Resumo: O presente trabalho teve como objetivo principal ampliar o conhecimento sobre a estrutura genética e obter informações para a conservação de pacu (Piaractus mesopotamicus Holmberg, 1887), provenientes de populações selvagens dos rios da Bacia do Alto Paraguai. Esta espécie possui grande valor comercial e imenso potencial para exploração em pisciculturas. Os marcadores moleculares do tipo microssatélite utilizados neste estudo, resultaram em muitas informações sobre a estrutura populacional desta espécie, permitindo uma possível caracterização dos bancos genéticos para esta espécie. Exemplares provenientes de nove sub-bacias do Pantanal Matogrossense foram coletados para a realização da extração de DNA visando à análise do material genômico. Para isto, foram retirados pequenos fragmentos de nadadeira de cada indivíduo. A amplificação dos locos microssatélites foi realizada num termociclador de PCR utilizando oligonucleotídeos marcados com fluorescência, conforme descrito na literatura. A genotipagem foi realizada no seqüenciador automático MegaBACETM 1000 (Amersham Biosciences), pertencente ao Centro de Estudos do Genoma Humano da Universidade de São Paulo. Os tamanhos dos alelos obtidos foram organizados para a montagem das matrizes de dados que foram submetidas aos programas computacionais para verificar a variabilidade genética nas populações. Os parâmetros que permitiram a determinação da diversidade genética intra e interpopulacional foram o número de alelos por loco, riqueza alélica, heterozigosidade observada e esperada, equilíbrio de Hardy Weinberg, índices Fst e Rst, análise de variância molecular (AMOVA), índice de fixação, desequilíbrio de ligacão e o número de migrantes. Também foi feita uma análise bayesiana para verificar a estrutura populacional e um dendrograma foi gerado a partir da matriz de distância baseada... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work aimed to obtain information about the genetic structure of pacu (Piaractus mesopotamicus Holmberg, 1887), from wild populations of Alto Paraguai Basin Rivers. This specie has a greatly commercial importance, with huge potential for hatcheries. The pacu has a wide distribution in the Prata Basin, formed by Paraguay, Paraná and Uruguay rivers and their tributaries Microsatellites markers offer relevant information about this specie, allowing the characterization of genetic stocks. Individuals from nine sampling sites in the Pantanal Matogrossense were analysed in this study. DNA extraction methods did not required killing the samples, we used fin clippings from each individual. The amplification of microsatellites loci was carried out via polymerase chain reaction (PCR) using oligonucleotide marked with fluorescent labels. The amplified fragments were analysed on the automatic DNA sequencer MegaBACETM 1000 (Amersham Biosciences), belonged to Centro de Estudos do Genoma Humano from Universidade de São Paulo. Allele sizes were organized in an input file that was submitted statistical analysis to verify the genetic variability of populations. The parameters used to estimate the genetic diversity intra and interpoulation were number of allele per locus, allele richness, observed and expected heterozygosities, Hardy Weinberg equilibrium, molecular variance analyses (AMOVA), fixation index, linkage disequilibrium and gene flow. Bayesian estimates were used to verify the populacional structure and a dendogram was calculated by the distances matrix based on chord distances values. The results showed no population structuring and intense gene flow. The most probable causes are the hight migratory capacityof this fish and the climatic and geographic characteristics of the Pantanal Matogrossense. / Orientador: Fausto Foresti / Coorientador: Daniela Calcagnotto / Banca: Alexandre Wagner Silva Hilsdorf / Banca: Emiko Kawakami de Resende / Banca: Jeffrey Frederico Lui / Banca: Claudio de Oliveira / Doutor

As abelhas da tribo Meliponini (abelhas sem ferrão) estão amplamente distribuídas pelas regiões tropicais do planeta, tendo um importante papel na polinização, sendo o gênero Melipona o que contém o maior número de espécies. A espécie Melipona marginata é uma das menores e mais ancestrais do grupo, e a exemplo de outras espécies nidifica em ocos de árvore. M. marginata, assim como outras espécies do gênero Melipona, vêm sofrendo com a destruição do seu ambiente natural, pelo desmatamento, sendo, aparentemente mais exigente que outras espécies quanto ao tamanho do fragmento florestal para se manter, devido a isso é encontrada apenas em fragmentos maiores, mais antigos e menos perturbados. Tendo em vista a perda de hábitat e o pouco conhecimento da biologia dessa espécie, este trabalho pretende analisar populações de M. marginata, através da técnica PCR+RFLP do DNA mitocondrial e marcadores microssatélites, visando contribuir para entendimento da estrutura populacional de M. marginata. Foram analisados 54 ninhos, provenientes dos estados de MG, SP, PR, SC e RS. Oito regiões do DNA mitocondrial foram amplificadas, e posteriormente digeridas com 12 enzimas de restrição. Foram detectados 14 haplótipos, sendo apenas um compartilhado. A população de SP apresentou o maior número de haplótipos. Os testes estatísticos demonstraram que as populações estão estruturadas e isoladas, não havendo fluxo gênico entre as populações. Já nas análises dos microssatélites foram analisados 4 locos, apresentando alta variabilidade genética, onde também foi verificado que as populações se encontram estruturadas. Os resultados obtidos podem ser explicados principalmente pela redução da área de floresta, mas, podem se dever a eventos antigos em conseqüência de mudanças climáticas ocorridas durante as últimas glaciações. / The tribe Meliponini (stingless bees) is present in all tropical regions of the world and has an important role in pollination. The genus Melipona has the highest number of species in the tribe. The specie Melipona marginata is considered the most ancestral within the genus, and like other species builds the nests in hollow of trees. Unfortunately several bee species have suffered with the devastation of their natural environment. M. marginata seems to be very dependent on the size of the forest fragment, being found only in the biggest, oldest and consequently more preserved ones. In view of the habitat loss and few biological knowledge about this specie, this research intended to analyse M. marginata populations molecularly, through mitochondrial DNA PCR-RFLP and microsatellite data., Fifty four colonies were analyzed, from MG, SP, PR, SC and RS states. Eight mitochondrial DNA regions were amplified and screened with 12 restriction enzymes. Fourteen haplotypes were verified and among them just one was shared. SP population showed the highest number of haplotypes. Statistic tests showed that the 5 populations were genetic structured and isolated, therefore not presenting gene flow. The 4 microsatellites loci analyzed showed high genetic variability. The statistics analysis indicated that the 5 populations are structure and isolated. These results can be explained mainly because the decrease of forest leading to population isolation, however we can not discard the hypothesis that this current scenario is a consequence of climatic changes occurred during the last glaciations.

O gênero Partamona compreende 33 espécies, distribuídas do sul do México ao sul do Brasil. O gênero tem sido amplamente estudado em diferentes níveis: citogenético, etológico e morfológico. O presente trabalho teve como objetivo contribuir com dados moleculares para o conhecimento do grupo, realizando estudos populacionais por meio da caracterização do DNA mitocondrial por PCR+RFLP e da análise de regiões de microssatélites do DNA genômico de duas espécies: P. mulata de distribuição restrita ao sul de Mato Grosso e norte do Mato Grosso do Sul, e P. helleri de distribuição mais ampla, do sul da Bahia até Santa Catarina. Foram detectados apenas dois haplótipos em P. mulata, os quais diferiram entre si por apenas um sítio de restrição. As análises estatísticas demonstraram não haver estruturação entre as populações sugerindo que esta espécie possa ter passado por recente afunilamento populacional. Em P. helleri foram observados dez haplótipos sendo alguns exclusivos e outros compartilhados. Análises estatísticas apontaram alta estruturação entre as populações e a distribuição filogeográfica observada sugere um possível isolamento por fragmentação da Mata Atlântica durante o Pleistoceno. A análise dos locos microssatélites mostrou baixa variabilidade genética em ambas espécies e discreta estruturação entre as populações, não relacionada com a distribuição geográfica das mesmas. Isto pode ser conseqüência de migração de machos entre populações visto que as rainhas são filopátricas ou, fragmentação dos habitats pela rápida degradação do cerrado e da Mata Atlântica, ou por alelos nulos causados pelo uso de primers heteroespecíficos. A análise de parentesco entre abelhas de um mesmo ninho apontou a existência de apenas uma patrilínea em P. mulata sugerindo monoandria para esta espécie. Foram encontradas duas patrilíneas em algumas colônias de P. helleri, o que pode ser resultante de fecundação por mais de um macho ou substituição recente da rainha. A caracterização parcial do DNAmt de duas espécies de Partamona poderá contribuir em estudos filogenéticos tanto do gênero quanto de outras espécies de Meliponini. A análise populacional mostrou o status da variabilidade genética das espécies, suas possíveis histórias evolutivas e a possível relação desta com degradação dos ambientes onde estas estão distribuídas. / The Partamona genus comprises 33 species distributed from south Mexico to south Brazil. This genus has been studied at different levels: cytogenetical, ethological and morphological. This work aimed at to contribute with molecular data for the knowledge about the group performing a population study employing the PCR+RFLP of mtDNA, and analysis of microsatellite loci from nuclear DNA of two species, P. mulata which is distributed in south Mato Grosso and north Mato Grosso do Sul, and P. helleri which geographic distribution is wider, from Santa Catarina to southern Bahia. It was detected two haplotypes in 58 colonies of P. mulata, each one differing by one single restriction site. The statistical analyses indicated no differentiation among populations suggesting that the species could have passed through a recent populational bottleneck. It was observed ten haplotypes in 47 colonies of P. helleri, some exclusive and others shared among populations. Statistical analysis pointed high population differentiation and the observed phylogeography distribution suggested a possible recent isolation probably by Atlantic Forest fragmentation during the Pleistocene. The microsatellite analysis showed low genetic variability in both species and discrete population structuring, not related to the geographic distribution. This might be consequence of migration of males, since the queens are highly phylopatric, or habitat fragmentation by degradation of savanna and Atlantic forest areas, or null alleles caused by the use of heterospecific primers. The relatedness investigation revealed only one patriline in nest mates of P. mulata that suggests monoandry for this species. It was found two patrilines in P. helleri that can be resulted from more than one mating or recent queen replacement. The partial characterization of the mtDNA of two Partamona species can contribute to further phylogenetic studies among bees of this genus or among other Meliponini species. The populational analysis showed the genetic variability status of the species, their putative evolutionary histories and the possible relation between the results and the environmental degradation in their distribution areas.

We developed a set of hypervariable microsatellite markers for the Pacific red snapper (Lutjanus peru), an economically important marine fish for small-scale fisheries in the west coast of Mexico. We performed shotgun genome sequencing with the 454 XL titanium chemistry and used bioinformatic tools to search for perfect microsatellite loci. We selected 66 primer pairs that were synthesized and genotyped in an ABI PRISM 3730XL DNA sequencer in 32 individuals from the Gulf of California. We estimated levels of genetic diversity, deviations from linkage and Hardy-Weinberg equilibrium, estimated the frequency of null alleles and the probability of individual identity for the new markers. We reanalyzed 16 loci in 16 individuals to estimate genotyping error rates. Eighteen loci failed to amplify, 16 loci were discarded due to unspecific amplifications and 32 loci (14 tetranucleotide and 18 dinucleotide) were successfully scored. The average number of alleles per locus was 21 (+/- 6.87, SD) and ranged from 8 to 34. The average observed and expected heterozygosities were 0.787 (+/- 0.144 SD, range 0.250-0.935) and 0.909 (+/- 0.122 SD, range 0.381-0.965), respectively. No significant linkage was detected. Eight loci showed deviations from Hardy-Weinberg equilibrium, and from these, four loci showed moderate null allele frequencies (0.104-0.220). The probability of individual identity for the new loci was 1.46(-62). Genotyping error rates averaged 9.58%. The new markers will be useful to investigate patterns of larval dispersal, metapopulation dynamics, fine-scale genetic structure and diversity aimed to inform the implementation of spatially explicit fisheries management strategies in the Gulf of California.

Apart from contributing to Computer Science, this research also contributes to Bioinformatics, a subset of the subject discipline Computational Biology. The main focus of this dissertation is the development of a data-analytical and theoretical algorithm to contribute to the analysis of DNA, and in particular, to detect microsatellites. Microsatellites, considered in the context of this dissertation, refer to consecutive patterns contained by genomic sequences. A perfect tandem repeat is defined as a string of nucleotides which is repeated at least twice in a sequence. An approximate tandem repeat is a string of nucleotides repeated consecutively at least twice, with small differences between the instances. The research presented in this dissertation was inspired by molecular biologists who were discovered to be visually scanning genetic sequences in search of short approximate tandem repeats or so called microsatellites. The aim of this dissertation is to present three algorithms that search for short approximate tandem repeats. The algorithms comprise the implementation of finite automata. Thus the hypothesis posed is as follows: Finite automata can detect microsatellites effectively in DNA. "Effectively" includes the ability to fine-tune the detection process so that redundant data is avoided, and relevant data is not missed during search. In order to verify whether the hypothesis holds, three theoretical related algorithms have been proposed based on theorems from finite automaton theory. They are generically referred to as the FireìSat algorithms. These algorithms have been implemented, and the performance of FireìSat2 has been investigated and compared to other software packages. From the results obtained, it is clear that the performance of these algorithms differ in terms of attributes such as speed, memory consumption and extensibility. In respect of speed performance, FireìSat outperformed rival software packages. It will be seen that the FireìSat algorithms have several parameters that can be used to tune their search. It should be emphasized that these parameters have been devised in consultation with the intended user community, in order to enhance the usability of the software. It was found that the parameters of FireìSat can be set to detect more tandem repeats than rival software packages, but also tuned to limit the number of detected tandem repeats. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Computer Science / unrestricted

Orientador: Anete Pereira de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T12:23:33Z (GMT). No. of bitstreams: 1 Laborda_PriandaRios_D.pdf: 3045492 bytes, checksum: d1a998a5b1c4300247471d90da1ee8da (MD5) Previous issue date: 2011 / Resumo: Drosophila mediopunctata, mosca não cosmopolita de distribuição neotropical e pertencente ao grupo tripunctata do subgênero Drosophila, é uma espécie utilizada por alguns grupos no Brasil como modelo em estudos com enfoque evolutivo. Abordagens que exploram a biologia da espécie, tais como a detecção de variação genética natural e a influência de inversões cromossômicas na dinâmica populacional são costumeiramente usadas. Todavia, essa espécie não possui disponíveis marcadores moleculares que facilitem a investigação das bases genéticas que norteiam os eventos explorados até agora. Os microssatélites são marcadores moleculares consagrados e a ferramenta de escolha no estudo de diversos organismos pela simplicidade de uso e de análise. Entretanto, para espécies que ainda não têm grande parte de seu genoma seqüenciado, a obtenção de marcadores desse tipo passa pela necessidade de desenvolvimento via bibliotecas genômicas. Esse trabalho objetivou desenvolver, caracterizar e mapear microssatélites em D. mediopunctata para que estudos de variação morfológica, de identificação de regiões genômicas associadas a fenótipos de interesse, de genética de populações entre outros possam ser realizados com o respaldo de dados moleculares. Uma biblioteca enriquecida em motivos repetitivos foi construída e, após o seqüenciamento de aproximadamente 2000 clones, 600 microssatélites foram identificados e 134 locos desenvolvidos. Os marcadores mostraram tamanho reduzido com relação ao número de repetições e preponderância de motivos AC/GT. A aplicação dos microssatélites de D. mediopunctata para amplificações heterólogas em outras trinta espécies foi feita com aproximadamente 50% de sucesso. Em adição, o uso de dados de presença/ausência nas espécies do grupo tripunctata recuperou algumas relações filogenéticas previamente conhecidas. Um mapa de ligação foi construído com 49 marcadores e revelou 450 cM de extensão. Os cinco principais cromossomos da espécie foram identificados por meio de comparações com o genoma de D. melanogaster e com os elementos de Müller. Essas comparações também confirmaram a grande sintenia prevista dentro do gênero Drosophila. Não houve concordância na ordem dos locos microssatélites nas duas espécies. No cromossomo II de D. mediopunctata foi mapeada uma região associada à característica "número de pintas abdominais" / Abstract: Drosophila mediopunctata, a non-cosmopolitan fly of Neotropical distribution that belongs to the tripunctata group of the Drosophila subgenus, was chosen by some Brazilian researchers as a model in evolutionary studies. Several approaches, such as the analysis of natural variation and the influence of chromosome inversions in population dynamics, are traditionally used. Nevertheless, molecular markers, which would enhance the investigation of the genetic bases of the already known phenomena, are still not available for the species. Microsatellites are celebrated molecular markers and the chosen tool for the exploration of various organisms due to their ease of use. Nonetheless, their application in species whose genomes have not yet been sequenced requires a prior development phase. This study intended to develop, characterize and map microsatellites for the species D. mediopunctata so that initiatives concerning morphological variation, identification of genomic regions linked to interesting phenotypes, population genetics, etc can be carried out in the light of molecular data. A repetitive DNA-enriched library was constructed and approximately 2000 clones were sequenced. Six hundred microsatellites were identified and 134 loci were developed. The loci are small in length, with reduced number of motif repetitions, and are mainly composed of AC/GT dinucleotides. The use of D. mediopunctata microsatellites for heterologous amplification in other thirty Drosophila species was done with a 50% success ratio. In addition, a clustering analysis carried out with binary data obtained from the tripunctata species recovered already known phylogenetic relationships. A linkage map was constructed with recombination data of 49 markers and is 450 cM in length. The five major species chromosomes were identified on the basis of comparisons with the D. melanogaster genome and the Müller elements. This strategy also confirmed the great synteny predicted for the genus Drosophila. It was not observed agreement in loci order between both species. A genomic region associated to the number of abdominal spots was mapped to the chromosome II of D. mediopunctata / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

The marsh rice rat (Oryzomys sp.) is a semiaquatic rodent native to wetlands in the southeastern United States. The northwestern-most part of the rice rat’s range extends to Illinois where rice rats are found in wetlands across the southern part of the state. Recent studies have shown that rice rats in the United States can be divided into two species: O. palustris and O. texensis, but the taxonomic status of rice rats in Southern Illinois is unclear. To resolve this, I sequenced cytochrome-b and the control region, two regions of mitochondrial DNA, for 16 rice rats and constructed a phylogeny using these new sequences and previously obtained O. palustris and O. texensis sequences. In contrast to previous morphological assessments, I found that rice rats in Southern Illinois should be classified as O. texensis. This would extend the range of O. texensis north and west from its current extent. Further investigation using nuclear loci will be needed to confirm this classification.

Marine protected areas (MPAs) have proliferated in the past decades to protect biodiversity and sustain fisheries. However, most of the MPA networks have been designed without taking into account a critical factor: the larval dispersal patterns of populations within and outside the reserves. The scale and predictability of larval dispersal, however, remain unknown due to the difficulty of measuring dispersal when larvae are minute (~ cm) compared to the potential scale of dispersal (~ km). Nevertheless, genetic approaches can now be used to make estimates of larval dispersal. The following thesis describes self-recruitment and connectivity patterns of a coral reef fish species (Centropyge bicolor) in Kimbe Bay, Papua New Guinea. To do this, microsatellite markers were developed to evaluate fine-scale genetics and recruit assignment via genetic parentage analysis. In this method, offspring are assigned to potential parents, so that larval dispersal distances can then be inferred for each individual larvae. From a total of 255 adults and 426 juveniles collected only 2 parentoffspring pairs were assigned, representing less than 1% self-recruitment. Previous data from the same study system showed that both Chaetodon vagagundus and Amphiprion percula have consistent high self-recuitment rates (~ 60%), despite having contrasting life history traits. Since C. bicolor and C. vagabundus have similar characteristics (e.g. reproductive mode, pelagic larval duration), comparable results were expected. On the contrary, the results of this study showed that dispersal patterns cannot be generalized across species. Hence the importance of studying different species and seascapes to better understand the patterns of larval dispersal. This, in turn, will be essential to improve the design and implementation of MPAs as conservation and management tools.

The use of candidate genes has become a widespread approach in the study of behavioral adaptations. Gene function is often maintained in very distant evolutionary lines. This approach allows us to extend knowledge about non-model species. I studied the influence of selection on candidate behavioral adaptation genes associated mainly with biorhythms. I was interested in comparing genetic variability between closely related species of songbirds from tropical and temperate zones. These environments differ mainly in the conditions stability. Tropical species live in very stable conditions with generally low seasonality, unlike temperate species. Timing of events of cirkanual cycle of temperate birds is essential because of the alternating of conditions of the environment. I therefore assumed a reduction in genetic variability in migrating temperate zone species compared to tropical species due to stabilizing selection. The study is based on analyzes of microsatellite loci in the exon region. I involved analysis of neutral microsatellites as a control for possible genetic variability reduction coming from different reasons than selection. Neutral microsatellites are expected to not be influenced by selection. In tropical species I found reduced genetic variability of neutral microsatellites. That might...

Hybridization in plants can be common, may lead to increased genetic variation, and in some instances the formation of new species. For endangered species, hybridization can introduce novel genetic variation and potentially increase genetic diversity. In contrast, hybridization can negatively affect an endangered species or population by introducing maladaptive alleles into locally adapted lineages or lead to the homogenization of once distinct lineages. We used microsatellites, AFLP markers, and morphological data to identify potential hybridization between the endangered Roan Mountain bluet (Houstonia purpurea var. montana) and its commonly occurring congener (H. purpurea var. purpurea). We used these data to clarify the taxonomic relationship of these varieties and to assess population structuring of the Roan Mountain bluet. The results showed that these lineages are distinct and few individuals exhibited admixture in several populations. Additionally, the results showed there is genetic population structure among the remaining populations of H. purpurea var. montana, which is consistent with other mountaintop species. Our results show that Roan Mountain bluet populations should be protected to maintain genetic diversity and monitored to assess future hybridization, and additional studies that comprise a greater population sampling would provide more information.