Recent improvements in sequencing technologies have caused various interesting problems to arouse. Having millions of read sequences as the final product of sequencing genome at a lower cost compared to micro array era, has encouraged scientists to enhance previous methods in various areas of bioinformatics. Genotyping and generating genetic maps to study inherited genotypes in order to analyze specific traits in a population is one of the fields of bioinformatics that involves generating different genetic markers and identify polymorphisms in different individuals of a population. Presence/absence markers are the main focus of this thesis. This is one type of Restriction site Associate DNA (RAD) markers which is present in some samples and absent in others and is the sign of variation in the cut site of a restriction enzyme. However, the counts of markers in an experiment are highly correlated and calling true absence and presence is not a straightforward task which means any marker with zero count is not necessarily absent in the sample under study. This is also the case for non-zero count markers which are not necessarily present. A good model that can fit the data is able to make true calls. We propose two different contexts for designing such models as a solution to this problem and investigate their performance. On the other hand, utilizing features of next generation sequencing technology in an even more efficient way, requires the ability to multiplex high number of samples in a single experiment run. In that case, appropriate barcoding, that is robust to various sources of noise in the machine, becomes paramount. Designing such barcodes in an efficient way is a challenging task which is addressed in detail as another problem of this thesis. We make two contributions. One, we propose an algorithm for barcoding multiplexed RADSeq samples. Two, we propose an algorithm for the statistical selection of presence/absence markers on the basis of RADSeq data on two related individuals. Operating characteristics of our methods are explored using both simulated and real data.

A key to understanding the origin and maintenance of the diversity of life is to understand how phenotypic and genetic variation is partitioned within and among populations. I characterize the spatial partitioning of phenotypic and genetic variation in an old Willamette Basin freshwater distribution of threespine stickleback (Gasterosteus aculeatus) and compare these results to younger populations. Phenotypic variation was measured using 14 phenotypic traits, and genetic variation was assessed using RADseq and Stacks software to identify tens of thousands of single nucleotide polymorphisms. The major partitioning of phenotypic and genetic variation in Oregon is along a stereotypical transition from oceanic to freshwater that has been seen in younger systems. Phenotypic and genetic variation is significantly partitioned between basin populations, and the genetic variation is geographically structured. This work suggests that parallel divergence between oceanic and freshwater forms originated before the end of the last glacial maximum.

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Agradecemos a compreensão. on 2016-06-15T19:04:14Z (GMT) / Submitted by MELINA AULINO CAMPOS DE LIMA null (mel_linaa@hotmail.com) on 2016-07-28T09:31:04Z No. of bitstreams: 1 Campos, versão final.pdf: 6669108 bytes, checksum: bf58ca3048946ca88e3c4dd8fb99500a (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-07-28T14:10:56Z (GMT) No. of bitstreams: 1 lima_mac_dr_bot.pdf: 6669108 bytes, checksum: bf58ca3048946ca88e3c4dd8fb99500a (MD5) / Made available in DSpace on 2016-07-28T14:10:56Z (GMT). No. of bitstreams: 1 lima_mac_dr_bot.pdf: 6669108 bytes, checksum: bf58ca3048946ca88e3c4dd8fb99500a (MD5) Previous issue date: 2016-05-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Degradação florestal, alterações ambientais antropogênicas e mudanças climáticas são fatores que podem modificar a dinâmica populacional de anofelinos. Anopheles (Nyssorhynchus) dar- lingi é o principal vetor de malária no Brasil e em outros países na América do Sul. Estudos observaram o aumento da abundância do vetor An. darlingi e número de casos de malária após desflorestação e demais modificações ambientais antropogênicas. Além de ter ampla distribuição geográfica, essa espécie possui plasticidade comportamental e diversidade morfo- lógica, biológica e genética. Tendo em vista essa heterogeneidade, o presente estudo avaliou a diversidade genética populacional em An. darlingi ligada a distribuição geográfica, dinâmica sazonal e comportamento hematofágico, através de marcadores microssatélites e SNPs. Espé- cimes de An. darlingi foram coletados em dois assentamentos rurais próximos e, em uma área urbana à aproximadamente 600 km de distância. Além disso, as coletas foram realizadas no primeiro e segundo semestre e, dentro e fora das casas, durante o período de atividade hema- tofágica do vetor, ou seja, das 18-6 horas. Os resultados apresentaram subpopulações de An. darlingi em aspecto geográfico, em escalas macro e microgeográficas, acessadas com mais pro- fundidade com os dados dos SNPs. Além disso, o estudo corroborou o prévio achado de duas subpopulações de An. darlingi relacionadas ao regime de chuvas. Por fim, pela primeira vez, a heterogeneidade genética em populações simpátricas desse vetor foi encontrada e relacionada com o fenótipo de comportamento hematofágico, endo e exofagia. Esses achados demonstram a importância do entendimento e vigilância entomológica, pois possuem potencial impacto na transmissão de malária. Dado que cada localidade possui características ambientais peculia- res e portanto, diferentes composições populacionais dos vetores, intervenções específicas em menor escala passam a ser abordagens interessantes no controle da malária. / Forest degradation, human environmental alteration and climate changes are all influence anopheline populations. Anopheles darlingi is the main vector of malaria parasite in Brazil and other countries of South America. Deforestation and others anthropogenic activities have been accompanied by sharp increases in both abundance of the primary malaria vector Anopheles darlingi and numbers of malaria cases. Besides it is widely distributed, this species display great behavioral plasticity and morphological, biological and genetic diversity. The aim of this study was to analyze population genetic diversity of An. darlingi related to geographical distribution, seasonal dynamics and hematophagic behavior, using microsatellites and SNPs markers. An. darlingi specimens were collected in two close rural settlements and in an urban area about 600 km away. In addition, collections were performed in both semesters of the year and, indoor and outdoor during the biting activity period of the vector, i.e., 6pm-6am. The results showed subpopulations of An. darlingi related to geographical aspect, in a macro and micro geographic scales, better accessed with SNPs dataset. Moreover, this study corroborated with a previous finding showing two subpopulations of An. darlingi related to rainfall. Finally, for the first time, genetic heterogeneity was found in sympatric populations of this vector, and it was associated with a phenotype of hematophagic behavior, endo and exophagy. These outcomes demonstrate that genetic heterogeneity may represent an important vector phenotypic variation with potentially highly significant consequences for malaria transmission. Since each site has unique environmental characteristics and therefore, different population compositions of vectors, specific interventions on a smaller scale become interesting approaches for optimal targeted malaria transmission interventions. / CAPES: BEX 9230/12-2 / FAPESP: 2014/09461-3 / FAPESP: 2012/04881-9

Orientador: Paulo Eduardo Martins Ribolla / Resumo: Degradação florestal, alterações ambientais antropogênicas e mudanças climáticas são fatores que podem modificar a dinâmica populacional de anofelinos. Anopheles (Nyssorhynchus) dar- lingi é o principal vetor de malária no Brasil e em outros países na América do Sul. Estudos observaram o aumento da abundância do vetor An. darlingi e número de casos de malária após desflorestação e demais modificações ambientais antropogênicas. Além de ter ampla distribuição geográfica, essa espécie possui plasticidade comportamental e diversidade morfo- lógica, biológica e genética. Tendo em vista essa heterogeneidade, o presente estudo avaliou a diversidade genética populacional em An. darlingi ligada a distribuição geográfica, dinâmica sazonal e comportamento hematofágico, através de marcadores microssatélites e SNPs. Espé- cimes de An. darlingi foram coletados em dois assentamentos rurais próximos e, em uma área urbana à aproximadamente 600 km de distância. Além disso, as coletas foram realizadas no primeiro e segundo semestre e, dentro e fora das casas, durante o período de atividade hema- tofágica do vetor, ou seja, das 18-6 horas. Os resultados apresentaram subpopulações de An. darlingi em aspecto geográfico, em escalas macro e microgeográficas, acessadas com mais pro- fundidade com os dados dos SNPs. Além disso, o estudo corroborou o prévio achado de duas subpopulações de An. darlingi relacionadas ao regime de chuvas. Por fim, pela primeira vez, a heterogeneidade genética em populações... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Forest degradation, human environmental alteration and climate changes are all influence anopheline populations. Anopheles darlingi is the main vector of malaria parasite in Brazil and other countries of South America. Deforestation and others anthropogenic activities have been accompanied by sharp increases in both abundance of the primary malaria vector Anopheles darlingi and numbers of malaria cases. Besides it is widely distributed, this species display great behavioral plasticity and morphological, biological and genetic diversity. The aim of this study was to analyze population genetic diversity of An. darlingi related to geographical distribution, seasonal dynamics and hematophagic behavior, using microsatellites and SNPs markers. An. darlingi specimens were collected in two close rural settlements and in an urban area about 600 km away. In addition, collections were performed in both semesters of the year and, indoor and outdoor during the biting activity period of the vector, i.e., 6pm-6am. The results showed subpopulations of An. darlingi related to geographical aspect, in a macro and micro geographic scales, better accessed with SNPs dataset. Moreover, this study corroborated with a previous finding showing two subpopulations of An. darlingi related to rainfall. Finally, for the first time, genetic heterogeneity was found in sympatric populations of this vector, and it was associated with a phenotype of hematophagic behavior, endo and exophagy. These outcomes demon... (Complete abstract click electronic access below) / Doutor

Orientador: Celso Luis Marino / Resumo: Os resultados obtidos durante o desenvolvimento deste trabalho estão apresentados na forma de capítulos. O primeiro capítulo apresenta uma análise dos parâmetros genéticos de uma população F1 obtida a partir do cruzamento de duas variedades comerciais de cana-de-açúcar oriundas do programa de melhoramento do Centro de Tecnologia Canavieira, Piracicaba/SP. Também avaliamos marcadores moleculares microssatélites buscando uma relação entre os mesmos com as características agronômicas aqui estudadas (Peso do bolo úmido, Peso do bolo seco, Fibra, Lignina e Celulose). No segundo capítulo, o qual está em formato de artigo científico que será submetido para a revista Genome, com o título “A SNP genetic map constructed using restriction site-associated DNA sequencing approach for sugarcane”, apresenta o primeiro mapa genético com marcadores SNPs (Single nucleotide polymorphisms) obtidos via RADSeq utilizando a população F1 de mapeamento obtida a partir do cruzamento de uma variedade comercial dos Estados Unidos com S. spontaneum, oriundos do programa de melhoramento da Texas A&M AgriLife Research, Weslaco, TX, USA. Durante o doutorado realizei estágio na Texas A&M AgriLife Research, Weslaco, TX, USA, sob orientação do Dr. Jorge A. G. da Silva, onde foram realizadas as análises dos componentes lignocelulósicos e o screening de marcadores SSR. Também houve o estágio na Texas A&M AgriLife Genomics and Bioinformatics Service (Texas A&M University, College Station, TX, EUA), sob orientação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The results obtained during this work are presented in chapters. The first chapter shows an analysis of genetic parameters of an F1 population obtained from the crossing of two commercial varieties of sugarcane derived from the Sugar Cane Technology Center breeding program, Piracicaba / SP. We also screened microsatellites in this population seeking a relationship between them with agronomic traits like the weight of wet cane, dry cane weight, fiber, lignin and cellulose content. In the second chapter, which is in scientific paper format to be submitted to Genome journal, entitled "The SNP genetic map constructed using restriction site-associated DNA sequencing approach for sugarcane," presents the first genetic map with SNP markers (single nucleotide polymorphisms) obtained by RADSeq using the F1 population of mapping obtained from the crossing of a commercial variety of the United States with S. spontaneum, coming from the breeding program at Texas A & M AgriLife Research, Weslaco, TX, USA. During the doctoral stage at Texas A & M AgriLife Research, Weslaco, TX, USA, under the supervision of Dr. Jorge A. G. Silva, were carried out the analysis of lignocellulosic components and the screening of SSR markers. There was also a stage at Texas A & M AgriLife Genomics and Bioinformatics Service (Texas A & M University, College Station, TX, USA), under the supervision of Dr. Charles Johnson, where the genotyping for SNPs discovery was made by RadSeq protocol. The bioinformatics a... (Complete abstract click electronic access below) / Doutor

Investigating secondary contact of historically isolated lineages can provide insight into how selection and drift influence genomic divergence and admixture. Here, we studied the genomic landscape of divergence and introgression following secondary contact between lineages of the Western Diamondback Rattlesnake (Crotalus atrox) to determine whether genomic regions under selection in allopatry also contribute to reproductive isolation during introgression. We used thousands of nuclear loci to study genomic differentiation between two lineages that have experienced recent secondary contact following isolation, and incorporated sampling from a zone of secondary contact to identify loci that are resistant to gene flow in hybrids. Comparisons of patterns of divergence and introgression revealed a positive relationship between allelic differentiation and resistance to introgression across the genome, and greater-than-expected overlap between genes linked to lineage-specific divergence and loci that resist introgression. Genes linked to putatively selected markers were related to prominent aspects of rattlesnake biology that differ between populations of Western Diamondback rattlesnakes (i.e., venom and reproductive phenotypes). We also found evidence for selection against introgression of genes that may contribute to cytonuclear incompatibility, consistent with previously observed biased patterns of nuclear and mitochondrial alleles suggestive of partial reproductive isolation due to cytonuclear incompatibilities. Our results provide a genome-scale perspective on the relationships between divergence and introgression in secondary contact that is relevant for understanding the roles of selection in maintaining partial isolation of lineages, causing admixing lineages to not completely homogenize.

Hedgehogs from the genus Erinaceus are an important model organism for studying the postglacial recolonisation of Europe and the processes that take place in the secondary contact zones of their areas of distribution. In this study, five individuals of white-breasted hedgehog (Erinaceus roumanicus), four individuals of western hedgehog (Erinaceus europaeus) and one estimated hybrid were analysed. Geographical distribution of individuals used in the study covers the region of the Central Europe, however in the further research expansion of analsysed individuals will be needed and the whole Palearct should be sampled. The main goal was to implement novel methods in research of hedgehogs, which will enable to map the population-genomic structure of the genus Erinaceus in western Palearct. The method RADSeq (Restriction site associated DNA sequencing) enables to obtain polymorphic markers, e.g., SNPs which we used (Single Nucleotide Polymorphisms) across the genome. In this work it was analyzed 16382 SNPs. Using the binary data which indicates the presence and absence of SNPs for each species, hypotheses raised under classical analyzes of genetic markers from previous studies have not been fully confirmed. In further research it will be necessary to verify possible occurrence of biases connected with...

O talha-mar Rynchops niger (Rynchopinae) e o trinta-réis-grande Phaetusa simplex (Sterninae), são aves aquáticas migratórias que se reproduzem simultaneamente em muitas praias fluviais da América do Sul. A taxonomia e a sistemática destas subfamílias foram objetos de poucos estudos. Além disso, possuem subespécies cuja delimitação e a caracterização ainda são confusas, além do que, uma revisão rigorosa da validade destes táxons nunca foi feita. E, como consequência dos poucos estudos, existe uma enorme imprecisão sobre a real área de distribuição de cada táxon, não havendo muita informação sobre os locais para onde se movimentam após o período reprodutivo. Assim sendo, este trabalho teve como objetivo caracterizar geneticamente e revisar a taxonomia destas duas espécies polítipas, definindo os seus táxons válidos e distribuição. Para tal, foram utilizados caracteres morfológicos (de plumagem e morfométricos), sequências do gene mitocondrial ND2 e de marcadores associados a sítios de restrição (ddRADseq), a fim de estimar a variação intra e interpopulacional, o fluxo gênico e a estrutura genética; visando também entender o padrão de migração destes táxons na América do Sul e a influência dos rios na taxonomia e na história evolutiva destas aves. De acordo com os resultados conclui-se: P. simplex passa a ser considerado um táxon monotípico, pois não é possível separar as subespécies entre si, nem morfologicamente e nem geneticamente. Apesar das variações genéticas entre as três subespécies de R. niger não serem significativas, estas continuam a ser consideradas como subespécies válidas, pois puderam ser plenamente diagnosticáveis quanto aos caracteres de plumagem e de distribuição. Não existem variações genéticas significativas entre as populações. As populações podem estar passando por um processo de expansão recente ou seleção positiva; ou podem estar se comportando como uma metapopulação. Os grandes rios sul-americanos, juntamente com o ciclo sazonal de precipitação da América do Sul (que altera a dinâmica destes rios), influenciam diretamente na distribuição, e, consequentemente, na evolução das aves aqui analisadas. Neste caso, os rios funcionam como vias de contato (e não como barreiras) entre os indivíduos, contribuindo para o intenso fluxo gênico dos táxons aqui apresentados / The Black Skimmer (Rynchops niger) and the Large-billed Tern (Phaetusa simplex) are migratory waterbirds that breed simultaneously on river beaches throughout South America. Few studies have been conducted on the taxonomy and systematics of these polytypic species and the delimitation and validity of each taxa described has never been studied in detail. As a result, the geographical distribution of both species is poorly understood and there is little information about the whereabouts of their non-breeding grounds. Therefore, the purpose of this study was to characterize genetically and revise the taxonomy and distribution of these two polytypic species. For this it was integrated morphological characters (plumage and morphometrics), mitochondrial sequences (ND2), and single nucleotide polymorphisms (SNPs) inferred from double digestion restriction associated DNA sequencing (ddRADseq) to estimate: 1) intra- and inter-populational variation, 2) gene flow, 3) population genetic structure, 4) migration patterns of these taxa within South America, and 5) assess the influence of rivers on the taxonomy and evolutionary history of these birds. The results lead to the following conclusions: P. simplex should now be considered a monotypic taxon because currently recognized subspecies are neither morphologically nor genetically diagnosable. Although genetic variation between the three subspecies currently recognized in R. niger is not significant, these taxa continue to be considered as valid subspecies because they are fully diagnosable in plumage characters and distributional patterns. There are no significant genetic variation between the populations of both species (R. niger and P. simplex). Populations may be undergoing a process of recent expansion or positive selection or they may be behaving like a metapopulation. Main South American rivers, together with the seasonal precipitation cycles of South America (which changes the dynamics of these rivers), have direct influence on the distribution, and, consequently, on the evolution of these birds. In this case, the rivers function as pathways of contact (and not as barriers) between individuals, contributing to the intense gene flow between these taxa

Une forte structure génétique a été décrite dans la littérature parmi les populations européennes de la coque de lagune Cerastoderma glaucum. Cependant, les limites géographiques entre les grandes divisions génétiques varient d'un marqueur à l'autre et leur faible nombre ne permettait pas de tester réellement les échanges génétiques entre ces groupes. Le but principal de cette thèse était donc d'estimer la structure des populations de C. glaucum à l'aide de marqueurs, en considérant les rôles respectifs de la divergence passée et de la fragmentation de l'habitat. Pour atteindre ses objectifs, des populations des côtes atlantiques et méditerranéennes ont été étudiées en utilisant : i) des marqueurs génétiques classiques (un EPIC et des marqueurs microsatellites déjà publiés); ii) des séquences associées à des sites de restriction (“restriction associated DNA sequences”) ou RADseq. Alors que les marqueurs génétiques classiques ont révélé le caractère divergent des populations de Méditerranée orientale, l'approche RADseq a permis de reconstruire les relations phylogénétiques entre groupes au sein de l'espèce avec une résolution jamais encore ateinte. Trois lignées profondément divergentes ont ainsi été identifiée au sein de C. glaucum: une en Mer Egée – Mer Noire, une en Mer Ionienne et la dernière largement distribuée de la Méditerranée à la Baltique. Cette dernière lignée c'est par ailleurs sous divisée en entités isolées en Méditerranée Occidentale, en Atlantique, en Mer du Nord et an Baltique. Comparée à l'espèce soeur C. edule, qui profite d'un habitat moins fragmenté, C. glaucum présente une beaucoup plus forte différenciation génétique entre populations. Il semble donc que des incompatibilités génétiques, conséquences de l'isolement géographique et de l'adaptation locale, aient pu être à l'origine d'un complexe d'espèces. Ces résultats soulignent par ailleurs le rôle évolutif particulier de l'habitat lagunaire, accélérateur de la diversification génétique. / Previous studies found a high level of genetic structuring among the European populations of the lagoon cockle Cerastoderma glaucum. However, the geographic locations of the major genetic divisions differed among markers and the small number of genetic markers used lacked the power to test for gene flow between divergent clusters. Therefore, the main aim of this thesis was to assess the population structure of C. glaucum using new genetic markers in the light of the respective role of past divergence and habitat fragmentation. To reach these objectives, populations from the Atlantic and Mediterranean coasts have been investigated using: i) classical genetic markers (EPIC together with previously published microsatellites); ii) restriction-site associated DNA sequencing (RADseq). When the classical genetic markers revealed the divergent character of Eastern Mediterranean populations, the RADseq approach allowed inferring phylogenetic relationships with an unprecedented resolution. Three deeply divergent lineages were described within C. glaucum: one in the Aegean-Black Sea region, one in the Ionian Sea, and the last one widely distributed from the Western Mediterranean to the Baltic Sea. This last lineage underwent further diversification with isolated entities detected in Western Mediterranean, Atlantic, North Sea and Baltic Sea. Compared to the sister species C. edule, which thrives in a less fragmented habitat, C. glaucum displayed a much stronger genetic differentiation among populations. Consequently, it seems that genetic incompatibilities, which emerged as a by-product of geographic isolation together with local adaptation, led to the origin of a species complex. These results highlight the evolutionary role of lagoon habitats, where genetic diversification can proceed very quickly.

L’anémone de mer symbiotique Anemonia viridis possède cinq morphes définis à l’aide de critères morphologiques. Premièrement, le statut taxonomique de trois des morphes d’A. viridis (var. rufescens, rustica et smaragdina) a été précisé à l’aide de marqueurs moléculaires basés sur des gènes de stress et des marqueurs RAD. Nous avons pu déterminer que ces trois morphes ne formaient qu’une seule espèce et mis en évidence quatre lignées génétiques indépendantes sur base de la géographie (trois en mer Méditerranée, une dans la Manche). Par l’utilisation des variations des séquences ITS2, nous n’avons pu détecter aucune implication du symbiote (Symbiodinium sp.) dans la différenciation des morphes, mais nous avons révélé une composition en symbiotes divergente entre les lignées génétiques indépendantes de l’hôte animal. Par ailleurs, A. viridis se développe dans des environnements particulièrement contrastés, faisant d’elle un modèle d’étude idéal pour l’étude de l’adaptation locale chez les Cnidaires. Par conséquent, l’adaptation locale chez A. viridis a été testée en comparant des populations venant de sites aux conditions environnementales contrastées (surface vs. profondeur et lagune vs. mer). Une recherche de loci outliers sur des marqueurs RAD et des marqueurs de gènes de stress n’a toutefois révélé aucun gène candidat dans l’adaptation locale par rapport aux conditions environnementales testées. Ce travail a donc permis de définir A. viridis comme un organisme extrêmement plastique capable de posséder un fort polymorphisme intrinsèque et de s’acclimater à des habitats contrastés. / The symbiotic sea anemone Anemonia viridis has five morphs described using morphological traits. First, the taxonomical status of three of the morphs of A. viridis (var. rufescens, rustica and smaragdina) was studied using stress gene markers and RAD markers. We revealed that the three morphs were not different species, but that A. viridis was split into four polymorphic independent genetic lineages based on geographical origin (three in the Mediterranean Sea, one in the English Channel). Using ITS2 sequence variation, we could not detect any implication of the symbiont (Symbiodinium sp) in the morph differentiation, but we revealed a divergence in symbiont composition among the geographic independent lineages of the animal host. If no effect of the symbiont was detected, a variable distribution of the ITS2 variants based on geography was revealed. Moreover, A. viridis lives in highly contrasted environments, making it an ideal species to study local adaptation. Thus, local adaptation was tested on A. viridis by comparing populations coming from contrasted environments (shallow vs. deep and lagoon vs. sea). Using RAD and stress genes markers in a search for outlier loci, we revealed no candidate adaptive genes under our environmental conditions. In conclusion, Anemonia viridis seems to be a very plastic organism, with a high intrinsic polymorphism and a high acclimation potential.