Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.

TITLE: Mutation and substitution rates in sexual and asexual forms: a clue to the persistence of sex in a model group of Cobitis? AUTOR: Jan Röslein DEPARTMENT: Ústav živočišné fyziologie a genetiky AVČR, v.v.i. SUPERVISOR: Mgr. Karel Janko, Ph.D. ABSTRACT: Subject of this thesis is to test several hypotheses about the evolution of asexual reproduction in model group of fish family Cobitis and its mutual competition among sexual and asexual forms, which touches one of the oldest unresolved issues of biology. Specifically, the work deals with the accumulation of non-synonymous mutations, which accelerated accumulation in the genome of clonal lineages theoretically leads to increased extinction compared with sexually reproducing populations (so-called. The theory of Muller's ratchet and Kondrashov's hatchet). This thesis is based on a normalized cDNA sequencing data from oocytes and liver tissue, which has served as a base matrix (generated based on non-normalized cDNA data) for transcriptome sequencing (RNAseq). Consequently, the RNAseq data have served as validation for acquired polymorphisms, detection of differential expression of allele- specific expression (ASE) hybrid biotypes. This diploma thesis balances among the edges of vast spectrum of hypotheses regarding the evolution of the genus hybrid...

The aim of this work was the construction of set of microsatellites for the Grey Partridge (Perdix perdix). I succeeded in isolation of seven new specific microsatellites. Another microsatellite markers were passed from close relative galliform species (Galliformes). On the whole I gained 18 microsatellite loci which were used for study of population charakteristics of three partridge populations from Stodůlky, Písecko and Milešín. The analysis showed conspicuous structuredness of these populations and surprisingly low values (less then 100) of their effective population sizes. Seven of the microsatellites were chosen for analysis of paternity. Indeed I found the evidence of extrapair paternity in this putatively monogamous bird. Finally I conclude, on the basis of sequenation of mitochondrial control region that our populations belong to the western linage of mitochondrial DNA.

>Magister Scientiae - MSc / As human beings we are curious about our origin and ancestry. A curiosity has led to an investigation of human evolution and expansion across the world by means of population genetics and phylo-genetics by evaluating a region in Southern Africa that is largely unknown. The objective of this study was to develop a quick, inexpensive and accurate hierarchical diagnostic screening system of the MtDNA phylogenetic tree, AI-SNPs in the mtDNA genome by using High Resolution Melting analysis to evaluate the population composition and ancestral haplogroups of Southern African populations in Limpopo. The admixture between the ‘Khoesan’ hunter-gatherers, herders and the Bantu speaking populations led to population growth and expansion in Limpopo. This has contributed to populations settling in Limpopo and has thus shaped the ancestral contemporary populations. No research on these individuals residing in Limpopo has been done before, thus an investigation of their ancestral origin was necessary. A total of 760 saliva samples were collected from individuals residing in Limpopo. Only 500 saliva samples were extracted by means of an optimized salting out technique. Five hundred extracted genomic samples were genotyped by means of a quick, inexpensive High-resolution melting analysis. Of the 500 samples, the genotyping results showed 95 individuals derived for the L3 haplogroup which gives a 19% ratio of individuals screened with Multiplex 1. Only 56 individuals were derived for the L1 haplogroup, which gives a percentage of 11%. A total of 249 individuals were derived for the L0 haplogroup, making up a 50% of the total individuals genotyped. Only 100 samples were derived for L0a, making up 20% of individuals screened with Multiplex 1. Of the 95 samples derived for the L3 haplogroup, the results showed 87 individuals to be ancestral for both M and N, making up 91.57% of individuals screened with Multiplex 2. http://etd.uwc.ac.za/. In population genetics using SNPs to infer population history and ancestral origin has become significant, this study allowed researchers to evaluate population groups by investigating their genetic markers and the application of the results allowed for downstream analyses. Finally, this study provides a quick and simple screening method for the selection of lineages that are of interest for further studies.

No description available.

<p>This work investigates the assignment of unknown sequences to their species of origin. In particular, I examine four questions: Is existing (GenBank) data reliable for accurate species identification? Does a segregating sites algorithm make accurate species identifications and how does it compare to another Bayesian method? Does broad sampling of reference species improve the information content of reference data? And does an extended model (of the theory of segregating sites) describe the genetic variation in a set of sequences (of a species or population) better? Though we did not find unusually similar between-species sequences in GenBank, there was evidence of unusually divergent within-species sequences, suggesting that caution and a firm understanding of GenBank species should be exercised before utilizing GenBank data. To address challenging identifications resulting from an overlap between within- and between species variation, we introduced a Bayesian treeless statistical assignment method that makes use of segregating sites. Assignments with simulated and <em>Drosophila</em> (fruit fly) sequences show that this method can provide fast, high probability assignments for recently diverged species. To address reference sequences with low information content, the addition of even one broadly sampled reference sequence can increase the number of correct assignments. Finally, an extended theory of segregating sites generates more realistic probability estimates of the genetic variability of a set of sequences. Species are dynamic entities and this work will highlight ideas and methods to address dynamic genetic patterns in species.</p> / Doctor of Philosophy (PhD)

La mitochondrie est un organite intracellulaire permettant la production d’énergie nécessaire à la survie de la cellule. Sa singularité passe par le fait qu’elle possède son propre génome (ADN mitochondrial ou ADNmt) distinct du génome nucléaire (ADNnu). Aussi, contrairement à l’ADNnu qui est transmis par les deux parents, l’ADNmt est lui hérité seulement par la mère chez les animaux. Exceptionnellement, un seul système connu va à l’encontre de cette « loi » d’hérédité. Il s’agit du système de double hérédité uniparentale (Doubly Uniparental Inheritance ou DUI) retrouvé chez plusieurs espèces de bivalves. Les espèces concernées possèdent ainsi 2 lignées d’ADNmt distinctes : une d’origine paternelle (ADNmt M) transmise seulement de pères en fils, et une d’origine maternelle (ADNmt F) transmise par la mère à la fois aux filles et fils. L’hypothèse privilégiée pour expliquer le maintien du système DUI au cours de l’évolution l’engage dans la détermination sexuelle chez les bivalves mais la vérification de cette hypothèse repose entre-autre sur une étude plus élargie de différents systèmes DUI. Jusqu’à maintenant, le DUI a été trouvée chez 46 espèces de bivalves, mais sa distribution chez ce groupe est certainement plus vaste étant donné le nombre d’espèces vivantes estimé à >20000. L’objectif de ce projet est d’étudier (et d’élargir) la distribution taxonomique du système DUI au sein du groupe des mollusques afin d’obtenir les prérequis indispensables à une meilleure compréhension de sa fonction mais également pour en retracer l’origine. Les résultats suggèrent l’absence du système DUI chez cinq espèces de gastéropodes et confirment sa découverte chez deux nouvelles espèces et familles de bivalves [i.e. Scrobicularia plana (Semelidae) et Yoldia hyperborea (Yoldiidae)]. En s’appuyant sur les résultats phylogénétiques, nous favorisons l’hypothèse d’une origine unique pour ce système. Finalement, la nouvelle distribution taxonomique proposée ici confirme la présence du système DUI chez 103 espèces de bivalves appartenant à 12 familles. / Mitochondria are semi-independent organelles, mostly known for their role in energy production necessary for cell survival. Several characteristics make them unique: they have their own genome, the mitochondrial DNA (mtDNA), and contrary to the nuclear genome (nuDNA), they are inherited uniparentally by Strict Maternal Inheritance (SMI) in animal species. Exceptionally, one model of mitochondrial inheritance found in some Bivalvia goes against the rule of SMI. It is called the Doubly Uniparental Inheritance (DUI) system. The species concerned possess two distinct mitochondrial lineages: one transmitted by the male (M mtDNA) to his sons only, and the other by the female to both sons and daughters. The most likely hypothesis to explain the retention of the DUI system in evolution involves him in sexual determination in bivalves but a widened study on different DUI systems is needed to verify this hypothesis. Until now, the DUI system has been described in 46 bivalve species but its distribution in this group might be broader given the total number of living species estimated to >20000. This project aimed to study (and broaden) taxonomic distribution of DUI within mollusks as a necessary prerequisite to a better understanding of its function and its origin. The results suggest the absence of DUI in five gastropods species and confirm its discovery in two new bivalves species and families [i.e. Scrobicularia plana (Semelidae) and Yoldia hyperborea (Yoldiidae)]. Based on phylogenetic data, we favor the hypothesis of a single origin of DUI. Finally, the new taxonomy proposed here confirms the presence of the DUI system in 103 bivalves species belonging to 12 families.

L'introgression est un processus fréquent et qui a d'importantes conséquences évolutives. L'objectif de ce travail était de tester un modèle neutre d'introgression chez des épicéas du Plateau tibétain et des régions voisines. Le travail a permis de mettre en évidence que la direction de l'introgression pouvait être prédite par la dynamique passée des populations d'arbres, et que l'importance de cette introgression était inversement proportionnelle à l'intensité des échanges génétiques au sein de l'espèce invasive, grâce à la comparaison de la structure génétique basée sur des marqueurs chloroplastiques (à hérédité paternelle) et mitochondriaux (à hérédité maternelle). / Introgression is a widespread phenomenon with potentially profound evolutionaryconsequences. Recently, significant progress in our understanding of introgression hasbeen made with the development of a neutral model. This model predicts that, whenone species invades an area already occupied by a related species, introgression ofneutral genes takes place mainly from the local species towards the invading ones. Inaddition, following a contact between two hybridizing species, the model predicts thatintrogression should be particularly frequent for genome components experiencinglittle gene flow. However, to date, there was no empirical example available, in whichone species expanded into the range of a closely related one and two markers withcontrasted rates of gene flow had been studied for both species. Only in such a casecould the two predictions outlined above be tested simultaneously. In addition, basedon these two predictions, species delimitation should be more efficient when usingmolecular markers experiencing high rates of gene flow. The present thesis was designed to test the hypotheses of this model. The biologicalmodel used was conifers, a group in which introgression and hybridization arecommon because of incomplete reproductive isolation. The species investigatedbelong to the genus Picea (spruce). We focused on two species complexes,represented by monographic clades in a phylogenetic study using the chloroplast genematK. All species studied occur in the Qinghai-Tibetan Plateau (QTP) and adjacenthighlands. The phylogeography of these species complexes was reconstructed usingorganelle markers (mitochondrial DNA, mtDNA and chloroplast DNA, cpDNA). Inconifers, mtDNA and cpDNA have contrasted modes of inheritance. The former ismaternally inherited, transmitted by seeds experiencing little gene flow while thelatter is paternally inherited, transmitted by both pollen and seeds experiencing highlevels of gene flow. Therefore, uniparentally inherited mtDNA and cpDNA markersexperience different rates of gene flow in such a group, providing an ideal model to test the relationship between rates of gene flow, introgression and species delimitation.Two mtDNA fragments (nad1intron b/c; nad5 intron1) and three cpDNA fragments(ndhK-C；trnL-trnF；trnS-trnG) were sequenced for nine species belonging to thePicea asperata and P. likiangensis species complex.(1) Nine mtDNA and nine cpDNA haplotypes were detected in 459 individualsfrom 46 natural populations in five species of P. asperata complex. As found in mostconifer species studied so far, low variation is present in the two mtDNA intronsalong with a high level of differentiation among populations (GST = 0.90). In contrast,higher variation and lower differentiation among populations was found at cpDNAmarkers (GST = 0.56). The cpDNA, although far from being fully diagnostic, is morespecies-specific than mtDNA: four groups of populations were identified usingcpDNA markers, all of them related to species or groups of species, whereas formtDNA, geographical variation prevails over species differentiation. A literaturereview shows that mtDNA variants are often shared among related conifer species,whereas cpDNA variants are more species-specific. Hence, increased intraspecificgene flow appears to decrease differentiation within species but not among species.[...]

My thesis aims at identifying both genetic and environmental causes of major depressive disorder (MDD), using a large case-control study: 6,000 Chinese women with recurrent MDD and 6,000 controls. One of the major challenges for conducting genetic research on MDD is disease heterogeneity. The first question addressed is how different MDD is from highly comorbid anxiety disorders. I examine how anxiety disorders predict clinical features of depression and the degree of heterogeneity in their predictive pattern. The second question addressed is whether clinically defined MDD is a single disorder, or whether it consists of multiple subtypes. Results are then compared with and interpreted in the context of Western studies. Furthermore, latent class analysis and factor analysis results are also used in association analysis to explore more genetically homogeneous subtypes. Genetic data were derived using a novel strategy, low pass whole genome sequence analysis. Using genotypes imputed from the sequence data, I show that a cluster of single nucleotide polymorphisms (SNPs) is significantly associated with a binary disease phenotype including only cases with = 4 episodes of MDD, suggesting that recurrence might be an indication of genetic predisposition. The third issue examined is the contribution of rare variants to disease susceptibility. Again using sparse sequence data, I identified exonic sequence variants and performed gene-based analysis by comparing the number of variants between cases and controls in every gene. Furthermore I performed gene enrichment test by combining P values of SNP association tests at different minor allele frequency ranges. Overall, I did not find convincing evidence that rare variants aggregately contribute to disease susceptibility. However, the gene-based analysis resulted in an unexpected finding: cases have an excess of variants in all thirteen-protein coding mitochondrial genes, which was due to copy number differences in the mitochondrial genome. Both human phenotypic data as well as mice experimental data show that the increase in the mitochondrial copy number in cases is due to chronic stress.

Mon étude vise à évaluer la propagation d’une zoonose en émergence au Québec, la maladie de Lyme, en conséquence du réchauffement climatique. Le pathogène responsable de cette infection, Borrelia burgdorferi, est transmis par l’intermédiaire d’une tique parasite, Ixodes scapularis, de plus en plus commune au Québec en raison de l’augmentation de la température moyenne du climat depuis les dernières décennies. Puisque la tique a une capacité de déplacement très restreinte, on s'attend à ce que sa dispersion soit liée à celle de son hôte primaire, soit la souris à pattes blanches (Peromyscus leucopus). Je décrirai donc d’abord les espèces impliquées, leur écologie et leur rôle dans ce système à trois niveaux (hôte/pathogène/vecteur). Puis, à l’aide de séquences d’ADN mitochondrial, je comparerai la phylogéographie des deux principales espèces de souris au Québec, la souris à pattes blanches et la souris sylvestre (P. maniculatus). Des analyses d’arbres et de réseaux d’haplotypes ont révélé des différences significatives dans la structure génétique et ainsi montré que les populations de P. leucopus seraient en expansion dans le sud du Québec. Cette étude nous a finalement permis d’émettre des hypothèses sur le patron d’établissement de la maladie de Lyme au Québec. / My study aims to assess the spread of an emerging zoonosis in Québec, Lyme disease, as a consequence of global warming. The pathogen responsible for this infection, Borrelia burgdorferi, is transmitted through a tick parasite, Ixodes scapularis, increasingly common in Québec due to the elevation of the average temperature of the atmosphere over the past decades. Since the tick has a very limited dispersal capacity, it is expected that its dispersion is linked to that of its primary host, the white-footed mouse (Peromyscus leucopus). I first described the species involved, their ecology and role in this three-level system (host / pathogen / vector). Then, using mitochondrial DNA sequences, I compared the phylogeography of the two main mouse species in Québec, the white-footed mouse and the deer mouse (P. maniculatus). Analyses of trees and haplotype networks revealed significant differences in the genetic structure, and thus showed that populations of P. leucopus are expanding in southern Québec. This study finally allowed making assumptions on the pattern of establishment of Lyme disease in Québec.