietéticos, e fatores internos, de natureza somática ou hereditária. Não está estabelecido se genótipos de polimorfismos de baixa penetrância em genes relacionados com o metabolismo do ácido fólico, como o metilenotetrahidrofolato redutase (MTHFR C677T e MTHFR A1298C), o metionina sintase (MTR A2756G), o metionina sintase redutase (MTRR A66G) e o timidilato sintase (TS 2R3R), estão associados com o risco de ocorrência da doença ou com suas manifestações clínicas. Portanto, o objetivo deste estudo foi verificar se esses polimorfismos gênicos influenciam o risco de ocorrência do adenocarcinoma colorretal esporádico (ACRE) e suas manifestações clínicas e biológicas em pacientes da região sudeste do Brasil. Foram avaliados 113 pacientes com ACRE e 188 controles, considerando os aspectos clínicos como a idade, o sexo, a raça, a localização, o grau de diferenciação do tumor, o estágio e os genótipos de cada gene. Os genótipos dos polimorfismos dos genes MTHFR, MTR, MTRR e TS foram avaliados por meio da reação em cadeia da polimerase (PCR) seguida ou não por digestão enzimática. O significado estatístico das diferenças entre grupos foi calculado por meio do teste da probabilidade exata de Fisher ou qui-quadrado. As determinações dos riscos de ocorrência do ACRE, a que pacientes e controles foram submetidos, foram obtidas por meio das razões das chances (ORs) e calculadas considerando um intervalo de confiança de 95%. Portadores dos genótipos MTRR 66AG+GG, do MTHFR 1298AC+CC+677CT+TT, do MTHFR 677CT+TT+MTR 2756AG+GG, do MTHFR 1298AC+CC + 677CT+TT + MTR 2756AG+GG e MTHFR 1298AC+CC + 677CT+TT + MTRR 66AG+GG apresentaram riscos 1,99, 3,26, 2,22, 10,92 e 14,88 vezes maiores, respectivamente, de desenvolver ACRE do que os outros. Além disso, os indivíduos com o genótipo MTHFR 677CT+TT e os genótipos MTR 2756AG+GG tiveram um risco de 2,12 e 1,42 vezes maior de desenvolver ACRE com idade menor do que 50 anos. Afro-Brasileiros com o genótipo GG do polimorfismo MTRR A66G tiveram risco 1,98 vezes maior de desenvolver ACRE, e indivíduos com o genótipo MTR 2756AG+GG e os genótipos MTHFR 677CT+TT estiveram sob risco 2,11 e risco 1,62 vezes maiores de ocorrência de tumores indiferenciados e avançados, respectivamente, do que os demais. Portadores dos genótipos MTHFR 1298AC+CC e MTHFR 1298AC+CC + MTRR 66AG+GG estiveram sob riscos 1,42 e 3,07 vezes maiores de tumor no reto, respectivamente, enquanto que portadores dos genótipos MTHFR 677CT+TT e MTHFR 677CT+TT + TS 2R3R+3R3R estiveram sob riscos 1,55 e 5,39 vezes maiores de tumor de cólon, respectivamente, do que portadores dos genótipos selvagens. Estes dados sugerem que polimorfismos dos genes MTHFR, MTR, MTRR e TS, que codificam enzimas que participam do metabolismo do ácido fólico, especialmente em combinação, têm papéis consistentes para o risco de desenvolver ACRE em indivíduos da região sudeste do Brasil / Abstract: The development of colorectal cancer (CRC) is the result of a complex interaction of variables, including external factors such as exposure to environmental agents and dietary factors and internal factors, whether somatic or hereditary. Is not been established genotypes with low penetrance polymorphisms in genes related to metabolism of folic acid such as methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G) and thymidylate synthase (TS 2R3R), are associated with the risk of the disease or its clinical manifestations. Therefore, the aim of this study was to determine whether these genetic polymorphisms influence the risk of sporadic colorectal adenocarcinoma (SCA) and their clinical and biological manifestations in patients from southeast Brazil. For this, we analyzed 113 patients with SCA and 188 controls, considering the clinical aspects such as age, sex, race, location, stage, degree of tumor differentiation and the genotypes of each gene described above. The genotypes of the polymorphisms of the MTHFR, MTR, MTRR and TS were assessed by polymerase chain reaction (PCR) and enzyme digestion. The statistical significance of differences between groups was calculated using the probability test of Fisher's exact or chi-square. Determination of the risks of SCA, the patients and controls were submitted, was obtained through the odds ratios (ORs) and calculated assuming a range of 95%. Carriers of the MTRR 66AG + GG, the MTHFR 1298AC+CC + 677CT+TT, the MTHFR 677CT+TT + MTR 2756AG+GG, the MTHFR 1298AC+CC + 677CT+TT + MTR 2756AG+GG, and the MTHFR 1298AC+CC + 677CT+TT + MTRR 66AG+GG genotypes had a 1.99, a 3.26, a 2.22, a 10.92 and a 14.88-fold increased risks for SCA than others, respectively. In addition, individuals with the MTHFR 677CT+TT and the MTR 2756AG+GG genotypes had a 2.12 and a 1.42-fold increased risks for SCA diagnosed under 50 years. African-Brazilians with the MTRR 66GG genotype had a 1.98-fold increased risk for SCA, and individuals with the MTR 2756AG+GG and the MTHFR 677CT+TT genotypes were under a 2.11 and a 1.62-fold increased risks for undifferentiated and advanced tumors, respectively, than others. Carriers of the MTHFR 1298AC+CC and the MTHFR 1298AC+CC + MTRR 66AG+GG genotypes had a 1.42 and a 3.07-fold increased risks for rectal tumor, respectively, while carriers of the MTHFR 677CT+TT and the MTHFR 677CT+TT + TS 2R3R+3R3R genotypes had a 1.55 and a 5.39-fold increased risks for colon tumor, respectively, than carriers of the wild genotypes. This data suggest that polymorphisms of genes MTHFR, MTR, MTRR and TS, which encode folate-dependent enzymes, particularly in combination, have consistent roles for SCA risk in southeastern Brazil / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências

>Magister Scientiae - MSc / Recent advances in the field of plant genetics and application of molecular technologies has lead to greater understanding of various crop genomes and their organization.The applications of these techniques include molecular markers which have been used to examine DNA variation within crop species. This allows for the creation of further genetic variation for new and favourable traits.Molecular markers or DNA markers are short fragments of DNA that can be used to locate desirable genetic traits in the genome or show specific genetic differences. The Maloideae subfamily includes fruit species such as pear. Pears (Pyrus communis L.) are large edible fruit that are grown in cool climates, native to coastal regions in Africa, Asia and Europe. The external appearance of this fruit plays a vital role on its rate of sale potential. Thus it is important for the appearances of the pear to meet the expectations of the consumer.External factors affecting the appearance of fruit, such as shape and colour, can have a large influence on the consumer’s first impression and opinion of what the fruit may taste like(Jaeger and MacFie, et al., 2001). The South African pear industry is the fourth largest in the fruit industry after apple, citrus and grape, exporting 3.8% to Europe (Ferrandi, et al., 2005).Increase in production and export of the pear is dependant on the variety of cultivars with desired traits. New cultivars, especially ranges of new cultivars, with harvest dates from early to late in the season, can fill gaps in the marketing strategy of exporters and in the local markets (Human, et al., 2005) Therefore, development of molecular markers allows for their possible use in maker-assisted selection and for the construction of a genetic linkage map thus leading to the location of favourable traits and ultimately the improvement of the quality of the pear.In this study high throughput genomic DNA extractions were performed. The Cetyltrimethyl ammonium bromide (CTAB) method was employed as the results proved to be most promising. Furthermore the screenings of molecular markers were conducted in order to obtain DNA variation. Molecular markers were used to locate specific genetic differences.Multiplexing PCR was conducted using fluorescent primers for further screening and results proved to be useful as many variations could be observed.

Orientadores: Maria Imaculada Zucchi, Ricardo Ribeiro Rodrigues / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T12:21:20Z (GMT). No. of bitstreams: 1 Schwarcz_KaiserDias_D.pdf: 5972760 bytes, checksum: 6089d106796adfd5fab684651bfb298e (MD5) Previous issue date: 2014 / Resumo: A degradação ecológica e o desflorestamento são processos que se iniciaram há muito tempo e cuja história confunde-se com a da agricultura. A Mata Atlântica é a segunda maior floresta tropical em ocorrência e importância na América do Sul, possuindo grande diversidade biológica e altos níveis de endemismo. A ocupação desordenada da Mata Atlântica causou sua redução a 11,26% de sua área original, com distribuição de forma fragmentada pelo território brasileiro. A destruição da Mata Atlântica tem resultado na eliminação de muitas populações e, potencialmente, na erosão da diversidade genética de diversas espécies. Essa combinação de alto endemismo e forte ameaça de extinção, faz com que a Mata Atlântica seja considerada um hotspot para a conservação. Nas últimas décadas a recuperação de ecossistemas degradados recebeu a atenção da comunidade científica, dando origem ao campo do conhecimento chamado Ecologia da Restauração, que se dedica aos estudos teóricos dos princípios, práticas, resultados e conseqüências de projetos de restauração. O estudo e monitoramento de áreas de restauração florestal é essencial para melhorar as técnicas de restauração em ecossistemas tropicais e subtropicais. Para que uma determinada espécie se perpetue em uma área em processo de restauração, é preciso que a mesma desenvolva todo o seu ciclo de vida e que gerem descendentes capazes de se desenvolver a ponto de substituir as árvores mães quando as mesmas entrarem em senescência. Por isso há a necessidade de se estudar a variabilidade genética de populações arbóreas dentro de áreas de floresta restaurada, assim como a ocorrência e efetividade do fluxo gênico entre estas áreas e os fragmentos de seu entorno. Neste trabalho, estudamos a variabilidade genética de Myroxylon peruiferum L. f., em duas diferentes áreas de restauração florestal e em duas áreas de remanescentes naturais de Floresta Estacional Semidecidual. Nossos resultados indicam que as restaurações florestais de Cosmópolis e Iracemápolis conservam diversidades genéticas HE e alélicas semelhantes às de remanescentes naturais. A principal diferença entre áreas naturais e restauradas foi a menor riqueza de alelos endêmicos nestas últimas o que é um efeito de amostragem que favorece a perda de alelos raros. A área de restauração florestal mais antiga em Cosmópolis apresentou uma estruturação genética espacial compatível com a de áreas naturais. O mesmo não ocorreu com a restauração mais recente de Iracemápolis. Observou-se a ocorrência de estruturação genética local nas áreas naturais e na área de restauração mais antiga e indícios de fluxo gênico entre os áreas nativas e restauradas. Um estudo adicional do efeito de amostragem sobre as freqüências alélicas demonstrou o fenômeno de perda de alelos com baixa freqüência em eventos de amostragem. O mesmo trabalho indicou que uma amostra de cerca de 30 indivíduos é capaz de representar adequadamente alelos com freqüências acima de 0,05; sendo este um bom número a se considerar na seleção de matrizes para fornecimento de mudas para restauração florestal / Abstract: Ecological degradation and deforestation are processes that started long ago and whose history is intertwined with that of agriculture. Atlantic Forest is the second largest rainforest in occurrence and importance in South America, having great biological diversity and high levels of endemism. Disordered occupation of Atlantic Forest caused its reduction to 11.26% of the original area, with distribution in forest fragments poorly conected across the Brazilian territory. Destruction of the Atlantic Forest has resulted in the elimination of many populations and potentially the erosion of genetic diversity of several species. This combination of high endemism and strong threat of extinction causes the Atlantic Forest to be considered a hotspot for conservation. In the last decades recovery of degraded ecosystems has received attention from the scientific community giving birth to an new area of knowledge called the Restoration Ecology. The study and monitoring of areas of forest restoration is essential to improve restoration techniques in tropical and subtropical ecosystems. For a given species to perpetuate itself in an area undergoing a restoration process, it needs to develop its whole life cycle and generates progeny capable of developing to the point of replacing mothers trees when they die. Therefore there is a need to study the genetic variability of tree populations within areas of restored forest, as well as the occurrence and effectiveness of gene flow between these areas and surrounding fragments. We studied the genetic variability of Myroxylon peruiferum L. f., in two different areas of forest restoration and in two areas of natural remnants of semideciduous forest. Our results indicates that restorations in Cosmopolis and Iracemápolis conserve genetic and allelic diversity HE similar to that of natural remnants. The main difference between natural and restored areas was the lowest richness of endemic alleles which is the result of a sampling effect that favors the loss of rare alleles. The area of older forest restoration in Cosmopolis presented a spatial genetic structure consistent with natural areas. This did not occur with the newer restoration in Iracemápolis. We observed the occurrence of local genetic structure in natural areas and in the area of older restoration and evidence of gene flow between native and restored areas. An additional study about the effect of sampling size on allele frequencies showed the phenomenon of loss of low frequency alleles in sampling events. The same study found that a sample of about 30 individuals are able to adequately represent alleles with frequencies above 0.05; this is a good number to consider in selecting matrix trees to supply seedlings for forest restoration / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Para estudar a epidemiologia e evolução do novo genótipo HRSVB denominado BA, caracterizado pela duplicação de 60 nt na proteína G, analisamos 4274 amostras clínicas coletadas de crianças hospitalizadas no Hospital Universitário/USP e Hospital da Santa Casa de Misericórdia, cidade de São Paulo entre os anos de 2001 e 2009. As amostras foram submetidas a RT-PCR seguido do sequenciamento da região G2 do gene G. A duplicação de 60 nt foi detectada em 104 (28.3%) das 367 amostras analisadas. De 2001 a 2004 a circulação do genótipo BA foi baixa, seguido de 85.4% (2005), 57.6% (2006), sem circulação (2007), 10% (2008) e 75% (2009) do total de amostras sequenciadas. As sequências foram comparadas com outras BA de diversos países do mundo. A análise filogenética preliminar dividiu as amostras brasileiras em 5 grupos (BA-I, BAII, BAIII, BAIV e BAVI), sendo que a maioria das amostras de 2005 a 2009 agruparam juntas na linhagem BA-IV, estabelecendo um grupo temporal e geográfico. / In order to study the epidemiology and evolution of the new genotype of HRSVB named BA characterized with a 60-nt duplication in the G protein we analyzed 4274 clinical samples collected from children hospitalized in University Hospital/USP and Santa Casa de Misericórdia Hospital, in São Paulo city, during 2001 to 2009. The samples were subject to RT-PCR followed by sequencing of the G2 region of the G gene. The 60 nt-duplication were detected in 104 (28.3%) of 367 sequencing samples. From 2001 to 2004 the circulation of the BA genotype was low, followed by 85.4% (2005), 57.6% (2006), no circulation (2007), 10% (2008) and 75% (2009) of total sequencing samples. Sequences were compared with G sequences with the 60 nt-duplication globally sampled. Preliminary phylogenetic analysis divided Brazilian samples into five clusters (BA-I, BAII, BAIII and BAVI and BAIV), and almost all samples from 2005 to 2009 were clustered together in BA-IV lineage, establishing temporal and geographical cluster.

Western redcedar (Thuja plicata Donn ex D. Don; redcedar), an indeterminate conifer in the Cupressaceae family, is vulnerable to maladaptation in the face of climate change. Assisted gene flow is one mitigation strategy and involves human-mediated migration of populations, where the projected climate of the area of deployment matches the source climate of the population. Despite the overall projections of warmer temperatures globally, in British Columbia (B.C.), the risk of seasonal frost events will remain and therefore the potential for cold damage and mortality of redcedar exists if the newly migrated populations cannot withstand these freezing events. Knowledge of redcedar's ability to withstand freezing temperatures (cold hardiness) is therefore crucial. Redcedar, like many Cupressaceae species, produces and accumulates the purple-coloured carotenoid rhodoxanthin during the winter. This was hypothesized to be correlated with cold hardiness. Assessment of variation in overall, fall and spring cold hardiness and associated rhodoxanthin concentrations were done through repeated, seasonal freeze testing of clonal grafts originating from across the range of redcedar, and seedling progeny from a subset of these clones. Cold damage was quantified using electrolyte leakage and rhodoxanthin concentrations were quantified using high performance liquid chromatography. Cold hardiness and rhodoxanthin were individually modelled using univariate and bivariate mixed effect models with clone/family as a random effect. Model outputs were compared to climatic variables associated with clonal origin to test for climatic relationships. This study found genetic variation in cold hardiness of redcedar with weak climatic clines. This indicates that assisted gene flow of redcedar should be done on a case-by-case basis, with no need for a climatic threshold. Overall heritability of cold hardiness was 0.17 ± 0.03. Novel findings included the positive genetic correlation between fall and spring cold hardiness (0.55 ± 0.33); lack of reciprocal or parental effect for overall cold hardiness; and weak climatic relationships between cold hardiness and predominantly temperature, with the strongest correlation between number of frost-free days in January (0.38, p < 0.01) in the location of origin and cold hardiness. All findings related to rhodoxanthin were novel. Rhodoxanthin varied with family/provenance and season with heritabilities of 0.30 ± 0.09 in fall, 0.42 ± 0.09 in winter and 0.28 ± 0.09 in spring. Winter and spring rhodoxanthin concentrations were phenotypically correlated (0.50, p < 0.01) and genetically correlated (0.76 ± 0.14). Surprisingly, rhodoxanthin was not detected in clonal grafts of redcedar in any season. Results also indicate that rhodoxanthin cannot be used to estimate cold hardiness. The absence of rhodoxanthin in the clonal grafts compared to the seedlings suggests that plant age impacts rhodoxanthin accumulation. / Graduate / 2021-12-14

As urban areas expand, agricultural lands become increasingly important habitat for many species. Compared to some types of agricultural land-use, ranchlands provide vast expanses of minimally modified habitat that support many threatened and endangered species. Conservation biologists can promote ecologically sound management approaches by quantifying the effects of agricultural practices on resident species. I examined the effects of pasture management, cattle grazing, and landscape characteristics on both adult and larval treefrogs in a ranchland in south-central Florida. I experimentally determined optimal deployment of artificial treefrog shelters constructed of polyvinylchloride (PVC) pipe to efficiently sample adult treefrogs (Chapter 1). Seventy-two shelters were hung on oak trees (Quercus virginiana) and cabbage palm trees (Sabal palmetto) with smooth trunks or boots (residual palm fronds), at all possible combinations of three heights (2, 3, and 4 m), four compass directions (N, S, E, and W) and two water levels (with or without 10 cm). Shelter residence was completely dominated by the Squirrel Treefrog, Hyla squirella (N = 65). Significantly fewer H. squirella were found in shelters on palms with boots than on smooth palms or oak trees (0.29 ± 0.21 [mean ± 1 SE hereinafter] versus 1.3 ± 0.21 and 1.1 ± 0.21, respectively), and shelters with water had slightly more H. squirella than those without (1.5 ± 0.19 versus 0.88 ± 0.19, respectively). Orientation and height did not affect the number of treefrogs encountered; thus, the optimal protocol is to deploy shelters on either smooth palms or oak trees, with water, at 2 m height for easy sampling, and in random compass orientations. I used this protocol to sample H. squirella in woodlots surrounding twelve wetlands and examined how time, frog stage and sex, and landscape features influenced treefrog survival, recapture and site fidelity (Chapter 2). I deployed 15 shelters/ha of woodlot within a 100 m buffer around each wetland. I sampled shelters three times during the fall breeding season, removed all shelters to force frogs to overwinter in natural refugia, and replaced shelters for the final spring sampling. During sampling periods, I sexed, measured, and individually marked each frog using visual implant elastomer (VIE) tags. I used Program MARK to build linear models that included either gender group (female, male or juvenile) or life history stage (adult, juvenile) and either time (sampling interval 1, 2, or 3) or season (fall, spring). I used the most informative model as a null model to assess effects of landscape covariates on survival and recapture. Females had higher survival than either males or juveniles, for which estimates were similar (0.867 vs 0.741 and 0.783, respectively). Survival did not vary over time, although there was some support for an effect of season, with lower survival during the final over-wintering period than in the fall intervals (relative variable importance: group = 0.730; stage = 0.134; time = 0.200; season = 0.310). Adults had higher recapture rates than juveniles (average recapture 0.214 vs 0.102), and recapture for both stages varied over time, with highest recapture in sampling interval two (relative variable importance: group = 0.262; stage = 0.514; time = 0.513; season = 0.229). Hyla squirella was extremely site loyal; no individuals moved between sampling sites, and 95% of recaptured individuals were in their original shelter. Strong terrestrial site fidelity calls into question the traditional "ponds as patches" metapopulation view of treefrog population dynamics. Area of woodlot within 250 m was the most important landscape variable in explaining additional variation in both survival and recapture. Frogs had higher survival and lower recapture in larger woodlots, indicating that intact, contiguous woodlots are higher quality habitat than more fragmented woodlots. Neither survival nor recapture varied with wetland grazing treatments or between pasture types. Finally, I experimentally assessed the effects of cattle grazing and pasture management on larval H. squirella. I selected four wetlands: two in semi-natural pastures (SN) and two in intensively managed pastures (IM). One wetland in each pasture type was fenced so that it was released from cattle grazing (R). I collected three clutches of H. squirella eggs (Clutches A, B, and C) and reared tadpoles in the laboratory until Gosner stage 25. In each wetland, I deployed a total of 50 tadpoles from each clutch into 105 L pens constructed of plastic laundry baskets and mesh window screening. Clutch significantly affected tadpole survival, with Clutch A having the highest percent survival, followed by Clutch B and finally Clutch C (41.66, 32.11 - 53.95 [mean, 95% confidence limits hereinafter]; 9.00, 6.76 - 11.88; 2.89, 2.02 - 4.01, respectively). Wetland type also affected survival, with SN wetlands supporting significantly higher survival than IM wetlands (SN-R: 53.95, 32.88 - 88.13; SN-G: 18.95, 11.30 - 31.36 vs IM-R: 7.32, 4.13 - 12.49; IM-G: 1.09, 0.29 - 2.39). Genetic variation in survival confirms the potential for H. squirella to adapt to rangeland management, but extremely low survival of some clutches indicates that few clutches may be able to survive in low quality wetlands, such as IM-G wetlands. Higher survival in SN pasture wetlands suggest this is a superior habitat and future management objectives should conserve semi-natural pastures and limit further modification of intensively managed pastures, including removing woodlots near wetlands.

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