Orientadores: Anete Pereira de Souza, Maria Imaculada Zucchi / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T01:07:14Z (GMT). No. of bitstreams: 1 Mori_GustavoMaruyama_D.pdf: 12793055 bytes, checksum: 6364bf6e873c229003959f7098e256c7 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The complete abstract is available with the full electronic document / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Eastern part of the African Sahel, connecting sub-Saharan Africa with North and East Africa, play an important role as a bidirectional corridor for vertically and horizontally migrations of populations. It is the strategic region to study human genetic diversity due to the presence of ethnically, linguistically, culturally and geographically diversity. This work is focused on the analysis of HVS-I and HVS-II segments of mtDNA. The work provides new information about genetic structure and migration activity of this region by analysis twelve populations belonging to three African linguistic families and different subsistent strategies. Analysis of mtDNA revealed the higher diversity of the populations of east Sudan and Horn of Africa, which is connected with the spreading of populations along the Nile River. It seems, that in this region linguistic factors have bigger impact on genetic diversity then the geografic ones. The opposite situation is observed in populations of Chad, where populations with similiar geografic location and different linguistic affilation revealed low genetic differentiation. The intra-population analysis shows the significant influence of genetic drift on the pastoralists living on the Red Sea Coast - Beja and Rashaida. In Beja is probably due to decrease of size of...

The ability to retrieve DNA from the skeletal remains of ancient humans has yielded many insights into the relationship between humans living today and our nearest evolutionary relatives, the Neandertals and Denisovans. Two important insights emerged from the first high-quality genome sequences of Neandertals and Denisovans: 1) these archaic humans had very low genetic diversity in comparison to most populations of present-day humans, and 2) there was gene flow from archaic humans into the ancestors of present-day people. In my thesis, I explored aspects of both these insights. In my first project, I analysed the consequences of low genetic diversity of archaic humans for immune genes, using genetic diversity in protein-coding genes (‘gene diversity’) as a proxy for functional diversity. I conclude that low gene diversity in archaic humans did not affect immune genes more severely than any other class of protein-coding genes. I then show that the MHC genes, that typical have high genetic diversity and are a component of the adaptive immune system, have substantially higher gene diversity than expected from the genome-wide gene diversity in archaic humans. Moreover, I find no detectable reduction in gene diversity between two Neandertals that lived more than 70,000 years apart. This is first evidence indicating that diversity in late Neandertals did not decrease over the last ~100,000 years of their existence, which would be expected if low gene diversity had played a considerable role in Neandertal extinction, as has been proposed. In my second project I analysed genomic regions depleted of both Neandertal and Denisovan ancestry in the genomes of humans living today (‘shared deserts’). It has been suggested that shared deserts reflect incompatibilities between archaic humans and the ancestors of present-day humans, and were created by negative selection against archaic alleles. By analysing archaic ancestry in almost 2,000 published present-day human genomes, including 155 published genomes from Oceania, I generated a further refined set of genomic regions that are most depleted of archaic ancestry. I discuss candidate variants in these regions that may underlie important phenotypic or functional differences between archaic and modern humans, such as in the brain-expressed genes CADM2 and KCND2, and propose this refined list as a set of candidates for future molecular testing.:Bibliographische Darstellung iii Table of contents iv Summary 8 Zusammenfassung 14 1. Introduction 21 1.1. A strange fossil and its genome 21 1.2. Archaic humans had low genetic diversity 26 1.3. Evidence of gene flow between archaic humans and AMH 29 1.3.1. Identification of archaic sequence and its impact on humans today 32 1.3.1.1. The distribution of archaic sequence in AMH is heterogeneous 34 1.3.1.2. Negative selection against introgressed archaic sequence 37 1.3.1.3. Adaptive introgression: Archaic sequence under positive selection in AMH 39 1.3.1.4. Association of introgressed variants with phenotypes of present-day people 41 1.3.2. Deserts: Gene flow left regions depleted of archaic introgression 43 2. Thesis outline 46 3. Methods 47 3.1. Methods for study of immune gene diversity 47 3.1.1. Data 47 3.1.2. Measure of gene diversity 47 3.1.3. Diversity in innate immune and MHC genes 48 3.1.4. GO enrichment analysis 49 3.2. Methods for study of deserts of archaic ancestry 50 3.2.1. Data sets and processing 50 3.2.2. Identification of introgressed haplotypes 51 3.2.2.1. Hidden Markov Model (HMM) 51 3.2.2.2. Probability cut-off for haplotypes to be archaic 51 3.2.3. Reanalysis of published deserts of archaic ancestry 52 3.2.3.1. Shared deserts 52 3.2.3.2. Sliding windows 52 3.2.3.3. Mean percentage introgression 53 3.2.3.4. Comparison to random regions 53 3.2.3.5. Definition of refined shared desert regions 54 3.2.3.6. Overlap of refined shared deserts with genes 54 3.2.3.7. Enrichment analyses in refined shared desert regions 55 3.2.3.8. Overlap with regions under ancient positive selection on the AMH lineage 55 3.2.3.9. Overlap with (nearly) fixed differences between present-day and archaic humans 56 4. Results 57 4.1. Immune gene diversity in archaic and present-day humans 57 4.1.1. Abstract 58 4.1.2. Introduction 59 4.1.3. Results 61 4.1.3.1. Archaic humans had lower overall gene diversity than present-day humans 61 4.1.3.2. Archaic humans had similarly low gene diversity in innate immune genes compared with non-immune genes 62 4.1.3.3. High MHC gene diversity in archaic humans 64 4.1.3.4. Genes with highest/lowest diversity show similar GO enrichments in archaic and present-day humans 66 4.1.4. Discussion 69 4.1.5. Supplementary results 71 4.1.6. Acknowledgements and author contributions 72 4.2. Refining deserts of archaic ancestry 73 4.2.1. Abstract 73 4.2.2. Introduction 75 4.2.3. Results 78 4.2.3.1. Genome-wide patterns of archaic introgression are consistent with previous maps 78 4.2.3.2. The published shared desert regions are not the most depleted regions in the genome 79 4.2.3.3. Levels of archaic introgression in shared deserts for the IGDP data set are comparable 82 4.2.3.4. Shared deserts unique to either the Vernot or Sankararaman set have lower mean percentage introgression 84 4.2.3.5. Refined shared deserts 84 4.2.3.6. Overlap of refined shared deserts with genes 87 4.2.3.7. Enrichment analyses 88 4.2.3.8. Overlap with regions under ancient positive selection on the AMH lineage 89 4.2.3.9. Overlap of refined shared deserts with (nearly) fixed differences (nFD) 89 4.2.4. Discussion 94 4.2.5. Acknowledgements 98 5. Discussion and outlook 99 5.1. Interpreting immune gene diversity in archaic humans 99 5.2. Implications from refined deserts of archaic ancestry 104 5.2.1. Comments on the origin of desert regions 106 5.2.2. Candidates for functional molecular testing in refined deserts 107 5.2.3. Future directions in the characterisation and definition of shared deserts 110 5.3. Future directions beyond shared deserts 115 6. Outlook: Molecular functional testing of candidate variants 118 7. Conclusions and final remark 122 8. Supplementary information (SI) 124 8.1. SI: Immune gene diversity in archaic and present-day humans 124 8.2. SI: Refining deserts of archaic ancestry 152 Index of figures 216 Index of tables 218 Index of supplementary data files 220 References 221 Abbreviations 240 Acknowledgements/Danksagungen 242 Curriculum vitae 244 Publications 248 Selected talks 249 Poster presentations 249 Selbstständigkeitserklärung 250 Nachweis über Anteile der Co-Autor:innen 251

<p>Due to Ireland’s secluded geographical location, its genetic structure is a popular topic of study. The indigenous inhabitants of Ireland remained undisturbed for a long period time, allowing for a distinct genetic population to be created. This peace was disrupted by conflict with invading forces, such as the Nordic Vikings and Anglo-Norman forces. However, these historical events helped to shape both the culture of Ireland and the ancestry seen in the Irish population today. In Ireland, quite like many countries around the world, the male’s surname is passed from father to son, just as the Y-chromosome. The relationship between Irish surnames and their corresponding Y-haplogroups was examined to determine if common and rare Irish surnames can be genetically linked to the historical invasions listed above. The surnames chosen for this study were selected based on their prevalence in Ireland, rare or common, and their proposed historical origin, Irish, Norse or British. To discover any possible patterns in surnames and Y-chromosomal DNA, Y-haplogroups were generated from the DNA of 630 Irish male subjects using an assay specifically developed for the region. The assay contains twenty single-nucleotide polymorphisms (SNPs) that were selected to further resolve the R1b-L21 Y-haplogroup for Irish ancestry, the most prevalent haplogroup in Western Europe, and Ireland in particular. Additional Y-STR data was also generated to examine recent surname history within the collected individuals. Each surname was examined to determine whether one haplogroup occurred more frequently and with this method, distinct patterns in Irish surnames and geographical locations were discovered. In addition to resolving Y-surname history patterns, it is also believed that this assay may be beneficial in determining if an unknown DNA sample is of Western European origin and even in some cases, if a more specific Irish origin can be predicted.</p>

The Western Massasauga (Sistrurus tergeminus) is a small, North American rattlesnake found west of the Mississippi River. Sistrurustergeminushas previously been divided into two putative subspecies, Desert (S. t. edwardsii) and Prairie Massasaugas (S. t. tergeminus) based upon qualitative variation in morphology, coloration, and habitat. The Desert Massasauga subspecies has been formally petitioned for federal listing under the U.S. Endangered Species Act. Ouroverarching goal was to evaluate genetic structure and genomic differentiation between specimens of the two putative subspecies in an effort to inform ongoing conservation assessments. To that end, we generated whole genome sequence data for both putativetaxa and then developed nearly 200 genetic markers from different fractions of the genome (~50 intergenic and ~50 genic markers from each of the two subspecies) to test for population structure across much of the Western Massasauga range. Mean genomic divergence between subspecies was only 0.0041 ± 0.0080 (Kimura’s 2-parameter distance) for nuclear sequences and 0.0175 ± 0.0031 for mitochondrial sequences, both exceedingly low values which approach the minimum of zero. Admixture analyses and F-statistics both indicated that regardless of how the markers were partitioned, genetic structure was oriented far more along a geographic axis (isolation-by-distance) than a taxonomic axis (i.e., between putative subspecies). Overall, our analyses provide little support that formal protection of the purported Desert Massasauga is warranted based on the homogeneity of the collective Western Massasauga gene pool.

The genetics of switchback evolution is largely unknown. While it is assumed that latent gene regulatory networks become reactivated to recreate an ancestral tissue, the details regarding this reactivation has yet to be elucidated. How has a network been maintained over the evolutionary history of this group? Are certain genes within these pathways more susceptible to suppression than other genes? In this study, I examined Euphilomedes carcharodonta, a member of the Sarsielloidea superfamily of ostracods, a clade which has demonstrated the loss and regain of the lateral eye multiple times over its evolutionary history. In particular, I looked at the genetic mechanisms for the development of the sexually dimorphic lateral eye, in which males have large, multifaceted image-forming eyes typical to those of other pancrustaceans (Hexapoda+Crustacea) while females do not. This sexual dimorphism is of particular interest because it allows me to study the genetic underpinnings of a regained trait using individuals of near-identical genetic backgrounds, as these organisms have a singlechromosome sex determination system (XXIXO). Examination of developmental eye genes ec-Dachshund, ec-Daughtless, ecChaoptic, ec-Shaven, and ec-Epidermal growth factor receptor showed differential gene expression patterns in which juvenile male eyes expressed these genes at a higher level than did juvenile female eyes. These genes are thus candidate genetic components of 6 switchback evolution, as this data is a first step towards comparative transcriptomics and gene expression studies comparing multiple species.

Sex determination is a key developmental process in several species regulated by sexrelated transcription factors. In many species a gene called doublesex and mab-3 related transcription factor 1 (dmrt1), plays an important role in sexual differentiation. I used African clawed frogs (Xenopus) to examine function of dmrt1 in two species: a diploid species, X. tropicalis, and an allotetraploid species, X. laevis. In both species, dmrt1 is an autosomal gene; Xenopus tropicalis has one copy of dmrt1 and X. laevis has two homeologous copies that by definition are derived from whole genome duplication: dmrt1.L and dmrt1.S in X. laevis. We generated knockouts of each of these genes to further examine their function in sexual differentiation. Histological examination showed testicular dysgenesis in X. tropicalis dmrt1 and X. laevis dmrt1.L null males whereas dmrt1.S null males presented no obvious difference in sperm density compared to wildtype males. X. tropicalis dmrt1 and X. laevis dmrt1.L null females were found to completely lack reproductive organs and are infertile whereas dmrt1.S null females appeared unaffected. The contrasting results between dmrt1.L and dmrt1.S in X. laevis provides evidence of both neofunctionalization and subfunctionalization following gene duplication and suggest that gene duplication is a major contributor to evolutionary change. Additional investigation of the transcriptome of these frogs and the role of dmrt1 in the secondary sex characteristic vocalization provides further evidence of the role of dmrt1 in development. Comprehensively, this investigation provides further knowledge of the role of dmrt1 and homeologs of this gene in sexual differentiation and introduces a novel aspect of this gene in female development. Future efforts are focused on generating double knockouts for dmrt1.L and dmrt1.S, further examining the role of dmrt1.S in somatic cell function and developing additional mutant lines in other Xenopus for comparative analysis. / Thesis / Master of Science (MSc) / In many species sexual differentiation is a crucial developmental event. Surprisingly, however, the systems orchestrating sexual differentiation are highly variable among species. The doublesex and mab-3 related transcription factor 1 (dmrt1) gene plays a role in sexual differentiation in many groups, but its specific roles in this process are incompletely characterized and potentially diverse. We used genetic engineering in two species of African clawed frog (Xenopus) to disable function of dmrt1 in order to explore effects on gonadal development and the development of secondary sex characteristics. We found that dmrt1 is required for normal ovary or testis development in both Xenopus species, and that functional divergence occurred following duplication of dmrt1 by whole genome duplication. Taken together, these findings identify previously uncharacterized roles of dmrt1 in Xenopus and provide evidence of dynamic functional evolution of this important gene.

A fundamental goal in biological research is to gain an understanding of the evolutionary processes and genetic elements that drive speciation. Genes responsible for reproductive isolation in young divergent lineages are particularly poorly known. In this thesis, the speciation genetics of carrion (Corvus (corone) corone) and hooded (C. (corone) cornix) crows were studied. These taxa differ strikingly in colouration and meet in a narrow hybrid zone in Europe, yet appear to be very similar genetically. A major component of reproductive isolation is social selection on colour differences. First, we investigated the genetic basis of plumage divergence between carrion and hooded crows using a candidate gene approach. Nucleotide divergence was confirmed to be low, while there was no evidence for any of the sequenced genes to be associated with colour differences. Second, we performed a simulation study to assess the performance of RNA-seq, a relatively novel approach that we later employed ourselves. We asked how variation in transcriptome complexity and bioinformatic workflow affected the accuracy of gene expression profiling. We generally found reassuring robustness and made a number of specific recommendations. Third, we compared the corticosterone stress response of carrion and hooded crows. In accordance with the hypothesis that the degree of melanization and physiological traits are correlated due to pleiotropy, we found a higher stress response in hooded crows, and detected possibly associated gene expression in pituitary. Fourth, we investigated genomic divergence by assembling a hooded crow reference genome followed by whole-genome resequencing of four European population samples. Northern European carrion crows were more similar to hooded crows than to Spanish carrion crows, pointing towards rampant introgression far beyond the hybrid zone. Nevertheless, several narrow genomic regions harboured high between-taxon divergence and were potentially associated with phenotypic traits. Fifth, we compared whole-transcriptome gene expression profiles between crows, focusing on skin with developing feathers. We used a design that allowed to differentiate between taxon-specific, colour-specific and body patterning effects. Widespread underexpression of genes in the melanogenesis pathway was associated with grey colour, and we detected several genes that may contribute to colour divergence in this system.

<div>Inferring genetic population structure in endangered, highly migratory species such as sea turtles is a necessary but difficult task in order to design conservation and management plans. Genetically discrete populations are not obvious in highly migratory species, yet require unique conservation planning due to unique spatial and behavioral life-history characteristics. Population structure may be inferred using slowly evolving mitochondrial DNA (mtDNA), but some populations may have diverged recently and are difficult to detect using mtDNA. In these cases, rapidly evolving nuclear microsatellites may better elucidate population structuring. Bayesian inference and ordination may be useful for assigning individuals to inferred populations when populations are unknown. It is important to carefully examine population inference results to detect hierarchical population structuring, and to use multiple, mathematically diverse methods when inferring and describing population structure from genetic data. Here I use Bayesian inference, ordination, and multiple genetic analyses to investigate population structure in Olive ridley sea turtles (ORs; Lepidochelys olivacea) nesting in northwestern Costa Rica (NWCR) and across the entire Eastern Tropical Pacific (ETP). Mitochondrial DNA did not show structure within NWCR, and existing data from prior studies are not appropriately published to compare NWCR to Mexican ORs. In NWCR, Bayesian inference suggested one population, but ordination suggested four moderately structured populations with high internal relatedness, and moderate to high levels of connectivity. In the ETP, Bayesian inference suggested a Mexican and Central American population, but hierarchical analysis revealed a third subpopulation within Mexico. Ordination revealed nine cryptic clusters across the ETP that primarily corresponded to Mexican and Central American populations but contained individuals from both populations, some from other, distant nesting sites. The subpopulation within Mexico was well-defined after ordination, and all clusters displayed high 10 internal relatedness and moderate genetic differentiation. Bottlenecks were detected in both putative populations, at seven Mexican and two Central American nesting beaches, and in six out of nine inferred clusters, including three out of four Mexican clusters. Bottleneck events may have played some role in cluster differentiation. Migration was significant from Mexico to Central America at multiple levels, but did not necessarily agree with potential migrants elucidated by ordination. Migration was generally lower between ordination-inferred clusters than between nesting sites or Bayesian-inferred clusters. Phylogenetic trees generally supported structuring by ordination, rather than by Bayesian inference. Structuring in ordination not tied to bottleneck events could be due to mating behaviors or patterns of nesting beach colonization dictated by environmental features. In this study, ordination provided a more practical and nuanced framework for defining MUs and DIPs in ETP ORs than did STRUCTURE. This may be due to hierarchical structuring within ETP ORs that may be present in other sea turtle populations and species. In the case of ETP ORs, hierarchical structure may be an artefact of recent population bottlenecks and subsequent recolonization of nesting beaches, or due to mating at foraging grounds or along migratory routes. Bayesian inference may not be the best method for population inference in highly migratory species such as sea turtles, which have a high potential for broad scale genetic connectivity, and therefore may display hierarchical population structuring not easily related to nesting sites. Future studies, and perhaps published studies, should incorporate Bayesian inference and ordination, as well as other measures of population divergence and descriptive statistics, when searching for population structure in highly migratory species such as sea turtles.</div>

Plants have spread to almost everywhere in the world. As they disperse, they meet many different environments to which they may be able to adapt. For a plant species to adapt to a new environment, genetic variation is needed. The individuals differ from each other in their genetic composition, which often means differences in phenotypes. Those individuals that manage to reproduce will form the next generation. With different conditions in different environments, it will not be the same phenotypes that reproduce everywhere. In that way, plant species will form into a mosaic of locally adapted populations varying genetically as the species disperses. After the last ice age plants have started to disperse away from the equators. With increasing latitudes come increasing challenges to migrating plants. As plant species disperse northwards along this gradient of varying conditions individuals are selected for cold adaptive traits like flowering time and freezing tolerance, acquired by cold acclimation. In this way, genetic variation from the original populations for these traits becomes sorted out along a latitudinal cline. The aim of this thesis was to understand how selection along a latitudinal gradient has shaped natural variation in cold adaptive traits in plants dispersing northwards, and specifically, to investigate what variation can be observed in phenotypes for these traits and how these traits correlate with genetic variation in genes known to be involved in cold acclimation. In this study significant variation was found in a sample of the model plan Arabidopsis thaliana accessions in cold adaptive traits flowering time and freezing tolerance. A clear latitudinal cline in the cold adaptive traits freezing tolerance for A. thaliana was observed. Analysis of nucleotide polymorphism for the cold responsive ICE1 (inducer of CBF expression 1) transcription factor revealed a haplotype structure with two allelic clades as well as unusually high levels of synonymous polymorphism. Nucleotide polymorphism analysis for the transcription factors CBF1, CBF2 and CBF3 (C-repeat binding factors) that play a key role in regulating the expression of a group of target genes known as the “CBF regulon” showed a distinct geographical haplotype structure. One haplotype was dominant in southern accessions while in the other northern accessions overrepresented. There was a significant effect of CBF haplotype on both freezing tolerance and flowering time even after correcting for latitude. Significant differences in CBF expression levels were found between the different CBF genes as well as between different accessions. Sequence variation at CBF was shown to have a significant effect on expression levels of CBF2. No clear correlations were found between CBF gene expression and freezing tolerance or temperature sensitivity for any of the accessions used in the study. This highlights the complex relationship between sequence variation in candidate genes and gene expression, and the problems associated with unraveling the genetic basis of ecologically important traits.