The laws for the inheritance of animal mitochondrial DNA differ from those revealed for nuclear DNA. In contrast to nuclear genes, animal mitochondrial DNA (mtDNA) is predominantly inherited through the maternal line and is typically assumed to be nonrecombining. The absence of both paternal transmission (hereafter: paternal leakage) and heterologous recombination of mtDNA are assumed to be key characteristics of mitochondrial DNA inheritance, which has enabled evolutionary models to be much simpler than those needed for the interpretation of nuclear DNA. However, recent revelations of paternal leakage in the animal kingdom challenge our current knowledge about mtDNA inheritance and the utility of mtDNA as a molecular marker. The occurrence of paternal leakage potentially introduces new haplotypes into populations and therefore impacts on the interpretation of mtDNA analysis. To date, it is unclear whether the documented cases of paternal leakage are exceptions to the general rule or if these events occur more frequently than so far believed. If this event occurred at a measurable frequency, it is vital to implement such data into models of mtDNA evolution to improve the accuracy at which evolutionary relationships and times of divergence are estimated. In this thesis, I aimed to provide an insight into the broader patterns of mtDNA inheritance using chinook salmon as a model organism. I first sought to delimit the frequency of paternal leakage in chinook salmon and further investigated two major mechanisms which are believed to limit paternal leakage: The many-fold dilution of paternal mtDNA by maternal mtDNA upon fertilization and the genetic bottleneck mtDNA is believed to be exposed to during early developmental stages. A screen of roughly 10.000 offspring did not reveal the presence of paternal mtDNA within these samples delimiting the maximum frequency of paternal leakage in this system to 0.18% (power of 0.95) and 0.27% (power of 0.99), suggesting that the occurrence of paternal leakage is most likely an exception to the general rule. To infer the dilution of paternal mtDNA upon fertilization, I employed real-time PCR and determined the mtDNA content of salmon spermatozoa and oocytes to be 5.73 ± 2.28 and 3.15x109 ± 9.98x108 molecules per gamete, respectively. Accordingly, the estimated ratio of paternal to maternal mtDNA in zygotes is 1:7.35x108 ± 4.67x108. This estimate is 3 to 5 orders of magnitude smaller than the ratio revealed for mammals. Consequently, and if the dilution acts as an efficient barrier against the transmission of paternal mtDNA, paternal inheritance of mtDNA per offspring will be much less likely in this system than in mammals. To estimate at what probability the diminutive contribution of paternal mtDNA in zygotes is potentially inherited to offspring, I determined the size of the bottleneck acting on mtDNA during both embryogenesis and oogensis by examining the transmission of mtDNA variants to offspring and oocytes within a pedigree of heteroplasmic individuals. The number of segregating units (mtDNAs) between a mother’s somatic tissue and oocytes was estimated to be 109.3 (median = 109.3; 62.4 < NeOog < 189.6; 95% confidence interval) and from a mother’s soma to offspring’s soma 105.4 (median = 105.4; 70.3 < NeEmb < 153.1; 95% confidence interval). Detected variances in allele frequency among oocytes were not significantly different from those in offspring, strongly suggesting that segregation of mtDNA occurs during oogenesis with its completion before oocyte maturation. However, considering a ratio of roughly 1:7.35x108 for paternal to maternal mtDNA in zygotes and that approximately 109.3 (NeOog) of the mitochondrial genomes present in zygotes are ultimately inherited to offspring, the probability for paternal mtDNA to be transmitted to offspring is in round terms 1.0x10-11/paternal mtDNA molecule. In summary, the results presented in this thesis document the presence of efficient barriers to prohibit the inheritance of minor allele contributions, such as paternal mtDNA, to offspring. These results strongly suggest that paternal leakage is an exception to the general rule. Furthermore, in comparison to studies undertaken in mammals, my results indicate that mechanisms in place to prevent paternal leakage may be unequally efficient among different animal taxa, reflecting differences in life traits, such as gamete morphology, gamete investment and reproductive strategies. Nonetheless, by the means of the dilution effect in zygotes and the genetic bottleneck during oogenesis, the occurrence of paternal leakage might be simply a quantitative phenomenon and cannot be excluded per se. The increasing number of documented cases of paternal leakage clarifies that its occurrence must be considered when applying mtDNA as a genetic marker. Furthermore, for species in which mtDNA inheritance can be confirmed to be purely random, theoretical frequencies of paternal leakage can be inferred and potentially implemented into models of mtDNA evolution.

No description available.

The genus Fucus (Phaeophyceae) has nine commonly accepted species, six of which inhabit North American shores: F. gardneri, F. serratus, F. distichus, F. evanescens, F. vesiculosus, and F. spiralis. Fucus inhabit the intertidal zones of the Atlantic, Pacific and Arctic Oceans and due to their highly plastic and morphologically simple phenotypes, are valuable candidates for molecular phylogenetic research. Furthermore, Fucus species are valuable for biogeographic investigations due to their relatively slow migration, limited dispersal range, and easily collected distribution. The closest ancestral species to the genus is a Pacific endemic Hesperophycus californicus supporting a North Pacific origin of the species (Serrão et al. 1999). However, a closer relationship between Atlantic and Arctic Fucus has been described casting doubt on the Pacific origins of the genus (Lindstrom 2001, Dunton 1992). The investigation of Fucus across this area is necessary to elucidate the apparent contradiction in the origin of the genus. Samples were collected from 79 locations across North American waters. The amplification of the divergent mtDNA spacer region was performed to assess taxonomic placement of 55 collected species and reconstruct the biogeography of North American Fucus species. Morphological analysis was also completed based on 21 measured characteristics in an attempt to support molecular analysis. Results of the study indicate two distinct lineages among collected samples. One lineage (F. spiralis and F. vesiculosus) with a distinct Atlantic origin and the second lineage (F. distichus and F. serratus) with a widely distributed F. distichus, and the Atlantic F. serratus. Morphological analysis and statistical support for these lineages was determined through discriminant analysis of the collected samples. Overall results determined biogeographic influences in the F. distichus species complex across Arctic, Pacific, and Atlantic regions with little apparent biogeographical influence within F. vesiculosus, F. serratus and F. spiralis. Closer phylogenetic relationships between Arctic and Atlantic samples were apparent as suggested by Lindstrom (2001) and Dunton (1992).

The genus Fucus (Phaeophyceae) has nine commonly accepted species, six of which inhabit North American shores: F. gardneri, F. serratus, F. distichus, F. evanescens, F. vesiculosus, and F. spiralis. Fucus inhabit the intertidal zones of the Atlantic, Pacific and Arctic Oceans and due to their highly plastic and morphologically simple phenotypes, are valuable candidates for molecular phylogenetic research. Furthermore, Fucus species are valuable for biogeographic investigations due to their relatively slow migration, limited dispersal range, and easily collected distribution. The closest ancestral species to the genus is a Pacific endemic Hesperophycus californicus supporting a North Pacific origin of the species (Serrão et al. 1999). However, a closer relationship between Atlantic and Arctic Fucus has been described casting doubt on the Pacific origins of the genus (Lindstrom 2001, Dunton 1992). The investigation of Fucus across this area is necessary to elucidate the apparent contradiction in the origin of the genus. Samples were collected from 79 locations across North American waters. The amplification of the divergent mtDNA spacer region was performed to assess taxonomic placement of 55 collected species and reconstruct the biogeography of North American Fucus species. Morphological analysis was also completed based on 21 measured characteristics in an attempt to support molecular analysis. Results of the study indicate two distinct lineages among collected samples. One lineage (F. spiralis and F. vesiculosus) with a distinct Atlantic origin and the second lineage (F. distichus and F. serratus) with a widely distributed F. distichus, and the Atlantic F. serratus. Morphological analysis and statistical support for these lineages was determined through discriminant analysis of the collected samples. Overall results determined biogeographic influences in the F. distichus species complex across Arctic, Pacific, and Atlantic regions with little apparent biogeographical influence within F. vesiculosus, F. serratus and F. spiralis. Closer phylogenetic relationships between Arctic and Atlantic samples were apparent as suggested by Lindstrom (2001) and Dunton (1992).

No description available.

The laws for the inheritance of animal mitochondrial DNA differ from those revealed for nuclear DNA. In contrast to nuclear genes, animal mitochondrial DNA (mtDNA) is predominantly inherited through the maternal line and is typically assumed to be nonrecombining. The absence of both paternal transmission (hereafter: paternal leakage) and heterologous recombination of mtDNA are assumed to be key characteristics of mitochondrial DNA inheritance, which has enabled evolutionary models to be much simpler than those needed for the interpretation of nuclear DNA. However, recent revelations of paternal leakage in the animal kingdom challenge our current knowledge about mtDNA inheritance and the utility of mtDNA as a molecular marker. The occurrence of paternal leakage potentially introduces new haplotypes into populations and therefore impacts on the interpretation of mtDNA analysis. To date, it is unclear whether the documented cases of paternal leakage are exceptions to the general rule or if these events occur more frequently than so far believed. If this event occurred at a measurable frequency, it is vital to implement such data into models of mtDNA evolution to improve the accuracy at which evolutionary relationships and times of divergence are estimated. In this thesis, I aimed to provide an insight into the broader patterns of mtDNA inheritance using chinook salmon as a model organism. I first sought to delimit the frequency of paternal leakage in chinook salmon and further investigated two major mechanisms which are believed to limit paternal leakage: The many-fold dilution of paternal mtDNA by maternal mtDNA upon fertilization and the genetic bottleneck mtDNA is believed to be exposed to during early developmental stages. A screen of roughly 10.000 offspring did not reveal the presence of paternal mtDNA within these samples delimiting the maximum frequency of paternal leakage in this system to 0.18% (power of 0.95) and 0.27% (power of 0.99), suggesting that the occurrence of paternal leakage is most likely an exception to the general rule. To infer the dilution of paternal mtDNA upon fertilization, I employed real-time PCR and determined the mtDNA content of salmon spermatozoa and oocytes to be 5.73 ± 2.28 and 3.15x109 ± 9.98x108 molecules per gamete, respectively. Accordingly, the estimated ratio of paternal to maternal mtDNA in zygotes is 1:7.35x108 ± 4.67x108. This estimate is 3 to 5 orders of magnitude smaller than the ratio revealed for mammals. Consequently, and if the dilution acts as an efficient barrier against the transmission of paternal mtDNA, paternal inheritance of mtDNA per offspring will be much less likely in this system than in mammals. To estimate at what probability the diminutive contribution of paternal mtDNA in zygotes is potentially inherited to offspring, I determined the size of the bottleneck acting on mtDNA during both embryogenesis and oogensis by examining the transmission of mtDNA variants to offspring and oocytes within a pedigree of heteroplasmic individuals. The number of segregating units (mtDNAs) between a mother’s somatic tissue and oocytes was estimated to be 109.3 (median = 109.3; 62.4 < NeOog < 189.6; 95% confidence interval) and from a mother’s soma to offspring’s soma 105.4 (median = 105.4; 70.3 < NeEmb < 153.1; 95% confidence interval). Detected variances in allele frequency among oocytes were not significantly different from those in offspring, strongly suggesting that segregation of mtDNA occurs during oogenesis with its completion before oocyte maturation. However, considering a ratio of roughly 1:7.35x108 for paternal to maternal mtDNA in zygotes and that approximately 109.3 (NeOog) of the mitochondrial genomes present in zygotes are ultimately inherited to offspring, the probability for paternal mtDNA to be transmitted to offspring is in round terms 1.0x10-11/paternal mtDNA molecule. In summary, the results presented in this thesis document the presence of efficient barriers to prohibit the inheritance of minor allele contributions, such as paternal mtDNA, to offspring. These results strongly suggest that paternal leakage is an exception to the general rule. Furthermore, in comparison to studies undertaken in mammals, my results indicate that mechanisms in place to prevent paternal leakage may be unequally efficient among different animal taxa, reflecting differences in life traits, such as gamete morphology, gamete investment and reproductive strategies. Nonetheless, by the means of the dilution effect in zygotes and the genetic bottleneck during oogenesis, the occurrence of paternal leakage might be simply a quantitative phenomenon and cannot be excluded per se. The increasing number of documented cases of paternal leakage clarifies that its occurrence must be considered when applying mtDNA as a genetic marker. Furthermore, for species in which mtDNA inheritance can be confirmed to be purely random, theoretical frequencies of paternal leakage can be inferred and potentially implemented into models of mtDNA evolution.

Made available in DSpace on 2014-06-12T18:06:18Z (GMT). No. of bitstreams: 2 arquivo6292_1.pdf: 2278888 bytes, checksum: 0ebd2a7a155939e0bf1c1540b30d92da (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / A análise das seqüências de DNA mitocondrial humano (mtDNA) tem sido uma ferramenta muito útil na genética forense devido às características especiais do mtDNA como herança materna, ausência de recombinação e alto número de cópias por célula. O objetivo deste trabalho foi determinar o polimorfismo das regiões hipervariáveis 1 e 2 do mtDNA humano na população de Alagoas, Brasil. Para isso, foram seqüenciados 167 mtDNAs de indivíduos não relacionados desta população para análise dos dois segmentos hipervariáveis, HV1 e HV2, da região controle. A heteroplasmia de comprimento, nas regiões de trechos de citosina repetidas em HV1/HV2, foi observada em 22% (37/167) e 11% (19/167) da amostra, respectivamente. Dos 123 segmentos de HV1 e HV2 restantes, um total de 110 haplótipos diferentes foram encontrados, sendo determinados por 128 posições variáveis. O haplótipo mais freqüente (16111, 16223, 16290, 16319, 16362, 73, 146, 153, 235, 263, 309.1C, 315.1C - haplogrupo A) foi econtrado em cinco indivíduos, seguido por um haplótipo compartilhado por três indivíduos e um haplótipo compartilhado por duas pessoas em sete ocorrências diferentes (frequência de 1,6%). A diversidade genética foi estimada em 0,997 e a probabilidade de dois indivíduos ao acaso possuírem mtDNAs idênticos foi de 0,011. Baseado nos resultados dos perfis de mtDNA, 45% das sequências puderam ser classificadas como haplogrupos africanos, 27% como nativo-americanos e 25% como europeus. Cerca de 3% dos haplótipos não puderam ser classificados em nenhum haplogrupo. A diversidade genética das regiões HV1 e HV2 indica a importância desses locos para a identificação humana na população de Alagoas

Doctor of Philosophy / A number of types of molecular polymorphisms can be used for studying genetic relationship and evolutionary history. Microsatellites are hypervariable and can be very useful tools to determine population structure, distinguish sibling species, as well as verifying parental relationships and pedigrees. However, while microsatellite polymorphisms are useful for solving relationships between populations within a species, relations among species or genera will probably be obscured due to a high degree of homoplasy —identity arising from evolutionary convergence not by descent. For long range evolutionary history, such as phylogeny from old world monkey to human, mtDNA markers may be better candidates. The aim of this thesis is to assess molecular polymorphisms of different types and their optimal use in different situations. Two widely separated taxa were used for testing –the green monkey Chlorocebus sabaeus, and the sibling dipteran flies Bactrocera tryoni and B. neohumeralis, known collectively as the Queensland fruit fly. In the present study a complete 16,550 bp mtDNA sequence of the green monkey Chlorocebus sabaeus is reported for the fist time and has been annotated (Chapter 2). Knowledge of the mtDNA genome contributes not only to identification of large scale single nucleotide polymorphisms (SNPs) (Chapter 4) or other mtDNA polymorphisms development, but also to primate phylogenetic and evolutionary study (Chapter 3). Microsatellites used for the green monkey paternity and pedigree studies were developed by cross-amplification using human primers (Chapter 5). For studies of population structure and species discrimination in Queensland fruit fly (Chapter 7), microsatellites were isolated from a genomic library of Bactrocera tryoni (Chapter 6) The total length of 16550 bp of complete mtDNA of the green monkey C. sabaeus, which has been sequenced and annotated here, adds a new node to the primate phylogenetic tree, and creates great opportunity for SNP marker development. The heteroplasmic region was cloned and five different sequences from a single individual were obtained; the implication of this are discussed. The phylogenetic tree reconstructed using the complete mtDNA sequence of C. sabaeus and other primates was used to solve controversial taxonomic status of C. sabaeus. Phylogenies of primate evolution using different genes from mtDNA are discussed. Primate evolutionary trees using different substitution types are compared and the phylogenetic trees constructed using transversions for the complete mtDNA were found close to preconceived expectations than those with transversions + transitions. The sequence of C. sabaeus 12SrRNA reported here agrees with the one published by ven der Kuyl et al. (1996), but additional SNPs were identified. SNPs for other regions of mtDNA were explored using dHPLC. Twenty two PCR segments for 96 individuals were tested by dHPLC. Fifty five SNPs were found and 10 haplogroups were established. Microsatellite markers were used to construct a genealogy for a colony of green monkeys (C. sabaeus) in the UCLA Vervet Monkey Research Colony. Sixteen microsatellites cross-amplified from human primers were used to conduct paternity analysis and pedigree construction. Seventy-eight out of 417 offspring were assigned paternity successfully. The low success rate is attributed to a certain proportion of mismatches between mothers and offspring; the fact that not all candidate fathers were sampled, the limitations of microsatellite polymorphisms; and weakness of the exclusion method for paternity assessment. Due to the low success rate, the pedigree is split into a few small ones. In a complicated pedigree composed of 75 animals and up to four generations with multiple links a power male mated with 8 females and contributed 10 offspring to the pedigree. Close inbreeding was avoided. Population structure within two species of Queensland fruit fly Bactrocera tryoni and Bactrocera neohumeralis (Tephritidae: Diptera) is examined using microsatellite polymorphisms. Queensland fruit flies B. tryoni and B. neohumeralis are sympatric sibling species that have similar morphological and ecological features. They even share polymorphism at the molecular level. Mating time difference is the main mechanism by which they maintain separate species. In the present study, 22 polymorphic and scorable microsatellites were isolated from B. tryoni and tested in the two species sampled from sympatric distribution areas. Pairwise genetic distance analysis showed explicit differentiation in allele frequencies between the two species, but very weak differences between conspecific populations. Gene flow is higher within B. tryoni than within B. neohumeralis, and gene exchange between the two species exists. An averaging linkage clustering tree constructed by UPGMA showed two major clusters distinguishing the two species, and it appears that population structure is highly correlated with geographic distance. The relationship between molecular markers, evolution, and selection are discussed using comparative studies within two large taxa: primate and insect. The degree of conservation and polymorphism in microsatellites varies between taxa, over evolutionary time.

<p>The legally binding international declaration of the Convention on Biological Diversity (signed by over 180 countries) recently acknowledged the importance of conserving genetic diversity within livestock species. This study aimed to help Canada assess molecular diversity in its horse and pony (<i>Equus ferus caballus</i>) genetic resources. Here, 24 populations were examined, with special focus on the native Canadian, Mountain and Moorland, and Nordic pony populations, using two well accepted molecular tools. Additional horse breeds and feral populations were also included in this project as some may have influenced the development of the three equine groups of interest. Altogether, 821 individuals were genotyped at 38 microsatellite loci, and 280 individuals were sequenced using a 421 base pair portion of the mitochondrial displacement Hypervariable Region I.</p> <p>Results from the microsatellite analyses indicated that 13.33% of genetic diversity arose from breed differences, whereas 84.60% and 2.07% of diversity arose from within and among individuals respectively. The New Forest and Welsh breeds were found to be the most diverse while having the highest average effective number of alleles and allelic richness (4.31 and 6.01; 4.33 and 5.87 respectively). The Eriskay and Lac La Croix breeds were found to have the lowest average effective number of alleles and allelic richness (2.51 and 3.98; 2.83 and 4.01 respectively). Expected heterozygosities were lowest in the Lac La Croix (0.61) and highest in the Welsh and New Forest (0.74) breeds, whereas observed heterozygosities were highest in the Kerry Bog (0.77) and lowest in the Exmoor (0.57) breeds. The genetic structure and admixture analyses suggested that the most probable number of unique genetic clusters was 21 as opposed to the 24 predefined populations.</p> <p>Results from the mitochondrial sequence data revealed that there were 36 informative sites producing 62 haplotypes, 20 of which were previously unreported. The Connemara was found to have the highest haplotype diversity of the pony breeds (0.89); however, the Highland pony was found to have the highest nucleotide diversity and pairwise difference (0.16 and 6.73 respectively). In contrast, the Fell pony had the lowest haplotype diversity (0.22), and the feral Sable Island population had the lowest nucleotide diversity and pairwise difference (0.01 and 0.29 respectively). Multiple phylogenetic trees were reconstructed and produced similar topologies. In general, the Mountain and Moorland and Nordic breeds were spread among the clades, whereas native Canadian populations were most frequent in the D and E clades. Interestingly, a large portion of ponies were found within the rare E clade as opposed to horses.</p> <p>Information gathered from this project can be incorporated with other available data into pre-existing conservation/breeding programs currently managed by the various breed societies to ensure that the most optimal and sustainable strategies are in place.</p>

El factor de terminación de la transcripción humano (mTERF) es una proteína codificada en el genoma nuclear, de 39 kDa, que reconoce una región de 28 pares de bases en el gen del tRNALeu(UUR) mitocondrial inmediatamente adyacente al gen del rRNA 16S. La unión de mTERF a esta región provoca terminación de la transcripción, de modo que esta proteína se considera un factor fundamental en el control de la síntesis de RNAs ribosómicos mitocondriales. A pesar de que el mTERF se une a DNA en forma monomérica, la presencia en su secuencia de tres zippers de leucina nos llevó a explorar la posibilidad de que mTERF establezca interacciones intermoleculares. Cuando un lisado mitocondrial de células HeLa fue sometido a cromatografía de gel filtración, mTERF eluyó en dos picos, detectados por Western blotting. El primer pico eluyó con un peso molecular compatible con la forma monomérica (41 +/- 2 kDa) y las fracciones de gel filtración que lo contenían mostraban capacidad de unión a DNA, tal como se demostró por experimentos de band-shift, Western blotting y cromatografía de heparina, así como actividad de terminación de la transcripción. El segundo pico eluyó con un peso molecular estimado de 111 +/- 5 kDa, y no presentaba capacidad de unión a DNA. Así pues, proponemos que mTERF existe en dos formas, un monómero activo y un polimero inactivo. El peso molecular estimado de la forma polimerica y el hecho de que mTERF purificado de células HeLa eluye de una columna de gel filtración con un peso molecular idéntico al polimero descrito sugiere que el polímero de mTERF es un homotrímero. / The human mitochondrial transcription termination factor (mTERF) is a nuclear-encoded 39 kDa protein that recognizes a 28 base pair region within the mitochondrial tRNALeu(UUR) gene immediately adjacent to and downstream of the 16S rRNA gene. Binding of mTERF to this site promotes termination of transcription, and so this protein is considered a key factor in the control of mitochondrial rRNA synthesis. Despite the fact that mTERF binds DNA as a monomer, the presence in its sequence of three putative leucine zipper motifs led us to explore the possibility of mTERF establishing intermolecular interactions. When a mitochondrial lysate from HeLa cells was submitted to gel filtration chromatography, mTERF eluted in two peaks, detected by immunoblotting. The first peak appeared at the expected molecular weight for the monomer (41 +/- 2 kDa) and the gel filtration fractions containing it showed DNA-binding activity, as tested by band-shift, immunoblotting of the shifted band and heparin chromatography, as well as transcription-termination activity. The second peak eluted at an estimated molecular weight of 111 +/- 5 kDa, and no mTERF-promoted DNA binding activity could be detected in the corresponding gel filtration fractions. Therefore, we propose that mTERF exists in two forms, an active monomer and an inactive polymer. The estimated molecular weight of the polymer and the fact that pure mTERF also elutes from a gel filtration column as a polymeric form of identical molecular weight, suggest that the inactive polymer of mTERF is a homotrimer.