Dissertations / Theses on the topic 'Population genetics analyses'

Many of the patterns of genetic variation we observe today have arisen via the complex dynamics of interactions and isolation of historic human populations. In this thesis, we focus on two important features of the genetics of populations that can be used to learn about human history: population structure and admixture. The Iberian peninsula has a complex demographic history, as well as rich linguistic and cultural diversity. However, previous studies using small genomic regions (such as Y-chromosome and mtDNA) as well as genome-wide data have so far detected limited genetic structure in Iberia. Larger datasets and powerful new statistical methods that exploit information in the correlation structure of nearby genetic markers have made it possible to detect and characterise genetic differentiation at fine geographic scales. We performed the largest and most comprehensive study of Spanish population structure to date by analysing genotyping array data for ~1,400 Spanish individuals genotyped at ~700,000 polymorphic loci. We show that at broad scales, the major axis of genetic differentiation in Spain runs from west to east, while there is remarkable genetic similarity in the north-south direction. Our analysis also reveals striking patterns of geographically-localised and subtle population structure within Spain at scales down to tens of kilometres. We developed and applied new approaches to show how this structure has arisen from a complex and regionally-varying mix of genetic isolation and recent gene-flow within and from outside of Iberia. To further explore the genetic impact of historical migrations and invasions of Iberia, we assembled a data set of 2,920 individuals (~300,000 markers) from Iberia and the surrounding regions of north Africa, Europe, and sub-Saharan Africa. Our admixture analysis implies that north African-like DNA in Iberia was mainly introduced in the earlier half (860 - 1120 CE) of the period of Muslim rule in Iberia, and we estimate that the closest modern-day equivalents to the initial migrants are located in Western Sahara. We also find that north African-like DNA in Iberia shows striking regional variation, with near-zero contributions in the Basque regions, low amounts (~3%) in the north east of Iberia, and as high as (~11%) in Galicia and Portugal. The UK Biobank project is a large prospective cohort study of ~500,000 individuals from across the United Kingdom, aged between 40-69 at recruitment. A rich variety of phenotypic and health-related information is available on each participant, making the resource unprecedented in its size and scope. Understanding the role that genetics plays in phenotypic variation, and its potential interactions with other factors, provides a critical route to a better understanding of human biology and population health. As such, a key component of the UK Biobank resource has been the collection of genome-wide genetic data (~805,000 markers) on every participant using purpose-designed genotyping arrays. These data are the focus of the second part of this thesis. In particular, we designed and implemented a quality control (QC) pipeline on behalf of the current and future use of this multi-purpose resource. Genotype data on this scale offers novel opportunities for assessing quality issues, although the wide range of ancestral backgrounds in the cohort also creates particular challenges. We also conducted a set of analyses that reveal properties of the genetic data, including population structure and familial relatedness, that can be important for downstream analyses. We find that cryptic relatedness is common among UK Biobank participants (~30% have at least one first cousin relative or closer), and a full range of human population structure is present in this cohort: from world-wide ancestral diversity to subtle population structure at sub-national geographic scales. Finally, we performed a genome-wide association scan on a well-studied and highly polygenic phenotype: standing height. This provided a further test of the effectiveness of our QC, as well as highlighting the potential of the resource to uncover novel regions of association.

Humans are dependent on the animals they raise and breed for food and secondary products. Archaeological and genetic investigations can provide critical insights into the history and development of these breeds and help understand human activities in the past. Furthermore, many well-adapted breeds are endangered and archaeological and genetic data can help inform future breed conservation choices. Utilising ancient DNA data could potentially permit detailed diachronic analyses of the development of animal breeds. Ancient DNA analyses have typically focussed on large-scale biogeographic patterns in time and space, such as the spread of domesticates or the movements of peoples. Few studies have attempted fine-scale diachronic analysis within single animal populations or breeds. This is largely due to restricted sample availability and the limited phylogenetic resolution provided by the mitochondrial genome, the most commonly used ancient DNA marker. In this thesis, I demonstrate that fine-scale diachronic analyses within single animal populations and breeds over short time scales are feasible. First, in order to address the limitations of sample size, I assessed three sample screening methods’ abilities (maximum mitochondrial DNA amplicon length, NanoDrop® spectrometry and collagen preservation) to select samples in which DNA was preserved and analysed the utility of parchment as a novel source of ancient and historic DNA. None of the screening methods accurately predicted DNA preservation, but collagen preservation was able to weed out extremely poorly preserved samples from further analysis. All but one of the tested parchments produced multiple sequences matching several different species. Parchment therefore was not appropriate for fine-scale diachronic analyses. Next, I assessed whether analysing the nuclear genome could permit fine-resolution diachronic genetic studies. Since single nucleotide polymorphisms are ideal candidate nuclear markers for diachronic DNA analyses, I assessed the accuracy of the nuclear SNP-typing methodology, SNaPshot™, by genotyping three coat colour markers for a sample of historic Thoroughbred horses for which both phenotypic and correct genotypic information were known from pedigree information in the General Stud Book. The SNaPshot™ protocol was found to provide accurate genotypic information in all cases. Finally, as a proof of method, I compared the diachronic information provided by the mitochondrial and nuclear genomes in Icelandic and Thoroughbred horses. Specifically, in the Icelandic horse, I analysed the mitochondrial D-loop and three coat colour genes in modern and historic populations. In the Icelandic horse, I found statistically significant evidence for genetic change in the mitochondrial genome over the last 150 years. I found no evidence for change in coat colour allele frequencies. Conversely, in the biased and small historic Thoroughbred dataset, the mitochondrial genome was insufficient to provide population-level information, but I was able to show that allele frequencies in the nuclear MSTN gene, a gene previously shown to influence racing performance, have changed significantly in the past century.

Paronychia virginica Spreng. (Caryophyllaceae) is a perennial evergreen herb of exposed, relatively xeric habitats. Approximately 10 mid-Appalachian populations remain in Virginia, West Virginia, and Maryland and are disjunct from populations located primarily in Texas, Oklahoma, and Arkansas. A study was conducted to test the hypothesis that eastern and western populations differ significantly and, therefore, represent at least two distinct taxa. Statistical analyses of 8 qualitative and 24 quantitative morphological characters indicated very highly significant (P < 0.001) variation between eastern and western populations of P. virginica. Characters differing most significantly included sepal pubescence, awn length, awn pubescence, awn curvature, length-width ratio of leaves, and shape of leaf apices. Starch gel electrophoresis was performed and six enzyme systems/nine loci (EST-2, EST-3, LAP, MDH-1, MDH-2, PGI, PGM-1, PGM-2, and SKDH) were identified as being consistently scorable and informative. Although gene flow between populations of P. virginica was shown to be restricted (mean FST = 0.353), populations are maintaining relatively high levels of genetic diversity. Genetic variability was quantified for each population and mean values for number of alleles per locus (A), percent loci polymorphic (P), and expected heterozygosity (HEXP) were found to be 1.95, 47.22%, and 0.204, respectively, exceeding those values reported for seed plants, widespread species, and endemic species. Hierarchical F statistics suggest higher levels of genetic variability within individual populations than among populations, regardless of geographic location. All statistically significant (P < 0.05) deviations from Hardy-Weinberg equilibrium indicated a deficiency in heterozygotes at the respective loci. Considering results from both the morphometric and allozyme analyses, the current author suggests recognizing two distinct subspecies, P. virginica subsp. virginica in the eastern U.S. and P. virginica subsp. scoparia in the south-central U.S. Conservation efforts should be focused on the maintenance of existing populations in both eastern and western regions of the U.S. in order to preserve the genetic and evolutionary potential of these taxa.
Master of Science

Basque people have been the focus of many studies in the last decades due to cultural and genetic characteristics that place them as an outlier population within the European landscape. Despite the existence of large amount of studies, several controversies exist in the population genetics field: their highest frequencies of Rh-negative, a strong genetic differentiation, a genetic substructure, and their connections with the main ancient European groups along history. Most of the previous studies focused on Basques have suffered from methodological flowbacks and weak study designs. In this thesis, these controversies are elucidated by using a more refined and accurate methodology, together with an exhaustive ethnolinguistic representation of the Basque population and surrounding groups. The main results show a high frequency of Rh-negative in agreement with previous studies, an internal genetic heterogeneity within Basques, together with a clear differentiation from the external populations, probably marked by a genetic continuity from the Bronze Age.
La población vasca ha sido objeto de estudio durante las últimas décadas, debido a características culturales y genéticas que parecen definirlos como una población aislada dentro del contexto europeo. A pesar de la existencia de numerosos estudios, diversas controversias han surgido en el área de genética de poblaciones: una de las frecuencias más altas de Rh-negativo, una fuerte diferenciación genética, una subestructura poblacional y la relación con las principales poblaciones antiguas en Europa. Los estudios previos han estado sujetos a una metodología y un muestreo limitados. En esta tesis, se abordan dichas controversias con una metodología más refinada y precisa, además de una representación exhaustiva de la población vasca y de poblaciones circundantes desde un punto de vista etnolingüístico. Los principales resultados muestran: una alta frecuencia de Rh-negativo similar a la observada en trabajos anteriores, una heterogeneidad genética interna, junto con una clara diferenciación de las poblaciones externas, marcada posiblemente por una continuidad genética desde la Edad de Bronce.

The advent of high-throughput DNA and RNA sequencing has made possible the assay of millions of nucleic acid molecules in parallel. This allows functional genomic elements to be identified from background in single-tube experiments. This dissertation discusses the development of two such functional screens as well as work implementing a third that was previously developed in my thesis laboratory. Restriction-Associated DNA sequencing (RAD-Seq) is a complexity reduction sequencing method that allows the same subset of genomic sequence to be read across multiple samples. Differences in sample collection and data analysis allow manifold applications of RAD-Seq. Here we use RAD-Seq to identify mutant genes responsible for altered phenotypes in Caenorhabditis elegans and to identify hyper-invasive alleles in trout population admixtures. Apart from acquiring genomic sequence data, massively-parallel sequencing can be used for counting applications that quantify activity across a large number of test molecules. This dissertation describes the development of a technique for simultaneously quantifying the activity of a restriction enzyme across all possible DNA substrates by linking digest of a sequenced genome to Illumina-sequencing in an unbiased fashion. Finally, a powerful approach to analyze transcriptional activation is described. This method quantifies output from millions of potential DNA transcriptional enhancers via RNA amplicon sequencing of covalently-linked randomer tags and is used in conjunction with RNA-Seq to provide a mechanistic view of hypoxic gene regulation in Drosophila. This dissertation includes previously published, co-authored material

Phylogeographic studies have relied on surveying neutral genetic variation in natural populations as a way of gaining better insights into the evolutionary processes shaping present day population demography. Recent emphasis on understanding putative adaptive variation have brought to light the role of epigenetic variation in influencing phenotypes and the mechanisms underlying local adaptation. While much is known about how methylation acts at specific loci to influence known phenotypes, there is little information on the spatial genetic structure of genome-wide patterns of methylation and the extent to which it can extend our understanding of both neutral and putatively adaptive processes. This research examines spatial genetic structure using paired nucleotide and methylation genetic markers in the Sonoran bark beetle, Araptus attenuatus, for which we have a considerable knowledge about its neutral demographic history, demography, and factors influencing ongoing genetic connectivity. Using the msAFLP approach, we attained 703 genetic markers. Of those, 297 were polymorphic in both nucleotide (SEQ) and methylation (METH) were assayed from 20 populations collected throughout the species range. Of the paired SEQ and METH locis, the METH were both more frequent (16% vs. 7%), maintained more diversity (Shannon IMeth = 0.361 vs. ISeq=0.272), and had more among-population genetic structure (ΦST; Meth = 0.035 vs. ΦST; Seq= 0.008) than their paired SEQ loci. Interpopulation genetic distance in both SEQ and METH markers were highly correlated, with 16% of the METH loci having sufficient signal to reconstruct phylogeographic history. Allele frequency variation at five loci (two SEQ and three METH) showed significant relationships with at-site bioclimatic variables suggesting the need for subsequent analysis addressing non-neutral evolution. These results suggest that methylation can be as informative as nucleotide variation when examining spatial genetic structure for phylogeography, connectivity, and, identifying putatively adaptive genetic variance.

The fixation of beneficial variants leaves genomic footprints characterized by a reduction of genetic variation at linked neutral sites and strong, localized allele frequency differentiation among subpopulations. In contrast, for phenotypic evolution the effect of adaptation on the genes controlling the trait is little understood. Theoretical work on polygenic selection suggests that fixations of beneficial alleles (causing selective sweeps) are less likely than small-to-moderate allele frequency shifts among subpopulations. This thesis encompasses three projects in which we have experimentally addressed the issue of selective sweeps vs. allele frequency shifts in the context of polygenic adaptation. We studied three X-linked QTL underlying variation in chill coma recovery time (CCRT), a proxy for cold tolerance, in Drosophila melanogaster from temperate (European) and tropical (African) environments. The analysis of these QTL was performed by means of selective sweep mapping and quantitative complementation tests coupled with expression assays. While the results of the selective sweep mapping approach identified a gene (CG4491) that is unlikely to be affecting CCRT, quantitative and gene expression analyses revealed two linked candidate genes (brk and CG1677) that appear to differ in their evolutionary histories. We found that the difference in expression of the gene brk between populations affects CCRT variation. Cold tolerant flies from the temperate zone have a lower expression of this gene than cold sensitive flies from the tropics. We found that a likely cause of this difference is variation in a cis-regulatory element in the brk 5’ enhancer region. Sequence variants in this element exhibit moderate frequency differences between populations from temperate and tropical environments, forming two latitudinal clines: one from the equator to the north and another one in opposite direction to the south. In contrast, the other gene within the same QTL (CG1677), which is linked to brk, showed no measurable effect on cold tolerance but is a likely target of strong positive selection leading to a selective sweep in the European population. These results are consistent with the aforementioned theoretical predictions about footprints of selection in polygenic adaptation. They are also proof of the conceptual bias incurred when identifying candidate genes within a QTL via selective sweep mapping, at least in naturally evolving populations. The challenge for the evolutionary genetics community in the coming years is to develop statistical tools that are as powerful and robust as those already available to map selective sweeps to identify sites in the genome where allele frequency shifts have occurred due to adaptive evolution at the phenotypic level. Finally, the last section of the results is a report of a new population genetics dataset. It consists of a collection of 80 inbred lines from a natural D. melanogaster population in Sweden and 19 full genome sequences derived from this sample. We hope this material will provide us with further insight into the processes underlying adaptation to novel and stressful environments.

The nurse shark, Ginglymostoma cirratum, is a sedentary shark species that inhabits coral reefs in the tropical and subtropical Atlantic Ocean and along the western coast of the Americas in the Pacific Ocean. Nurse shark tissue samples were collected from the Bahamas, Belize, Brazil, and Dry Tortugas National Park in Florida. 186 individuals were genotyped at 11 microsatellite loci, the control region of the mitochondrial genome was sequenced in 190 individuals, and 89 individuals from the Bahamas, Belize, and Dry Tortugas were genotyped at the major histocompatibility complex (MHC) class IIα locus. An analysis of molecular variance (AMOVA) for the microsatellite loci indicated significant subdivision only between the Bahamas and Dry Tortugas populations. An AMOVA for the mitochondrial control region sequences indicated significant subdivision between all population pairs. The AMOVA for MHC class IIα locus indicated significant subdivision between two population pairs: the Bahamas population and the Dry Tortugas population and the Belize population and the Dry Tortugas population. The nurse shark has the lowest mitochondrial DNA nucleotide diversity (π=0.0125%) and haplotype diversity (h=0.2402) of any shark species to date. There were 14 MHC alleles from 39 polymorphic sites; ten were the same as published alleles (Kasahara et al. 1993; Ohta et al. 2000). This study was the first study to use MHC class IIα genes as a marker for population genetics in sharks. Our results showed that MHC class IIα locus behaves as a diploid locus and is a powerful tool for determining population genetic structure between populations.

Le Liban est un pays qui constitue un carrefour des civilisations. Depuis le temps des pharaons, ses ressources forestières ont été exploitées, notamment pour le commerce du bois. Le Liban, connu pour son cèdre, possède une autre espèce emblématique, le sapin de Cilicie, dont les forêts sont en majorité non protégées. Historiquement, le bois de sapin a été exploité durant le Nouvel Empire égyptien ancien pour la construction des temples et des navires. Il représentait ainsi un signe de pouvoir du pharaon, en formant notamment la barque d'Amon. De même, le sapin a été coupé pour la construction du temple de Jérusalem, ainsi que des instruments de musique et de guerre. La fragmentation des sapinières au Liban n'est pas ancienne mais cette faible divergence se traduit cependant par un dème Nord-Est englobant 11 populations et un dème Sud-Ouest englobant 4 populations qui semblent être le résultat de deux processus démographiques consécutifs ou simultanés durant l'histoire du sapin au Liban. Le premier est un phénomène de migration en altitude en réponse à des changements dans l'environnement ou le climat. La reconstruction de la dynamique passée du sapin au Liban a montré que le sapin a subi des fluctuations importantes dans sa taille, depuis le Tardiglaciaire, il y a 14,000 ans. Notamment, le sapin a connu des périodes d'absence du registre pollinique qui pourrait être liées à la fragmentation anthropique de l'habitat ou à des extinctions locales ou contraction de l'aire de répartition. De même, il a connu des périodes d'expansion notamment au cours des événements de sécheresse dans le climat, notamment à 4090 cal. BP, à 5010 cal. BP et de 7800 à 8090 cal. BP. La richesse en allèles privés dans le dème Nord-Est indique la présence de plusieurs micro-refuges glaciaires de basses et de hautes altitudes, ainsi que des zones de suture issues de la recolonisation. Dans le dème Sud-Ouest, une recolonisation postglaciaire en altitude à partir du seul micro-refuge glaciaire détecté est probable. Le deuxième phénomène est lié à une migration asymétrique des populations génétiquement diversifiées du centre du dème vers les populations marginales génétiquement peu diversifiées. Ce processus, qui semblait être le résultat de la faible taille des populations cibles, pourrait permettre de retarder l'extinction des populations marginales, localement menacées. La superformance de la migration sur la dérive génétique et la dispersion sur des longues distances de 15 à 20 km constituent les effets médiateurs de ces processus démographiques. L'empreinte de cette dynamique démographique est une réduction historique de la taille effective des populations sur le long terme avec un signal ancien plutôt que récent, et une diversité génétique et richesse allélique basses. Cette diversité génétique semble être façonnée par les effets anthropiques ainsi que par les changements dans l'environnement ou le climat. La conservation in situ et ex situ de ces sapinières est nécessaire pour préserver leur patrimoine historique et génétique
The Lebanon is a country that constitutes a crossroads of civilizations. Back in the time of pharaohs, fir forests in Lebanon were exploited, particularly for the timber trade. Lebanon, known for its cedar, has another emblematic species, the Cilician fir, whose forests are in majority unprotected. Historically, the fir was used during the ancient Egyptian New Kingdom rule over Phoenicia for the construction of temples and ships. Notably, it represented a sign of power of the pharaoh, forming the sacred barque of Amun. Similarly, this tree was cut from Lebanon to build the temple of Jerusalem, as well as for making instruments of music and war. The fragmentation of the fir populations in Lebanon is not ancient but their low divergence, however, is marked by a Northeastern Ridge including 11 populations and a Southwestern Ridge including 4 populations that seem to be the result of two consecutive or simultaneous demographic processes during the history of fir in Lebanon. The first is a phenomenon of altitudinal migration in response to changes in the environment or climate. The reconstruction of the past dynamics of fir in Lebanon showed that it has undergone significant fluctuations in size, since the Late Glacial, 14,000 years ago. In particular, the tree has experienced periods of absence from the pollen record that could be related to anthropogenic habitat fragmentation or to local extinctions or contraction of the range of distribution. Similarly, there have been periods of expansion especially during periods of drought in the climate, at 4090 cal. BP, at 5010 cal. BP and between 7800 and 8090 cal. BP. The private allelic richness in the Northeastern Ridge indicated the presence of multiple glacial microrefugia of low and high elevations, as well as suture zones issues from recolonization. In the Southwestern Ridge, postglacial altitudinal recolonization from single microrefugial population is moslty probable. The second phenomenon is related to an asymmetric Northeast-Southwest migration from genetically diverse populations towards marginal and less genetically diverse populations. This process, which seems to be the result of the small size of the target populations, could help delay the extinction of marginal populations, locally threatened. The outperformance of migration over genetic drift and the dispersal over long distances of 15 to 20 km constitute the mediating effects of these demographic processes. The footprint of these population dynamics is a historic reduction in the effective population size on the long-term rather than on the short term, and weak genetic diversity and allelic richness. This genetic diversity seems to be shaped by anthropogenic effects as well as by changes in the environment or climate. In situ and ex situ conservation of fir populations in Lebanon is necessary to preserve their historical and genetic heritage

Les familles de gènes impliqués dans le cancer et autres maladies génétiques se sont beaucoup élargies via deux Duplications Globales de Génome (DGG) qui ont eu lieu à l'origine des vertébrés. La rétention des copies de ces gènes implique une susceptibilité plus grande aux maladies génétiques et constitue une énigme du point de vue de l'évolution. Dans cette thèse, nous avons généralisé des modèles classiques de génétique des populations pour révéler le mécanisme non-adaptatif qui a conduit à cette conservation de gènes potentiellement délétères chez les vertébrés. Nous avons résolu un modèle déterministe haploïde, nous avons étendu ce modèle à des génomes diploïdes et nous avons analysé les effets de taille finie des populations et de la sélection positive par une approche stochastique. Les résultats montrent, en accord avec les données génomiques du cancer chez l'homme, que les copies DGG susceptibles aux mutations délétères dominantes sont conservées indirectement via la sélection de purification dans les espèces post-DGG, qui présentent nécessairement une incompatibilité de ploïdie avec la population pre-DGG. Les résultats obtenus en étendant des méthodes avancées d'inférence bayésienne, quantifiant les effets causaux directs, soutiennent l'hypothèse d'une influence directe de la susceptibilité aux mutations délétères dominantes sur la rétention des copies DGG. Ces résultats révèlent le mécanisme d'évolution non-adaptatif responsable de la rétention de gènes DGG susceptibles aux mutations délétères dominantes et notre extension de méthodes d'inférence bayesienne ouvre la voie à la quantification des relations causales directes dans un large ensemble de problématiques
Gene families implicated in cancer and other genetic diseases have been greatly expanded through two rounds of whole-genome duplication (WGD) that occurred at the onset of jawed vertebrates. However, such gene duplicates are expected to lead to an enhanced susceptibility to genetic diseases, and thus their retention represents an evolutionary puzzle from a natural selection perspective. In this thesis, we have expanded classical population genetics models to reveal the non-adaptive mechanism through which such potentially deleterious ohnologs (WGD-duplicated genes) were retained in the vertebrate genomes. We have solved a deterministic haploid model, we have considered extensions to diploid genotypes, and we have analyzed population size effects and the impact of positive selection through a stochastic approach. The results demonstrate, consistently with available human cancer genome data, that ohnologs prone to dominant deleterious mutations are indirectly selected through purifying selection in post-WGD species, arisen through the ploidy incompatibility between post-WGD individuals and the rest of the pre-WGD population. Extending advanced Bayesian inference methods to quantify direct and indirect causal effects, we have found further supporting evidences for the direct role of the gene susceptibility to deleterious mutations on ohnolog retention. Our findings rationalize the evolutionary mechanism responsible for the expansion of ohnologs prone to dominant deleterious mutations, highlighting the role of WGD-induced speciation. Our extension of Bayesian inference methods paves the way for the identification of direct causal relationships in a huge variety of problems

Human genome project published their first human whole genome sequence on 2001 at the cost of billions of dollars. Since, the cost of sequencing is decreasing faster than Moore’s law. Now, we not only have sequenced thousands of modern humans’ whole genome, we also obtained whole genome sequences of extinct hominin and other ancient modern humans with relatively good quality. These sequences granted us some unexpected results: like how recently modern humans left Africa and populated around all over the world (which is called recent African origin model) while doing so how they have admixed with multiple hominin populations. Until now modern biology (unlike physics) always dominated by empirical results compared to theoretical concepts, which forces people to perceive biology as a descriptive science. As we are obtaining more and more data every day, it is now time to push our theoretical concepts before empirical results in biology. Here in this thesis, we provided deeper knowledge about ancestry of Indian, Asian and Pacific populations. We were also able to reveal an unknown hominin population existed even before it is sequenced. In addition to these, we demonstrated strong natural selection could change human morphology drastically in a short period.
El projecte del genoma humà va publicar la primera seqüència completa del genoma humà el 2001 amb un cost de milers de millions de dòlars. Després d'això, el cost de la seqüenciació està disminuint més ràpid que la llei de Moore. Actualment no només tenim la seqüència de del genoma humà, sinó que tenim la de molts humans i d’homínids extingits amb una qualitat relativement bona. L’estudi de les seqüències de molts genomes humans varen proporcionar la base per postular que els humans moderns es varen originar a Àfrica, i en la sortida d’Àfrica (Out Of Africa) varen poblar la resta del món, amb una certa barreja amb diferents poblacions d'homínids. La base del treball en biologia i en genòmica evolutiva ha estat fonamentalment empírica (a diferència de la física), però actualment la disponibilitat de moltes dades permet empenyer la recerca cap a aspectes molt més analítics: aquest és l’enfocament del nostre treball en seqüències de DNA. Aquí, en aquesta tesi, hem proporcionat un coneixement més profund sobre l’origen i l'ascendència de poblacions indígenes, d’Àsia i del Pacífic, centrant-nos en la India continental i especialment en les Illes Andaman. També hem estat capaços de revelar l’existència d’una població d'homínids desconeguts que es va barrejar amb els ancestres d’aquestes poblacions. A més, hem demostrat que una forta selecció natural pot canviar dràsticament la morfologia humana en un curt període de temps i que explicaria la morfologia pigmea del pobladors de les illes Andaman.

How populations differentiate and become new species is a foundational question to the field of evolutionary biology and has important implications for the generation of both local and global patterns of species-level biodiversity. Ernst Mayr emphasised the importance of geographical isolation as a driver of speciation: “populations in separate locations begin a process of differentiation, and once differentiation is sufficient the populations have become two species”. In marine environments, the lack of obvious physical barriers would suggest that panmixia, especially in highly mobile species, or isolation-by-distance, in other cases, will prevail. However, there is some counterintuitive evidence of fine-scale differentiation among populations and species in a number of mobile marine organisms, a phenomenon that has been described as the “marine species paradox”. Seabirds of the order Procellariiformes present some of the most extreme examples of this paradox. On the one hand, Procellariiformes are highly mobile pelagic seabirds with a high dispersal ability and perform some of the longest animal migrations on Earth. On the other hand, they show high philopatry to their breeding grounds, which is expected to limit gene flow and therefore reinforce genetic differentiation. This thesis aims to gain insights into the patterns and processes that contribute to genetic and phenotypic diversification, speciation and dispersal across multiple evolutionary timescales. To this end, I focus on shearwaters (Calonectris, Puffinus and Ardenna), a globally distributed and threatened group of Procellariiformes. Through an integrative approach combining two types of phylogenomic markers, which evolve at different nucleotide substitution rates, and state-of-the-art phylogenetic and introgression analyses, I inferred a robust phylogeny. This approach allowed to discover that the majority of the phylogenetic conflict in shearwaters is generated by high levels of incomplete lineage sorting (ILS) due to rapid speciation events. Divergence time estimation analyses highlighted a severe impact of the Pliocene marine megafauna extinction on shearwaters, probably caused by a sudden reduction in the availability of coastal habitat. Subsequently, the late Pliocene-early Pleistocene was inferred as a period of high and rapid speciation and dispersal, probably promoted by Pleistocene climatic shifts. Biogeographic analyses showed that surface ocean currents promote species dispersal and founder events are a main mode of speciation in shearwaters. Our analysis, combining genomic data with morphological and ecological evidence, did not support any of the current taxonomic classifications for the North Atlantic and Mediterranean Puffinus shearwaters, and so I propose a more accurate taxonomy for the group. Moreover, the detection of fine-scale genetic structure within Puffinus shearwater species, highlights the need for management of evolutionary significant units below the species level. Population genomics analyses identified genetic drift as the major process shaping the genomic landscapes of divergence. In conclusion, the marriage of these various investigations identifies a prevalence of ILS across different timescales, highlights the important role of paleoceanographic events in promoting diversification, and demonstrates the importance of neutral evolution at driving population differentiation in pelagic seabirds. Overall, this thesis showcases the use of multiple genomic approaches, leveraging phylogenetic and population genetic analyses across multiple timescales, to shed light on the evolutionary history of shearwaters.
Una de les preguntes fundacionals del camp de la biologia evolutiva és com es diferencien les poblacions i esdevenen noves espècies, i té importants implicacions en l’establiment dels patrons locals i globals de biodiversitat específica. Ernst Mayr va subratllar la importància de l’aïllament geogràfic com a mecanisme promotor d’especiació: “les poblacions en localitats aïllades comencen un procés de diferenciació i, quan aquesta diferenciació és suficient, les poblacions esdevenen dues espècies”. En l’ambient marí, la manca de barreres físiques evidents suggereix que la panmixi (en espècies amb gran capacitat de moviment) i l’aïllament per distància (en espècies de mobilitat reduïda) són els patrons prevalents. En canvi, s’ha detectat diferenciació a petita escala entre diferents poblacions i diferents espècies en un elevat nombre d’organismes marins mòbils, un fenomen que es coneix com la “paradoxa de les espècies marines”. Els ocells marins de l’ordre dels Procellariiformes representen un dels exemples més extrems d’aquesta paradoxa. Per una banda, els Procellariiformes són ocells marins amb gran capacitat de moviment i alta capacitat dispersiva que duen a terme algunes de les migracions més llargues del planeta. Per altra banda, són espècies molt fidels a les seves zones de cria, fenomen que probablement limiti el flux gènic i reforci la diferenciació genètica. Aquesta tesi té com a objectiu caracteritzar els patrons i processos que contribueixen a la diversificació genètica i fenotípica, a l’especiació i a la dispersió a diferents escales evolutives. Per assolir aquest objectiu, m’he focalitzat en les baldrigues (Calonectris, Puffinus i Ardenna), un grup amenaçat de Procellariiformes distribuït per tot el planeta. Mitjançant una aproximació que combina dos tipus de marcadors filogenòmics que evolucionen amb diferents taxes de substitució nucleotídica, i anàlisis filogenètiques i de detecció d’introgressió infereixo una filogènia ben resolta. Aquesta aproximació ha permès descobrir que la majoria del conflicte filogenètic en les baldrigues és producte dels alts nivells de sorteig incomplet de llinatges (incomplete lineage sorting) degut a esdeveniments ràpids d’especiació. L’anàlisi dels temps de divergència ha demostrat un gran impacte de l’extinció de megafauna marina del Pliocè en les baldrigues, probablement a causa de la sobtada reducció en la disponibilitat d’hàbitats costaners. Posteriorment, el Pliocè tardà i el principi del Pleistocè van ser períodes de molta i ràpida especiació i dispersió, probablement a causa dels canvis climàtics del Pleistocè. Les anàlisis biogeogràfiques han demostrat que els corrents marins promouen la dispersió i que l’efecte fundador és un mecanisme important d’especiació en les baldrigues. Les nostres anàlisis, combinant dades genòmiques amb evidència morfològica i ecològica, no han donat suport a les classificacions taxonòmiques actuals del grup de les baldrigues del gènere Puffinus del nord de l’Atlàntic i del Mediterrani i, per tant, proposo una classificació taxonòmica més adequada pel grup. A més a més, la detecció d’estructura genètica a petita escala en les espècies d’aquest grup de baldrigues destaca la necessitat de gestió d’unitats evolutives significatives per sota del nivell d’espècie. Les anàlisis de genòmica de poblacions han identificat la deriva genètica com a principal promotor de divergència en el genoma. Per concloure, el conjunt de les investigacions d’aquesta tesi identifiquen la prevalença del sorteig incomplet de llinatges al llarg de diferents escales evolutives, destaquen l’important paper dels esdeveniments paleoceanogràfics com a promotors de diversificació i indiquen la importància de l’evolució neutra com a causa de diferenciació poblacional en ocells marins pelàgics. Globalment, aquesta tesi demostra la utilitat de diferents aproximacions genòmiques, mitjançant anàlisis filogenètiques i de genètica de poblacions en diferents escales evolutives per proporcionar nou coneixement sobre la història evolutiva de les baldrigues.

The present thesis covers two aspects of statistical analysis applied to the genetics of human diseases. First, the significance of LOD-score results for the confirmation of linkage is addressed, with special emphasis on small pedigrees. A new analytical approach is presented for the linkage analysis of heterogenetic traits, using hereditary spastic paraplegia as a model, a disease well suited for the analyses. The critical significance values for confirmation of linkage are evaluated using Bayesian statistics, and empirical P-values for LOD score results are calculated using computer simulation methods. The presented analytical approach resulted in conclusive linkage analyses on small to medium-size families, under the restrictions of genetic heterogeneity.
The second part addresses linkage-disequilibrium based fine mapping in the French Canadian population. The performance of five linkage-disequilibrium based fine-mapping methods is evaluated using French Canadian chromosomes with one of three diseases found in this population: oculopharyngeal muscular dystrophy (OPMD), hidrotic ectodermal dysplasia (HED), and sensorimotor polyneuropathy with or without agenesis of the corpus callosum (ACCPN). The gene for OPMD was recently mapped and cloned, allowing us to evaluate the performance of the methods with the OPMD results, and to make predictions about the ACCPN and HED putative gene positions. In addition, a new approach to linkage-disequilibrium based fine mapping is presented using FrenchCanadian ascending genealogies. The method involves two steps. First, the likely founding couple of a mutation-bearing chromosome is identified using a computerised randomisation statistic. Then, using a delete-d jackknife resampling scheme, the distribution of gene mapping estimates is calculated from the count of ancestral recombinants and ancestral meioses joining the identified founding couple to the disease gene carriers. Gene mapping estimates are calculated from each marker individually, and confidence intervals of the estimates are derived from the jackknife distributions. The method, when applied to French Canadian families with OPMD, successfully confirmed the localisation of PABP2 responsible for OPMD and performed better than other linkage disequilibrium-based mapping models.

Discriminant analysis is a procedure for identifying the relationships between qualitative criterion variables and quantitative predictor variables. Data bases of genetic polymorphisms are currently available that group such polymorphisms by ethnic origin or nationality. Such information could be useful to entities that base financial determinations upon predictions of disease or to medical researchers who wish to target prevention and treatment to population groups. While the use of genetic information to make such determinations is unlawful in states and confidentiality and privacy concerns abound, methods for human “redlining” may occur. Thus, it is necessary to investigate the efficacy of the relationship of certain genetic information to ethnicity to determine if a statistical analysis can provide information concerning such relationship. The use of the statistical technique of discriminant analysis provides a tool for examining such relationship.

In Chapter 2, a directed linkage scam for loci involved in body weight and carcass composition traits is performed in a commercial Charollais sheep population. Five chromosomes were investigated based on prior evidence for major genes effecting the studied traits in other breeds. A maximum likelihood variance component analysis using identity-by-descent values estimated by Markov chain Monte Carlo methods was performed on a complex pedigree containing a total of 570 sheep. Of the total of nine QTL detected, the estimated position of only one overlapped with the regions showing major genes that were used in chromosome selection. During the analysis of the Charollais sheep population, a region of the genome showing a significant deviation from the published sheep linkage map was detected. This region is examined in more detail in Chapter 3, with the addition of further microsatellite markers as well as the investigation of this region in two further sheep breeds. With the inclusion of the published linkage map, this demonstrated a total of three linkage maps across four populations. Such heterogeneity in linkage maps across sheep breeds has important consequences for the design and analysis fine-mapping studies. The significance of a QTL linkage peak is not readily evaluated with general pedigrees. The extension of permutation methodology that is commonly used with structured pedigrees to more general pedigrees is investigated in Chapter 4. Chapter 5 examines the population dynamics of a well studied wild Soay sheep population. A unified statistical framework is developed for all major aspects of the life cycle of the sheep. This forms the basis of a simulation model of the population that is used to predict the amount of linkage disequilibrium in the population (Chapter 6) and the effective population size of the population (Chapter 7). The examination of the linkage disequilibrium structure in a population is an important step in the design of studies with the aim of fine-mapping quantitative trait loci. The simulated population showed significant decline of linkage disequilibrium with genetic distance and low levels of background linkage disequilibrium, indicating that the Soay sheep population is a viable resource for linkage disequilibrium fine mapping. Through the use of the simulation model, the effective population size of the Soay sheep population was estimated to be approximately 0.17 of its census population size. This is approximately half the value obtained with the use of a general predictive equation.

This thesis consists of four papers on various aspects of inbreeding, effective population sizes and genetic differentiation in structured populations, that is, populations that consist of a number of subpopulations. Three of the papers concern age structured populations, where in the first paper we concentrate on calculating the variance effective population size (NeV) and how NeV depends on the time between measurements and the weighting scheme of age classes. In the third paper we develop an estimation procedure of NeV which uses age specific demographic parameters to obtain approximately unbiased estimates. A simulation method for age structured populations is presented in the fourth paper. It is applicable to models with multiallelic loci in linkage equilibrium. In the second paper, we develop a framework for analysis of effective population sizes and genetic differentiation in geographically subdivided populations with a general migration scheme. Predictions of gene identities and gene diversities of the population are presented, which are used to find expressions for effective population sizes (Ne) and the coefficient of gene differentiation (GST). We argue that not only the asymptotic values of Ne and GST are important, but also their temporal dynamic patterns. The models presented in this thesis are important for understanding how different age decomposition, migration and reproduction scenarios of a structured population affect quantities, such as various types of effective sizes and genetic differentiation between subpopulations.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Submitted.

[Truncated abstract] This thesis investigates novel methods for the genetic association analysis of haplotype data in samples of unrelated individuals, and applies these methods to the analysis of coronary heart disease and related phenotypes. Determining the inheritance pattern of genetic variants in studies of unrelated individuals can be problematic because family members of the studied individuals are often not available. For the analysis of individual genetic loci, no problem arises because the unit of interest is the observed genotype. When the unit of interest is the linear combination of alleles along one chromosome, inherited together in a haplotype, it is not always possible to determine with certainty the inheritance pattern, and therefore statistical methods to infer these patterns must be adopted. Due to genotypic heterozygosity, mutliple possible haplotype configurations can often resolve an individual's genotype measures at multiple loci. When haplotypes are not known, but are inferred statistically, an element of uncertainty is thus inherent which, if not dealt with appropriately, can result in unreliable estimates of effect sizes in an association setting. The core aim of the research described in this thesis was to develop and implement a general method for haplotype-based association analysis using multiple imputation to appropriately deal with uncertainty haplotype assignment. Regression-based approaches to association analysis provide flexible methods to investigate the influence of a covariate on a response variable, adjusting for the effects of other variables including interaction terms. ... These methods are then applied to models accommodating binary, quantitative, longitudinal and survival data. The performance of the multiple imputation method implemented was assessed using simulated data under a range of haplotypic effect sizes and genetic inheritance patterns. The multiple imputation approach performed better, on average, than ignoring haplotypic uncertainty, and provided estimates that in most cases were similar to those observed when haplotypes were known. The haplotype association methods developed in this thesis were used to investigate the genetic epidemiology of cardiovascular disease, utilising data for the cholesteryl ester transfer protein gene (CETP), the hepatic lipase (LIPC) gene and the 15- lipoxygenase (ALOX15) gene on a total of 6,487 individuals from three Western Australian studies. Results of these analyses suggested single nucleotide polymorphisms (SNPs) and haplotypes in the CETP gene were associated with increased plasma high-density lipoprotein cholesterol (HDL-C). SNPs in the LIPC gene were also associated with increased HDL-C and haplotypes in the ALOX15 gene were associated with risk of carotid plaque among individuals with premature CHD. The research presented in this thesis is both novel and important as it provides methods for the analysis of haplotypic associations with a range of response types, while incorporating information about haplotype uncertainty inherent in populationbased studies. These methods are shown to perform well for a range of simulated and real data situations, and have been written into a statistical analysis package that has been freely released to the research community.

Abstract Sensorineural hearing impairment (SNHI) is a well-recognized manifestation of mitochondrial diseases and occurs either in a non-syndromic form or as a part of a syndrome. Mitochondrial deafness is bilateral, usually progressive and is inherited maternally. Approximately 70% of patients with the most common syndromes, Kearns-Sayre, MELAS or MERRF, have SNHI. Several mutations in mitochondrial DNA (mtDNA) have been found to cause non-syndromic SNHI, including 1555A>G, 7445T>C, 7472insC and 7511T>C. In order to estimate prevalences of pathogenic mtDNA mutations in population-based cohorts of patients with SNHI, we obtained samples from 133 patients with SNHI, reportedly representing 117 separate maternal lineages. We found five patients with the 3243A>G mutation and three with the 1555A>G mutation, whereas the other point mutations associated with SNHI were absent. The frequencies of the mutations in the cohort were thus 4.3 % for 3243A>G and 2.6 % for 1555A>G, suggesting a total frequency of 6.9 % for mtDNA mutations known to be associated with hearing impairment. We found a mutation 10044A>G, which has been reported as pathogenic, in our patients with SNHI, but we also found it among the controls. Our results show it to be a homoplasmic polymorphism associated with a fairly rare haplotype within mtDNA haplogroup H which has recently been confirmed as subcluster H4. These results highlight the difficulty in determining the pathogenicity of a mtDNA mutation when it is identified only in one family. Therefore, in addition to the previously published criteria, we suggest that a sufficient number of haplotype-specific controls should be screened before the pathogenic nature of a mtDNA mutation can be verified. We determined the complete mtDNA sequences for 121 Finns, and after complementing our recent data, for a total of 192 Finns, and were able to construct a phylogenetic network based on complete mtDNA sequences, the largest set of complete sequences available at that time. These mtDNAs provide a rich source of information for studies in population genetics and a potential tool for analysing new substitutions and genotypes that entail a risk of mitochondrial disease. We used the phylogenetic network to find new pathogenic mutations or risk genotypes for SNHI. The entire coding region sequences of mtDNA were determined in 32 patients with SNHI and compared with the network. The patients were found to harbour more rare polymorphisms and haplotypes than the controls and to show increased variation in their mtDNA sequences, suggesting mildly deleterious effects for these substitutions. Two of the new mutations were suggested as putatively pathogenic.

La diversité intra-spécifique, mobilisée à l'échelle de la parcelle (populations et associations variétales) devrait jouer un rôle important dans la transition agroécologique, en contribuant à l’adaptabilité et à la résilience aux stress des systèmes agricoles à bas niveaux d'intrants. Toutefois, la complexité des interactions entre plantes dans ces peuplements soulève de nombreuses questions sur leur conception et leur gestion, pour lesquelles l'approche par modélisation est particulièrement pertinente, mais peu développée à ce jour. Cette étude porte ainsi sur la modélisation structure-fonction et son utilisation pour étudier l'impact de l'architecture aérienne des plantes sur la performance et l'évolution de peuplements hétérogènes de blé. Ce projet de thèse est basé sur l'utilisation de WALTer : un modèle de plante structure-fonction préexistant qui représente le développement d'une parcelle de blé du semis à maturité et simule la plasticité du tallage (propriété de ramification) en réponse à la compétition pour la lumière. Dans un premier temps, des adaptations ont été effectuées sur WALTer pour réduire le coût computationnel des simulations, augmenter le réalisme du modèle et permettre la simulation de peuplements hétérogènes complexes. Le fonctionnement de cette nouvelle version de WALTer a ensuite été caractérisé en analysant l'impact de l'aléatoire sur les simulations et en réalisant une analyse de sensibilité sur des parcelles homogènes. D'autre part, une méthode de calibration automatique, peu développée pour ce type de modèle, a été appliquée à WALTer sur la base de données expérimentales afin d'estimer les valeurs des paramètres précédemment identifiés comme étant importants par l'analyse de sensibilité. WALTer a ensuite été utilisé pour identifier, via une analyse de sensibilité sur des mélanges binaires équilibrés, les traits de l'architecture aérienne (décrits par leurs valeurs moyennes dans le mélange et les valeurs de différences entre les 2 variétés) qui ont le plus d'impact sur la performance des mélanges. L'analyse a ainsi révélé le rôle prépondérant de la capacité de tallage et des dimensions foliaires comme déterminants de la performance des mélanges simulés. Grâce à une phase d'optimisation, les combinaisons de traits les plus favorables pour la performance des mélanges simulés ont été identifiées. Les résultats des simulations ont ainsi souligné l'importance de sélectionner soigneusement les combinaisons de traits associés en mélange, en montrant l'intérêt potentiel d'une diversité d'architecture dans les mélanges, mais également des effets défavorables possibles d'une telle diversité. Enfin, WALTer a été couplé à un modèle de génétique des populations afin de simuler l'évolution de peuplements hétérogènes de blé au cours de générations de resemis. Le couplage a été utilisé pour simuler une expérience de gestion dynamique d'une population présentant une grande diversité de hauteur. L'évolution génétique et phénotypique de la population sur 20 générations a ainsi pu être comparée à des données expérimentales pour confirmer le bon fonctionnement du couplage. Cette thèse a permis de développer des méthodes et outils pour la modélisation structure-fonction des peuplements hétérogènes de blé. Les simulations réalisées ont également apporté des éléments de compréhension sur l'impact de l'architecture aérienne sur la compétition pour la lumière dans ces peuplements. À terme, ces travaux seront utiles pour la conception et la gestion des peuplements hétérogènes de blé en champ
Intra-specific diversity, mobilized at the scale of the field (cultivar mixtures and composite populations) should play an important role in the agroecological transition, contributing to the adaptation and resilience of low input cropping systems, more prone to stresses. However, many questions are raised by the complex plant-to-plant interactions occurring in such heterogeneous canopies, and there is a growing need to develop modelling approaches to improve their design and management. Thus, our study focuses on the use of functional-structural plant modelling to study the impact of the aerial architecture of plants on the performance and the evolution of heterogeneous wheat populations. The thesis project is based on WALTer : a pre-existing functional-structural plant model that simulates the development of a wheat field from sowing to maturity and the plasticity of tillering (i.e. branching) in response to competition for light. In a first step, modifications were made to WALTer to reduce the computational cost of simulations, enhance its realism and improve its ability to simulate complex heterogeneous stands. The functioning of this new version of WALTer was then characterized by analysing the impact of stochasticity on the simulations and by performing a sensitivity analysis on homogeneous plots. Furthermore, on the basis of field observations, an automatic calibration method, which is rarely developed for this type of model, was applied to WALTer in order to estimate the values of the parameters previously identified as important by the sensitivity analysis. Thanks to a sensitivity analysis of balanced binary mixtures, WALTer was then used to identify the traits of the aerial architecture (described by their mean values in the mixture and the values of the differences between the 2 varieties) that have the most impact on the performance of the plots. The analysis thus revealed the preponderant role of tillering capability and dimensions of the leaves as determinants of the performance of the simulated mixtures. Thanks to an optimization phase, the combinations of traits that are the most favourable for the performance of the simulated mixtures were identified. The results of the simulations highlighted the importance of carefully selecting combinations of traits to associate in mixtures, showing the potential interest of a diversity of architecture in heterogeneous stands, but also the possible adverse effects of such diversity. Finally, WALTer was coupled to a population genetics model to simulate the evolution of heterogeneous wheat stands along generations of resowing. The coupling was used to simulate the dynamic management of a population presenting a large diversity of plant height. The genetic and phenotypic evolution of the population over 20 generations of resowing could thus be compared with experimental data to confirm the good functioning of the coupling. This thesis allowed the development of methods and tools for the functional-structural modelling of heterogeneous wheat stands. The simulations carried out have also improved our understanding of the impact of the aerial architecture on the competition for light in these stands. Ultimately, this work will be useful for the design and management of heterogeneous wheat stands adapted to farmers' needs

Una de les forces selectives més fortes que han afectat a les poblacions humanes en la història més recent és el paràsit de la malària: Plasmodium falciparum, que és la causa de varis exemples d'adaptació induïda per patògens en els éssers humans. Una forma especial de malària és l'associada a l'embaràs, que es caracteritza per l'acumulació d'eritròcits infectats en la placenta, i que pot arribar a causar fins a 200.000 morts maternoinfantils cada any. L'objectiu d'aquest treball és descriure com aquesta forma peculiar de malària ha afectat la variació genètica humana. Amb aquesta finalitat, hem utilitzat mètodes tant de la genètica evolutiva com de l'epidemiologia molecular, resultant en la primera investigació a gran escala de la base genètica de la malària placentària. Els resultats ofereixen una nova visió sobre els gens que modulen el risc d'infecció, ,així com de la selecció natural actuant sobre les vies cel·lulars implicades en la patogènesi de la malaltia. Finalment, també aportem noves dades sobre l'estructura genètica de les poblacions sub-saharianes analitzades.
One of the strongest selective forces affecting human populations in recent history is the malaria parasite Plasmodium falciparum, which is the cause of a variety of well-established examples of pathogen-induced adaptation in humans. A special form of malaria is pregnancy-associated malaria, which is characterised by the accumulation of infected erythrocytes in the placenta, and causes up to 200,000 maternal and infant deaths every year. The aim of this work is to characterise how this particular form of malaria has shaped human genetic variation. To that end we use methods of both evolutionary genetics and molecular epidemiology, reporting the first large-scale investigation of the genetic basis of placental infection. Our results provide new insights into genes modulating the risk of infection, as well as natural selection acting on cellular pathways involved in the pathogenesis of the disease. Finally, we also provide new data on the genetic structure of affected populations in Sub-Saharan Africa.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009.
Dissertation presented for the degree of Doctor of Philosophy in Medical Biochemistry at Stellenbosch University.
ENGLISH ABSTRACT: The infectious disease tuberculosis (TB) still represents a global threat due to its devastating effect on health and the subsequent high mortality rate. Previous studies have indicated that host genetic factors are implicated in host susceptibility to TB. Since TB is a complex disease, it can be assumed that susceptibility to M. tuberculosis has multiple genetic causative factors (as well as environmental causes). The current study focussed on a number of South African Coloured (SAC) individuals, some of whom were TB cases and others controls. Population substructure was tested in the admixed SAC population as it can be a strong confounding factor for association studies. Our results using the programme STRUCTURE indicated no population substructure in the SAC population. We further investigated the population structure of the SAC group using Affymetrix 500k SNP chip data which showed that the SAC population group has 4 major ancestral components: the Khoesan, European, African and Asian (Indian). A number of candidate polymorphisms in eight genes, previously indicated to play an important role in TB susceptibility, were tested in case-control associations studies. We found statistically significant associations between IFNGR1, IL-8, IL-1Ra and NRAMP1 polymorphisms and TB susceptibility in the SAC population. It has become increasingly evident that gene-gene interactions play a far more important part in an individual’s susceptibility to a complex disease than single polymorphisms would on their own. The importance of epistasis was clearly identifiable in this study with only four associations found between the individual variants and TB susceptibility, but eight instances of statistically significant gene-gene interactions. A combined data set consisting of 106 variants constructed from our database and also used for gene-gene interaction analysis yielded numerous statistically significant interactions. The interaction between the genotype of the human host and the bacterial strain genotype was also investigated and yielded interesting results. Owing to various polymorphisms in several cytokine genes, the protein levels of the main modulators of the immune system, cytokines and chemokines, are changed in several diseases such as infectious diseases and may affect susceptibility or resistance to TB. The functional polymorphisms or haplotype patterns in some of these cytokine genes might be vital for protective immune responses and may serve as biomarkers of protection or susceptibility to TB. The present study investigated 18 cytokines including pro-inflammatory, anti-inflammatory and chemokine factors in healthy (mantoux positive or negative) children using the Linco-plex immunoassay, and investigated potential interactions. The basic research will one day contribute to personalised genetics which may benefit infectious diseases such as TB. If individuals can be identified as potentially more vulnerable, they may require different vaccination strategies, a higher index of suspicion if exposed to TB, and prophylactic treatment.
AFRIKAANSE OPSOMMING: Die infektiewe siekte tuberkulose (TB) is steeds ‘n gevaar wat die hele wêreld bedreig weens die groot impak op gesondheid en die gevolglike hoë mortaliteit. Vorige studies het bevind dat die gasheer se genetiese faktore wel betrokke mag wees by die gasheer se vatbaarheid vir TB. Aangesien TB ‘n komplekse siekte is, kan dit aanvaar word dat vatbaarheid tot M. tuberculosis veelvuldige genetiese oorsaaklike faktore (sowel as omgewingsoorsake) het. Hierdie studie het gefokus op ‘n aantal Suid-Afrikaanse Kleurling (SAC) individue, waarvan sommige TB pasiënte en ander kontroles was. Die gemengde SAC populasie is getoets vir populasie-stratifikasie, aangesien stratifikasie ‘n sterk verwarrende invloed op pasiënt-kontrole studies kan hê. Ons resultate is verkry met behulp van die program STRUCTURE en het aangedui dat daar geen populasie sub-struktuur tussen die pasiënte en kontroles was nie. Ons het ook die populasiesamestelling van die SAC groep ondersoek met data verkrygbaar van die Affymetrix 500k enkel nukleotied polimorfisme mikroskyfie. Hierdie data het getoon dat die SAC populasie uit 4 hoof voorouerlike komponente bestaan naamlik die Khoesan, Europeërs, Afrikane en Asiate (Indiërs). ‘n Aantal kandidaat polimorfismes in agt gene, wat volgens vorige studies ‘n belangrike rol in TB vatbaarheid te speel, was in hierdie pasiënt-kontrole assosiasie studie bestudeer. Ons het statistiese beduidende verwantskappe tussen IFNGR1, IL-8, IL-1Ra en NRAMP1 polimorfismes en TB vatbaarheid in die SAC populasie gevind. Dit het al hoe meer duidelik geword dat geen-geen interaksies ‘n baie belangriker rol in ‘n individu se vatbaarheid vir ‘n komplekse siekte speel as enkel polimorfismes op hul eie. Die belang van epistase kon duidelik in hierdie studie geïdentifiseer word met slegs vier assosiasies wat tussen die individuele variante en TB vatbaarheid gevind is, in vergelyking met agt statisties beduidende geen-geen interaksies. ‘n Gekombineerde datastel wat uit ons databasis saamgestel is en wat 106 variante bevat is ook in ‘n aparte geen-geen interaksie analise gebruik, wat verskeie statisties beduidende interaksies getoon het. Die interaksie tussen die menslike gasheer genotipe en die bakteriese stam genotipe is ook in hierdie studie ondersoek en het interessante resultate opgelewer. Veranderde proteïen uitdrukking van die hoofmoduleerders van die immuunsisteem, sitokine en chemokine, kom voor in verskeie siektes soos infektiewe siektes weens verskillende polimorfismes in verskeie sitokien-gene. Sulke polimorfismes kan ook vatbaarheid vir of weerstandigheid teen TB beïnvloed. Die funksionele polimorfismes of haplotipe patrone in sommige van hierdie sitokien-gene mag noodsaaklik wees vir beskermende immuunresponse en mag ook as biomerkers vir beskerming teen of vatbaarheid vir TB dien. Hierdie studie het 18 sitokiene (insluitend pro-inflammatoriese-, anti-inflammatoriese- en chemokiene faktore), sowel as potensiële interaksies in gesonde (mantoux positiewe of negatiewe) kinders, ondersoek met behulp van die Linco-plex immuno-analise. Hierdie basiese navorsing sal eendag in die toekoms bydrae tot persoonlike genetiese analises wat tot voordeel kan wees vir infektiewe siektes soos TB. Indien individue as potensieël meer vatbaar vir TB geïdentifiseer kan word, kan sulke persone ander vaksineringstrategieë sowel as voorkomende behandeling vereis.

Thesis (MScAgric)--University of Stellenbosch, 2001.
ENGLISH ABSTRACT: DNA micro satellite loci express extensive allelic variation making them convenient markers for research in many fields employing population genetic tools, including aquaculture and conservation genetics. Twelve Oreochromis mossambicus populations from wild, captive and introduced sources in Southern Africa were screened for genetic variation at ten CA repeat micro satellite loci. Three of the loci - UNHI04, UNHlll, and UNH123 - were sufficiently well resolved to screen extensively and were interpreted according to a model of Mendelian inheritance. Data was analyzed in terms of genetic structure and levels of genetic variation, the effect of management regime in captivity through successive generations on genetic diversity, and the nature of phylogenetic relationships present between populations. Exact tests, carried out using Monte Carlo type multiple resampling techniques, and F-Statistics were used to detect and quantify genetic structure among the twelve populations. The Exact test X2 (P < 0.001), a FST of 0.27 (P < 0.001), eST of 0.26, RsT of 0.28, and a ST of 0.17 all indicated significant structuring among the populations. The evident genetic structuring endorsed the practice of maintaining the populations as separate genetic stocks, in separate tanks, in order to preserve unique genetic material for aquaculture strain development. Populations also exhibited some significant deviations from Hardy Weinberg equilibrium characterised by an overall reduced heterozygosity across the loci. In microsatellite studies, null alleles are often suggested as major contributors to heterozygote deficits. To test for null alleles, two controlled crosses of 0. mossambicus were made. The progeny from each cross were examined for expected parental allelic ratios at the UNHI04, UNHlll and UNH123 loci. All three loci presented evidence of possible null alleles. Accelerated inbreeding and genetic drift through successive generations in captivity can reduce heterozygosity and gene diversity. To investigate loss of diversity a sample taken from the Bushmans population in 1999 (N = 25) was compared with a Bushmans 2000 sample (N = 36). The comparison highlighted altered allele frequencies, a significant increase in average observed heterozygosity and a non-significant change in average expected heterozygosity using the UNHI04 and UNH123 loci. Calculation of genetic distances and phylogenetic comparisons between the populations provided insight into the degree of management required in conserving genetic diversity in natural populations of Mozambique tilapia. UPGMA and Neighbour-Joining techniques were used to construct phylogenetic trees using Dm and ({)~)2 distance matrices. Clustering of populations appeared to reflect geographic locality of the source populations, however certain populations were not congruent with geography. Mantel tests were used to expose a possible association between genetic distance matrices generated from each individual locus. An association would support a geographic background to population genetic structure. The Mantel tests did not provide conclusive evidence. Mantel tests for association between the combined locus Dm and (81l)2 genetic distance matrices and a geographic distance matrix were similarly non-significant. Multi-dimensional scaling (MDS) plots of Euclidean distance values for Dm and (81l)2 matrices presented a two-dimensional view of the genetic distance data. The degree of similarity with the UPGMA and Neighbour-Joining tree-clustering pattern was higher for the (81l)2 than for the Dm MDS plots. Scatter plots indicated a reliable non-linear correlation between Euclidean distance and genetic distance for the two-dimensional MDS. The micro satellite markers employed in this research provided molecular information needed for complimenting a co-study on quantitative genetic evaluation of the twelve populations. The quantitative co-study provided measures of average length and weight gain indices for the populations based on progeny growth trials. No significant correlation of average heterozygosity (gene diversity) with either average weight or length gain was found. The significant genetic diversity and structure present between the twelve populations provided rationale for implementing strategies to conserve natural 0. mossambicus populations as genetic resources, and manage captive populations for long term maintenance of genetic diversity.
AFRIKAANSE OPSOMMING: Die verstaffing van groot alleliese variasie deur DNA mikrosateliete maak van hulle gerieflike merkers vir navorsing in 'n verskeidenheid van velde wat gebruik maak van populasie genetiese gereedskap, ingesluit akwakultuur en bewarings genetika. Twaalf 0. mossambicus populasies wat verkry was vanuit die natuur, in gevangeneskap en ingevoerdes, van Suidelike Afrika was getoets vir genetiese variasie by tien verskillende CA-herhalende mikrosateliet loci. Drie van die loci - UNHI04, UNHlll en UNH123 - is op grootskaal getoets en volgens In model van Mendeliese oorerwing geinterpreteer. Die data was ontleed volgens genetiese struktuur en vlakke van genetiese variasie, die effek wat bestuur strategie in gevangeneskap op genetiese diversiteit in opeenvolgende generasies uitgeoefen het, so wel as die aard van die filogenetiese verhoudings wat teenwoordig is tussen die populasies. "Exact" toetse is uitgevoer deur gebruik te maak van Monte Carlo tipe veelvuldige hermonsterinsamelings tegnieke en F-statistieke is gebruik vir die deteksie en kwantifisering van die genetiese struktuur tussen die twaalfpopulasies. Die Exact toets X2 (P < 0.001), 'n FST van 0.27 (P < 0.001), SST van 0.26, RsT van 0.28, en 'n