Glucosinolates are amino acid derived secondary metabolites found in the order Capparales. It is an important class of phytochemicals involved in plant-microbe, plant-insect, plant-animal and plant-human interactions. It is, therefore, important to understand genetic mechanism of glucosinolate biosynthesis in Brassica for efficient manipulation. In this study, QTL mapping of leaf and seed glucosinolates was performed in B. rapa using two RIL populations, SR-RILs and BU-RILs. QTL mapping was performed using SR-RILs developed from a cross of Chinese cabbage and turnip rapeseed and a genetic map in B.rapa. Genetic map was developed using a total 1,579 molecular markers including 9 markers specific to glucosinolate genes, GSL-ELONG, GSL-PRO, GSL-FMOOX1, and GSL-AOP/ALK. Several QTL for progoitrin, gluconapin, glucoalyssin, glucobrassicanapin, 2-methylpropyl and 4-hydoxyglucobrassicin glucosinolates were identified with phenotype variance between 6 and 54%. Interestingly, a major QTL for 5C aliphatic glucosinolates was co-localized with a candidate Br-GSL-ELONG locus on linkage group A3, displayed co-segregation with co-dominant SCAR marker BrMAM1-1. The Br-GSL-ELONG locus was identified to regulate 20 µmole/g seed 5C glucosinolate biosynthesis. BU-RILs derived from a cross of yellow sarson and USU9 was segregated for glucoerucin, gluconapin and progoitrin 4C aliphatic glucosinolates with 4-hydoxyglucobrassicin. Phenotyping was performed in controlled and field environments for seed glucosinolates and controlled environments for leaf glucosinolates. Genetic map was developed using SRAP markers and glucosinolate gene, GSL-ELONG and GSL-PRO specific 4 loci were integrated on map. Four and three QTL were identified for seed glucoerucin and gluconapin, respectively in both environments with phenotypic variance up to 49%. Additionally, genetic manipulation of glucosinolates was performed by backcross with MAS in B. rapa. Resynthesized B. napus line was backcrossed with B. rapa genotypes, RI16, BAR6 and USU9 for replacement or introgression of glucosinolate genes, GSL-ELONG- and GSL-PRO+. In RI16 genotype, 15 to 25 µmole/g seed 5C glucosinolates reduced in 15 BC3F2 lines those were positive with GSL-ELONG- marker and negative with the A-genome and gene specific marker BrMAM1-1. This suggests that the functional allele has replaced by non-functional from B. oleracea. GSL-PRO+ positive backcross lines in RI16 genotype displayed sinigrin 3C aliphatic glucosinolate in B. rapa. This suggests introgression of GSL-PRO+ in B. rapa.

Glucosinolates are amino acid derived secondary metabolites found in the order Capparales. It is an important class of phytochemicals involved in plant-microbe, plant-insect, plant-animal and plant-human interactions. It is, therefore, important to understand genetic mechanism of glucosinolate biosynthesis in Brassica for efficient manipulation. In this study, QTL mapping of leaf and seed glucosinolates was performed in B. rapa using two RIL populations, SR-RILs and BU-RILs. QTL mapping was performed using SR-RILs developed from a cross of Chinese cabbage and turnip rapeseed and a genetic map in B.rapa. Genetic map was developed using a total 1,579 molecular markers including 9 markers specific to glucosinolate genes, GSL-ELONG, GSL-PRO, GSL-FMOOX1, and GSL-AOP/ALK. Several QTL for progoitrin, gluconapin, glucoalyssin, glucobrassicanapin, 2-methylpropyl and 4-hydoxyglucobrassicin glucosinolates were identified with phenotype variance between 6 and 54%. Interestingly, a major QTL for 5C aliphatic glucosinolates was co-localized with a candidate Br-GSL-ELONG locus on linkage group A3, displayed co-segregation with co-dominant SCAR marker BrMAM1-1. The Br-GSL-ELONG locus was identified to regulate 20 µmole/g seed 5C glucosinolate biosynthesis. BU-RILs derived from a cross of yellow sarson and USU9 was segregated for glucoerucin, gluconapin and progoitrin 4C aliphatic glucosinolates with 4-hydoxyglucobrassicin. Phenotyping was performed in controlled and field environments for seed glucosinolates and controlled environments for leaf glucosinolates. Genetic map was developed using SRAP markers and glucosinolate gene, GSL-ELONG and GSL-PRO specific 4 loci were integrated on map. Four and three QTL were identified for seed glucoerucin and gluconapin, respectively in both environments with phenotypic variance up to 49%. Additionally, genetic manipulation of glucosinolates was performed by backcross with MAS in B. rapa. Resynthesized B. napus line was backcrossed with B. rapa genotypes, RI16, BAR6 and USU9 for replacement or introgression of glucosinolate genes, GSL-ELONG- and GSL-PRO+. In RI16 genotype, 15 to 25 µmole/g seed 5C glucosinolates reduced in 15 BC3F2 lines those were positive with GSL-ELONG- marker and negative with the A-genome and gene specific marker BrMAM1-1. This suggests that the functional allele has replaced by non-functional from B. oleracea. GSL-PRO+ positive backcross lines in RI16 genotype displayed sinigrin 3C aliphatic glucosinolate in B. rapa. This suggests introgression of GSL-PRO+ in B. rapa.

Doctor of Philosophy / Before the early 2000s, many producers were heavily selecting for production traits without accounting for the negative relationship between many production traits and fertility. The large decrease in fertility that occurred as a result of these selection practices put a heavy burden on producers. Replacement heifer development is the third largest cost incurred by producers, behind feed and labor. Consequently, the failure of a heifer to conceive in her first breeding season translates directly into financial loss for the producer and lasting consequences on the animal's longevity and performance in subsequent breeding seasons. To make improvements in a trait, or traits, of interest, producers often selectively breed two animals with desirable characteristics. However, the complex nature of fertility traits limits the effectiveness of this method. As a result, researchers have been attempting to identify biological molecules whose abundance differs between cattle of differing fertility potential, termed molecular markers, that could be used to identify superior cattle earlier and more accurately. While a good amount of research has been conducted in mature cows and in a laboratory setting, very few studies have attempted to identify molecular markers of fertility in heifers. Therefore, the objective of this work was to identify different biological molecules (genomic variants, genes, proteins, and metabolites) whose abundance differed between heifers with varying fertility potential. Investigation into DNA variations led to the identification of three variants that were associated with fertility, 16 variants that were associated with health, and 29 variants that were associated with both health and fertility. Given that some variants can impact a trait by changing gene expression, we attempted to identify variations in RNA that were having this effect on heifer fertility. Although some variants were found to influence gene expression, we were unable to correlate these changes with fertility differences. However, in a different study, we were able to identify two genes (APMAP and DNAI7), as well as one protein (alpha-ketoglutarate-dependent-dioxygenase FTO), that differed significantly between fertile and sub-fertile heifers. Importantly, the results of this study allowed us to create a biological profile that was capable of accurately distinguishing 21/22 heifers based on their fertility potential. Finally, investigation of the metabolite profile revealed one metabolite (2-dehydro-D-gluconate) that was differentially abundant between fertile and sub-fertile heifers. Overall, this work sheds light on the complex nature of heifer fertility and provides several potential molecular that could be used to distinguish between heifers of varying reproductive potential.

L'évolution des plantes à fleurs est remarquable par la vitesse et l'étendue de la diversification qui l'accompagne. La variabilité génétique qui découle de cette diversification pourrait provenir de nombreux processus. Parmi ceux-ci, les éléments transposables (ET) ont été considérés comme l'un des agents les plus importants. Les ET peuvent constituer de grandes proportions des génomes de plantes (>80%) et joueraient un rôle important dans l'établissement de la diversité génétique. Notre travail contribue à la compréhension du rôle des ET dans l'évolution des génomes d'espèces du genre Coffea, genre qui appartient à la famille des Rubiacées. Plus précisément, l'objectif de l'étude était d'étudier l'impact d'ET actifs sur le génome de certaines espèces du genre Coffea. Les données obtenues ont été divisées en deux parties qui composent les deux chapitres de la thèse. Dans le premier chapitre, dix rétrotransposons (LTR-RTs) ont été identifiés et annotés dans le génome de C. canephora. Le profil de leur polymorphisme d'insertion a été analysé à l'aide des approches IRAP et REMAP chez plusieurs génotypes des espèces C. canephora (18) et C. eugenioides ( 5), qui sont les espèces parentales de l'allotétraploïde, C. arabica (21). Les résultats soulignent la dynamique évolutive de ces éléments dans ces espèces, ainsi que leur transmission. Nos résultats montrent également des modifications de la structure du génome de l'hybride. Dans la seconde partie est constitué par une analyse complète de la répartition et de l'évolution d'un rétrotransposon particulier : Copia25. Ces analyses, menées in silico et in vitro, ont montré que ce LTR-RT est largement distribué au sein de la famille des Rubiacées et qu'il est également présent chez d'autres espèces proches ou bien plus éloignées phylogénétiquement (Asterides, Rosides ou Monocotylédones). Une situation particulière est constituée par la relation étroite qui existe entre les séquences de Copia25 identifiées dans le genre Musa, monocotylédone, et dans le genre Ixora, dicotylédones de la famille des Rubiacées. Nos résultats révèlent la complexité de la dynamique évolutive de Copia25 chez les angiospermes qui implique plusieurs processus incluant un mécanisme de conservation de la séquence, un « turnover » rapide, des pertes stochastiques et le transfert horizontal.L'article présenté dans le dernier chapitre a été accepté avec modifications par la revue « Plant Molecular Biology ». il est actuellement en cours de révision pour être renvoyé prochainement en vue de son acceptation définitive. / The evolutionary history of the flowering plants is remarkable for its rapid and extensive diversification. The background of the genetic variability for this diversification is originated by numerous processes, among them the transposable elements (TEs) have been considered as one of the most important agents. TEs may compose large amounts of plant genomes and might play important roles in the promotion of genetic diversity. Our study contributes for the understanding of TEs impact on the genome of Coffea species. Coffea is a genus that belongs to the Rubiaceae family. The goal of the study was to investigate the occurrence and diversity of active TEs in Coffea species and the data obtained were divided in two parts that compose the two chapters of the thesis. In the first chapter, ten retrotransposons with LTRs (LTR-RTs) were annotated in the C. canephora genome and had their insertion polymorphism profile analyzed, using the IRAP (inter-retrotransposon-amplified polymorphism) and REMAP (Retrotransposon-microsatellite amplified polymorphism) methods, in genotypes the progenitor species, C. canephora (18) and C. eugenioides (5), and the allotetraploid hybrid, C. arabica. The results outline the evolutionary dynamics of these elements in the species, as well as their inheritance. Our results also suggest the occurrence of genomic structural changes mediated by the LTR-RTs in the hybrid. And, the second part constitutes a wide analysis of the distribution and evolution of a particular LTR-RT, Copia25. In silico and in vitro analyses showed that Copia25 is widely distributed among the Rubiaceae family and that it is also present in other distantly related species belonging to asterids, rosids and monocots. A particular situation is the closer relationship between the Copia25 sequences of Musa, a monocot, and Ixora, a dicot species (Rubiaceae). Our results disclose the complexity of the evolutionary dynamics of Copia25 in angiosperm involving several processes including sequence conservation, rapid turnover, stochastic losses and horizontal transfer. The article presented in the last chapter was accepted for publication after modifications in “Plant Molecular Biology”, it is at the present under correction to be soon sent back for final approval.

O cerrado brasileiro é um dos biomas de maior riqueza e endemismo de plantas, mas com alto índice de desmatamento nas últimas décadas, o que resultou na fragmentação dos habtats e na ameaça de extinção de centenas de espécies vegetais. Dentre estas espécies ameaçadas está Anacardium humile, conhecida como cajuzinho-do-campo, uma planta caméfita de ampla distribuição pelo país, servindo de alimento para o homem e para alguns animais e apresentando propriedades medicinais. Estudos sobre a estrutura de populações de Anacardium humile são escassos na literatura, e seu entendimento é fundamental para a preservação e conservação da espécie. Dessa forma, o presente trabalho teve dois objetivos: 1) em duas fitofisionomias de cerrado distintas, estimar a abundância de ramets da espécie, seu padrão de distribuição espacial em macro e microescala, e a influencia da porcentagem de abertura do dossel na determinação deste padrão; 2) a construção de uma biblioteca genômica enriquecida de microssatélites para a espécie e o desenho de primers a partir desta biblioteca, a fim de isolar locos com potencial para uso como marcadores genéticos. Com relação ao primeiro objetivo, três parcelas de 0,5 ha, divididas em 200 subparcelas, foram instaladas (duas num fragmento de cerrado típico à cerradão e uma num fragmento de cerrado aberto), e todos os ramets da espécie foram amostrados por contagem, sendo discriminados os com e sem ataque de Contarinia sp. (Diptera: Cecidomyiidae). Fotografias foram tiradas do centro de cada parcela para determinar a porcentagem de abertura do dossel. A abundância de ramets foi maior nas áreas mais abertas, mas a incidência de ataque de Contarinia sp foi maior no fragmento mais fechado. As análises de macroescala (Índice de Dispersão) e de microescala (Autocorrelação Espacial) mostraram que os ramets da espécie apresentam padrão agregado em ambas as áreas, mas que essa agregação, em microescala, não está relacionada à porcentagem de abertura do dossel, embora haja diferenças significativas para este ultimo fator entre os grids, indicando que o padrão é devido à sua forma de vida. Já em relação ao segundo objetivo, a construção da biblioteca genômica enriquecida de microssatélites resultou em 180 clones, dos quais 84 foram seqüenciados, sendo detectadas 23 sequências contendo microssatélites, o que representa um enriquecimento da biblioteca de 27,38%. Foram desenhados 15 pares de primers dentre os 34 microssatélites obtidos, mas apenas 7 pares amplificaram. Destes, cinco pares foram visualizados em acrilamida, e um loco polimórfico foi observado. O número de clones obtidos está dentro do observado em estudos com outras espécies da família Anacardiaceae, mostrando a eficiência do protocolo. Problemas com a otimização de reagentes podem ter impedido a amplificação de alguns primers, uma vez que os procedimentos para o desenho dos mesmos foram adequados. Os resultados de polimorfismo são preliminares, devido ao número baixo de indivíduos avaliados. Ao menos um loco apresenta potencial como marcador genético. / Brazilian cerrado is one of the richest and plants endemism biomes, but with a high deforestation in the last decades, resulted in habitats fragmentation and extinction threat of hundreds of plant species. Among these threatened species is Anacardium humile, known as cajuzinho-do-campo, a camephyth plant with a wide distribution through the country, which serves as aliment to man and some animals and presents some medical properties. Studies about Anacardium humiles populations structure are very scarce in the literature, and its understanding is fundamental to the preservation and conservation of the species. Thus, the present work had two objectives: 1) in two distincts cerrado plant physiognomies, estimate the abundance of species ramets, its spatial distribution pattern in macro and microscale, and the influence of canopy openness percentage in the determination of this pattern; 2) construction of a genomic enriched library with microsatellites to the species and primers design from this library, to isolate loci with potential to be used as genetic markers. In relation to the first objective, three 0,5 ha quadrats, divided in 200 contiguous quadrats of 25 m2, were installed (two in a typical cerrado to cerradão fragment and one in an open cerrado fragment), and all species ramets were sampled by count, being differentiated in with and without Contarinia sp attack ((Diptera: Cecidomyiidae). Photographies of the center of each parcel were taken to determinate the percentage of canopys openness. The abundance of ramets were higher in the most opened areas, but the incidence of Contarinia sp attack were higher in the closest fragment. Macroscale (Dispersion Index) and microscale analysis (Spatial Autocorrelation) showed that species ramets present an aggregated pattern in both areas, but this aggregation is not related to the percentage of canopys openness, although there are significant differences to this last factor among the grids, indicating that pattern is due to its way of life. In relation to the second objective, the construction of genomic library enriched with microsatellites resulted in 180 clones, which 84 were sequenced, being detected 23 sequences containing microsatellites, representing a librarys enrichment of 27,38%. 15 primers pairs were designed among the 34 obtained microsatellites, but only 7 pairs amplified. From these, five pairs were visualized in acrylamide, and one polymorphic loco was observed. The number of obtained clones is in accordance with the observed in studies with another species from Anacardiaceae family, showing protocols efficiency. Problems with the optimization of reagents may have impeded the amplification of some primers, once that procedures to their design were suitable. Polymorphism results are preliminaries, due to low number of evaluated individuals. At least one loco presents potential as genetic marker.

The Crimson-spotted rainbowfish (Melanotaenia fluviatilis) from the Murray-Darling basin of Australia is a common indicator species in Australian ecotoxicology. Biochemical changes have been investigated in this species, but not molecular markers of ecotoxicological interest. In this study genes of M. fluviatilis were isolated using a cDNA library and sequences analysed. Of 345 randomly selected clones, 94 shared similarity with 26 different genes in other organisms in public databases. Amongst these, reproductive genes coding for vitellogenin, retinol binding protein, sialyltransferase and zona pellucida protein were considered of interest in ecotoxicology. The vitellogenin gene was selected for study as it has been widely used as a molecular marker of exposure to 17â-estradiol (E2) in teleosts. Gene expression was examined via northern blot, RT-PCR and Real-Time PCR relative to the housekeeping gene (18S rRNA). The expression of vitellogenin mRNA was observed a t 12 hours post-exposure, peaked at 48 hours according to northern blot analysis; and cleared within 4 days, partly consistent with RT-PCR. However, Real-time PCR yielded an inconclusive result, probably due to differences between pooled and individual samples. Vitellogenin in blood plasma was confirmed by western blot, found to be significantly increased and retained in the plasma in fish treated with E2 compared to controls. It was concluded that vitellogenin mRNA is a molecular marker of exposure to 17â-estradiol in the rainbowfish, and could potentially be used as a marker of exposure to environmental estrogenic chemicals. Further investigations of the expression of genes in the cDNA library, could establish other molecular markers of ecotoxicological interest in M. fluviatilis.

Made available in DSpace on 2014-06-11T19:26:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-09-30Bitstream added on 2014-06-13T19:33:19Z : No. of bitstreams: 1 utsunomiya_ath_me_jabo.pdf: 1152493 bytes, checksum: 3f817736ec374e78cf3c1f57eaeab65b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Um grande volume de informações de marcadores SNPs vem sendo produzidos e aplicados a metodologias de avaliação genética animal. A quantidade de informações disponíveis faz-se um desafio, pois armazená-las e processar-las é demandante computacionalmente. Então, propôs-se com este trabalho 2 algoritmos (MLOCO e MSNP) para simular dados SNPs como alternativa de minimizar a demanda por processamento e consumo de memória computacional. MLOCO simula SNP como um loco que possui configuração de alelos, posição no genoma e efeitos sobre a expressão de fenótipos (nulos para SNP). MSNP simula SNP apenas como loco que possui configuração de alelos e posição no genoma. Ao nível de cromossomo, para MLOCO, poligenes, QTLs e SNPs são armazenados em um único vetor de acordo com suas localizações. Para MSNP, poligenes, QTLs e SNPs também são armazenados de acordo com suas localizações, porém em vetores específicos para cada tipo de loco. Considerando o consumo de memória, MLOCO é menos eficiente porque armazena um número de variáveis maior (efeito do loco, mesmo que seja zero). MSNP não armazena esta variável. Quanto à velocidade de processamento, MLOCO é mais eficiente porque os locos são armazenados de maneira seqüencial, facilitando a amostragem dos alelos devido a variável “posição”, para formação de um gameta e consequentemente um indivíduo. Como o fator limitante na realização de simulações e utilizações de dados é a memória RAM, o MSNP é a melhor alternativa para simular dados SNPs / A large amount of information of SNPs markers have been produced and applied methodologies in genetic evaluation. The amount of information available makes it challenging, for storing and processing them is computationally expansive. So, it was proposed in this paper two algorithms (MLOCO and MSNP) to simulate data SNPs as an alternative to minimize the demand for processing and consumption of computer memory. MLOCO simulates SNP as a locus that has configuration of alleles, position in the genome and its effects on the expression phenotypes (null for SNP). MSNP SNP simulates only locus that has position and configuration of alleles. At the level of the chromosome to MLOCO, polygenes, QTLs and SNPs are stored in a single vector according to their locations. To MSNP, polygenes, QTLs and SNPs are also stored according to their locations, but in specific vectors for each locus type. Whereas the memory consumption, MLOCO is less efficient because stores a greater number of variables (locus effect, even if it is zero). MSNP does not store this variable. As for processing speed, MLOCO is more efficient because the loci are stored sequentially, facilitating the sampling of alleles due to the variable position to form a gamete and hence an individual. As the limiting factor in simulations and use data is the RAM memory, the MSNP is the best alternative for simulating SNPs data

O cerrado brasileiro é um dos biomas de maior riqueza e endemismo de plantas, mas com alto índice de desmatamento nas últimas décadas, o que resultou na fragmentação dos habtats e na ameaça de extinção de centenas de espécies vegetais. Dentre estas espécies ameaçadas está Anacardium humile, conhecida como cajuzinho-do-campo, uma planta caméfita de ampla distribuição pelo país, servindo de alimento para o homem e para alguns animais e apresentando propriedades medicinais. Estudos sobre a estrutura de populações de Anacardium humile são escassos na literatura, e seu entendimento é fundamental para a preservação e conservação da espécie. Dessa forma, o presente trabalho teve dois objetivos: 1) em duas fitofisionomias de cerrado distintas, estimar a abundância de ramets da espécie, seu padrão de distribuição espacial em macro e microescala, e a influencia da porcentagem de abertura do dossel na determinação deste padrão; 2) a construção de uma biblioteca genômica enriquecida de microssatélites para a espécie e o desenho de primers a partir desta biblioteca, a fim de isolar locos com potencial para uso como marcadores genéticos. Com relação ao primeiro objetivo, três parcelas de 0,5 ha, divididas em 200 subparcelas, foram instaladas (duas num fragmento de cerrado típico à cerradão e uma num fragmento de cerrado aberto), e todos os ramets da espécie foram amostrados por contagem, sendo discriminados os com e sem ataque de Contarinia sp. (Diptera: Cecidomyiidae). Fotografias foram tiradas do centro de cada parcela para determinar a porcentagem de abertura do dossel. A abundância de ramets foi maior nas áreas mais abertas, mas a incidência de ataque de Contarinia sp foi maior no fragmento mais fechado. As análises de macroescala (Índice de Dispersão) e de microescala (Autocorrelação Espacial) mostraram que os ramets da espécie apresentam padrão agregado em ambas as áreas, mas que essa agregação, em microescala, não está relacionada à porcentagem de abertura do dossel, embora haja diferenças significativas para este ultimo fator entre os grids, indicando que o padrão é devido à sua forma de vida. Já em relação ao segundo objetivo, a construção da biblioteca genômica enriquecida de microssatélites resultou em 180 clones, dos quais 84 foram seqüenciados, sendo detectadas 23 sequências contendo microssatélites, o que representa um enriquecimento da biblioteca de 27,38%. Foram desenhados 15 pares de primers dentre os 34 microssatélites obtidos, mas apenas 7 pares amplificaram. Destes, cinco pares foram visualizados em acrilamida, e um loco polimórfico foi observado. O número de clones obtidos está dentro do observado em estudos com outras espécies da família Anacardiaceae, mostrando a eficiência do protocolo. Problemas com a otimização de reagentes podem ter impedido a amplificação de alguns primers, uma vez que os procedimentos para o desenho dos mesmos foram adequados. Os resultados de polimorfismo são preliminares, devido ao número baixo de indivíduos avaliados. Ao menos um loco apresenta potencial como marcador genético. / Brazilian cerrado is one of the richest and plants endemism biomes, but with a high deforestation in the last decades, resulted in habitats fragmentation and extinction threat of hundreds of plant species. Among these threatened species is Anacardium humile, known as cajuzinho-do-campo, a camephyth plant with a wide distribution through the country, which serves as aliment to man and some animals and presents some medical properties. Studies about Anacardium humiles populations structure are very scarce in the literature, and its understanding is fundamental to the preservation and conservation of the species. Thus, the present work had two objectives: 1) in two distincts cerrado plant physiognomies, estimate the abundance of species ramets, its spatial distribution pattern in macro and microscale, and the influence of canopy openness percentage in the determination of this pattern; 2) construction of a genomic enriched library with microsatellites to the species and primers design from this library, to isolate loci with potential to be used as genetic markers. In relation to the first objective, three 0,5 ha quadrats, divided in 200 contiguous quadrats of 25 m2, were installed (two in a typical cerrado to cerradão fragment and one in an open cerrado fragment), and all species ramets were sampled by count, being differentiated in with and without Contarinia sp attack ((Diptera: Cecidomyiidae). Photographies of the center of each parcel were taken to determinate the percentage of canopys openness. The abundance of ramets were higher in the most opened areas, but the incidence of Contarinia sp attack were higher in the closest fragment. Macroscale (Dispersion Index) and microscale analysis (Spatial Autocorrelation) showed that species ramets present an aggregated pattern in both areas, but this aggregation is not related to the percentage of canopys openness, although there are significant differences to this last factor among the grids, indicating that pattern is due to its way of life. In relation to the second objective, the construction of genomic library enriched with microsatellites resulted in 180 clones, which 84 were sequenced, being detected 23 sequences containing microsatellites, representing a librarys enrichment of 27,38%. 15 primers pairs were designed among the 34 obtained microsatellites, but only 7 pairs amplified. From these, five pairs were visualized in acrylamide, and one polymorphic loco was observed. The number of obtained clones is in accordance with the observed in studies with another species from Anacardiaceae family, showing protocols efficiency. Problems with the optimization of reagents may have impeded the amplification of some primers, once that procedures to their design were suitable. Polymorphism results are preliminaries, due to low number of evaluated individuals. At least one loco presents potential as genetic marker.

<p>Microbial metagenomics is the exploration of taxonomical diversity of microbial communities in environmental habitats using large, exhaustive DNA sequence datasets. However, due to inherent limitations of sequencing technology and the complexity of environmental genomes, current analytical approaches do not reveal the existence of all microbes that may be present. In this study, a new classification approach is proposed based upon unique proteins that are specific for different clades of Alphaproteobacteria to predict the presence and absence of species from these groups of bacteria in published metagenomic datasets. In this work, 264 previously–identified, published conserved signature proteins (CSPs) characteristic of individual taxonomic clades of Alphaproteobacteria are used as probes to detect the presence of bacteria in metagenomic datasets. Although public genome sequence information has increased manifold since these CSPs were initially identified 6 years ago, results indicate that nearly all of these CSPs (259 of 265) are specific for their previously characterized clades. Furthermore, they are confirmed to be present in the newly–identified and sequenced members of these clades. In view of their specificity and predictive ability in different monophyletic clades of Alphaproteobacteria, the sequences of these CSPs provide reliable probes to determine the presence or absence of these Alphaproteobacteria in metagenomic datasets. In this work, CSPs are used to determine the presence of Alphaproteobacteria diversity in 10 published metagenomic datasets (bioreactor, compost, wastewater, activated sludge, groundwater, freshwater sediment, microbial mat, marine, hydrothermal vent and whale fall metagenomes), which cover diverse environment and ecosystems. It is indicated that the BLAST searches with these CSPs can be used to efficiently identify Alphaproteobacteria species in these metagenome dataset and substantial differences can be determined in the distribution and relative abundance of different Alphaproteobacteria species in the tested metagenome datasets. Thus the CSPs, which are specific for different microbial taxa, provide novel and powerful means for identification of microbes and for their taxonomic profiling in metagenomic datasets.</p> / Master of Science (MSc)

A caracterização e o entendimento da diversidade e estrutura genética de acessos armazenados em bancos de germoplasma é importante para a sua efetiva utilização em programas de melhoramento. Neste trabalho, 192 acessos japoneses de arroz pertencentes ao Banco de Germoplasma do Departamento de Genética da ESALQ/USP foram caracterizados por 23 descritores agromorfológicos (13 variáveis contínuas e dez variáveis categóricas) e 24 marcadores microssatélites (12 SSRs genômicos e 12 EST-SSRs). As variáveis contínuas que apresentaram maior contribuição para a variabilidade entre os acessos foram avaliadas novamente em um segundo experimento para considerar os efeitos de interação genótipo por ano na diversidade. Os acessos apresentaram variabilidade para ambos os tipos de variáveis, e o tipo de variável utilizada na avaliação e a interação genótipo por ano implicaram em diferentes padrões de agrupamento entre os acessos, sendo que o agrupamento mais consistente foi o realizado considerando todas as variáveis e os dois anos agrícolas. Os 24 marcadores microssatélites detectaram um total de 181 alelos, 38 dos quais foram exclusivos. A média do número de alelos por loco foi de 7,54 e a diversidade gênica foi de 0,59 para os SSRs genômicos e de 0,46 para os EST-SSRs. As análises caracterizaram os acessos japoneses como 98,4% pertencentes à subespécie Japônica. A resposta dos acessos para estresse por frio na germinação foi avaliada em três experimentos; I: os 192 acessos e três testemunhas tolerantes foram avaliados sob duas condições: 13ºC por 28 dias (estresse por frio) e 28ºC por sete dias (condições ótimas); II: dezessete acessos selecionados entre os que apresentaram melhores desempenhos na temperatura de 13ºC foram avaliados juntamente com as três testemunhas em gradiente de temperatura de 11ºC, 13ºC, 15ºC, 17ºC e 28ºC para estudar a interação genótipo por temperatura; III: os genótipos do experimento II foram avaliados para germinação e estabelecimento inicial de plântulas a 15ºC em areia. A diminuição da temperatura teve efeito significativo no desempenho dos acessos, e a interação genótipo por temperatura foi significativa para comprimento do coleóptilo e índice de velocidade de germinação. Entre os acessos estudados foi observada variação genética para as características relacionadas com a germinação em baixa temperatura e encontraram-se acessos com reduções nos comprimentos de coleóptilo e radícula devido ao frio comparáveis com a das testemunhas, embora com velocidade de crescimento menor, inclusive em temperatura ótima. Uma coleção nuclear constituída por 52 acessos e representativa da diversidade fenotípica e molecular observada nos 192 acessos foi obtida. / Germplasm characterization and the knowledge of its diversity and population structure are important to effective utilization of genetic resources in breeding programs. In this work, 192 Japanese rice accessions maintained at Germplasm Bank of Genetics Department at ESALQ/USP were characterized using 23 agromorphological descriptors (13 continuous and ten categorical variables) and 24 SSR markers (12 genomic SSRs and 12 EST-SSRs). The continuous variables presenting more contribution in the divergence among accessions were evaluated again in a second harvest year to account interaction between genotype and year in the diversity. Japanese accessions presented variability for continuous and categorical variables and the type of variable and the genotype by year interaction resulted in a different pattern of clustering of accessions. The most consistent cluster was that considering both continuous and categorical variables and the two harvest years. A total of 181 alleles were detected by the microsatellite markers, 38 of them were private. Allele per locus mean was 7.54 and gene diversity was 0.59 for genomic SSRs and 0.46 for EST-SSRs. Accessions were classified as 98.4% belonging to japonica subspecies. Accessions response to cold stress at the germination was evaluated in three experiments; I: 192 accessions and three coldtolerant controls were evaluated under two conditions: 13ºC during 28 days (cold stress) and 28ºC during seven days (optimal temperature); II: seventeen accessions selected among those presenting better performance in the first experiment were evaluated along with three cold-tolerant controls under five temperatures: 11ºC, 13ºC, 15ºC, 17ºC e 28ºC to study genotype by temperature interaction; III: germination and plantule initial development in sand at 15ºC of genotypes used in the second experiment were evaluated. It was detected variability among the accessions to germination under cold stress. Temperature decrease had significant effect for germination velocity index and coleoptile and radicle length, and the interaction between genotype and temperature was significant for germination velocity index and coleoptile length. The accessions presented genetic variability for cold tolerance germination and accessions presenting coleoptile and radicle reductions due to cold comparable with cold-tolerant controls were detected. A core collection containing 52 accesions representing the phenotypic and molecular diversity of the 192 accessions was obtained.