In most tropical river systems there has been a lack of integrated ecological research to investigate the dynamics and impacts of invasive species on recipient river systems. This is in sharp contrast to temperate river systems. This thesis investigated the nature, extent, and impact of Nile tilapia, Oreochromis niloticus (Linnaeus, 1758), on indigenous congenerics within the Limpopo River basin in northern South Africa. An integrated approach was adopted to gain a better understanding of factors that allow Nile tilapia to be a successful invader and also to gain an insight into its invasion rate and conservation implications within South Africa. Morphometric and genetic variation between Nile tilapia, indigenous congenerics and their associated hybrids were determined. Intermediate meristic characters obscured the identification of hybrid specimens from pure morpho-specimens and species identity was only confirmed through mtDNA analysis. Preliminary evidence points to unidirectional hybridization among Oreochromis congeners in the Limpopo River system. The hypothesis that bigger Nile tilapia males may have a competitive advantage over spawning grounds and in female mate choice is proposed. The trophic ecology of Nile tilapia was investigated using both stomach contents and stable isotope analysis. A high similarity in stomach contents was observed but interspecific differences were revealed in the isotopic composition of diets that suggest fine scale patterns of resource partitioning that could be achieved by the ability of fish to selectively feed on what is immediately available and the ability to perceive the dynamics that determine food resource availability. Ecological niche models were used to determine the potential invasive range of Nile tilapia and revealed broad invasive potential over most river systems in southern Africa that overlapped the natural range of endemic congenerics. It was noted that model performance and the degree of niche conservatism varied significantly with variable selection and spatial extent of study area. This implied that the spatial distribution of suitable and unsuitable environmental variables varied between the native and introduced ranges of Nile tilapia and also indicated the ability of Nile tilapia to survive in conditions incongruent with its native range. The extreme hardiness and adaptive life history characteristics of Nile tilapia have probably predisposed it to be a successful invader in novel systems within southern Africa. Lastly, a qualitative risk assessment method was developed as a potential application to determine the risk of establishment and spread of the invasive Nile tilapia. Results showed that in the absence of quantitative data on ecosystem structure and functioning, habitat suitability analysis in terms of known physiological tolerance limits to minimum water temperature, presence or absence of dams, seasonality of river flows and the presence of indigenous fish species of concern could be adequate for identifying vulnerable river systems. The model developed also provides an objective method that is easy to implement, modify and improve on as new data become available. Furthermore, the model can be applied to highlight areas of uncertainty where future research should be directed. / Thesis (PhD)--University of Pretoria, 2012. / Zoology and Entomology / Unrestricted

In the present study, haplogroup composition of five native Turkish sheep breeds, (Karayaka, Akkaraman, G&ouml / k&ccedil / eada, Dagli&ccedil / , Morkaraman) and two sheep breeds from neighboring countries (Herik from Iran, samples from Azerbaijan) were determined by single strand length polymorphism (SSCP) analysis of mitochondrial DNA (mtDNA) NADH dehydrogenase subunit 4 (ND4) region and restriction fragment length polymorphism (RFLP) analysis of mtDNA control (CR) region. Results of the SSCP and RFLP approaches were found to be 96,82% consistent. Most of the 3,18% inconsistency was due to unidentified band patterns of 9 individuals. SSCP method could identify haplogroups A, B and C, but not D and E. Similarly RFLP method could identify haplogroup A, B and possibly D, but not E and C. Data of the present study were compared with those of the previous studies to test the robustness of results under different samplings and were found to be homogenous with a previous study with similar sampling strategy. Neighbor joining tree, principal component analysis (PCA), Delaunay network analysis and analysis of molecular variance (AMOVA) were employed to analyze the haplogroup frequencies and breeds were separated in four groups according to the genetic barriers between breeds from different geographical locations. Strongest differentiation was present between two groups which were eastern breeds (Morkaraman, Herik-Iran and Azerbaijan) and western breeds (G&ouml / k&ccedil / eada, Akkaraman, Karayaka and Dagli&ccedil / ). Additionally, Azerbaijan was proposed as the entrance point of the haplogroup A and the Iran was proposed as the entrance point of haplogroup C to Anatolia with the Spearman rank correlation test.

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.

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.

>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.

<p>In many forensic cases, such as mass disasters reconstruction cases, the recovered DNA is highly degraded. In such incidences, typing of STR loci has become one of the most powerful tools for retrieving information from the degraded DNA. However, as DNA degradation proceeds, three phenomena occur consecutively: loci imbalance, allele dropout and no amplification. To solve the problem of degraded DNA, redesigned primer sets have been developed in which the primers were positioned as close as possible to the STR repeat region. These reduced primer sets were called Miniplexes. Unfortunately, a few of the CODIS STR loci cannot be made into smaller amplicons. For this reason non-CODIS miniSTRs have been developed. The present study was undertaken for the population genetic analysis of microsatellite variation in four South African populations / Afrikaner, Xhosa, Mixed Ancestry and Asian Indian using eight non-CODIS miniSTR loci. These miniSTRs loci were characterized within the populations by estimating the levels of diversity of the markers, estimating the population genetic parameters, and studying the inter-population relationships. All of the miniSTRs were amplified successfully and the genetic variability parameters across all loci in Afrikaner, Mixed Ancestry, Asian Indian and Xhosa were estimated to be in the range of 3 (D4S2364) to 12 (D9S2157) alleles, the total number of alleles over all loci ranged from 100 to 204, the allelic richness ranged from 3.612 to 10.307 and the heterozygosity ranged from 0.4360 to 0.8073. Genetic distance was least between Afrikaner and Asian Indian and highest between Xhosa and Mixed Ancestry. Deviations from Hardy-Weinberg equilibrium were not observed for most of the loci. The low mean FIS (-0.027) and FIT (-0.010) and FST (0.017) values across the populations indicated low level of inbreeding within (FIS) and among (FST) the populations. The Asian Indian population showed higher levels of the inbreeding coefficient, indicating less gene exchange between it and other populations. These 8 markers can be used for genetic investigations and assessing population structure. The study contributed to the knowledge and genetic characterization of four South African populations. In addition, these MiniSTRs prove to be useful in cases where more genetic information is needed.</p>

Der plötzliche Kindstod (engl. Sudden Infant Death Syndrome-SIDS) ist die häufigste Todesursache bei Säuglingen innerhalb des ersten Lebensjahres. Die zugrundeliegenden pathophysiologischen Veränderungen sowie die genaue Todesursache sind bis dato ungeklärt. Viele Forschungsbereiche setzen sich intensiv mit der Klärung dieses „Phänomens“ auseinander. Ein Schwerpunkt liegt auf dem genetischen Gebiet und der Betrachtung verschiedener Polymorphismen. Ein Fokus wird hierbei auf die genetischen Polymorphismen der mitochondrialen DNA (mtDNA) gesetzt. In der vorliegenden Arbeit wurden daher drei Polymorphismen der mtDNA und mögliche Risikofaktoren im Bezug zu SIDS-Fällen untersucht. Die Folge eines mitochondrialen Polymorphismus kann beispielsweise die verminderte Genexpression der Untereinheiten der Atmungskette zur Folge haben. Daraus kann ein Defizit in der ATP (Adenosintriphosphat)-Produktion resultieren. Der physiologische Kreislauf einer menschlichen Zelle ist durch dieses Defizit nur eingeschränkt gewährleistet. Im Rahmen der Forschungsarbeit wurden die SNPs G3010A, T16519C und C7028T der mtDNA in Hinblick auf einen möglichen Zusammenhang mit dem SIDS untersucht. Schon 2003 untersuchten Divne et al. (2003) einen möglichen Zusammenhang der SNPs G3010A und C7028T im Zusammenhang mit SIDS, jedoch ohne signifikantes Ergebnis. Boles et al. (2010) konnten eine Assoziation zwischen den Polymorphismen G3010A und T16519C mit dem plötzlichen Kindstod herstellen. Da bislang jedoch keine ausführliche Publikation zu dieser Frage vorliegt, wurde mit der vorliegenden Arbeit die Rolle der Polymorphismen G3010A und T16519C in Bezug auf den plötzlichen Kindstod gemeinsam mit der (bei Europäern) häufigsten Variation C7028T untersucht. Die DNA von 176 SIDS-Fällen und einer Kontrollgruppe von 113 Erwachsenen wurde mittels Singleplex-PCR und RFLP-Analyse genotypisiert. Anhand der Genotypisierung konnten die SNPs quantifiziert und im Hinblick auf einen möglichen Unterschied zwischen SIDS-Fällen und der Kontrollgruppe untersucht werden. Bei Betrachtung der einzelnen SNPs G3010A, T16519C und C7028T lassen sich keine signifikanten Unterschiede zwischen den SIDS-Fällen und der Kontrollgruppe feststellen. Das gehäufte Vorliegen einer erhöhten Mutationsrate in einem Individuum bei SIDS-Fällen im Vergleich zur Kontrollgruppe, sowie die von Opdal et al. (1999) geäußerte Annahme, dass beim Vorliegen einer Mutation in der D-Loop-Region weitere Mutationen im kodierenden Bereich vorkommen, konnten durch diese Arbeit bestätigt werden.

<p>In many forensic cases, such as mass disasters reconstruction cases, the recovered DNA is highly degraded. In such incidences, typing of STR loci has become one of the most powerful tools for retrieving information from the degraded DNA. However, as DNA degradation proceeds, three phenomena occur consecutively: loci imbalance, allele dropout and no amplification. To solve the problem of degraded DNA, redesigned primer sets have been developed in which the primers were positioned as close as possible to the STR repeat region. These reduced primer sets were called Miniplexes. Unfortunately, a few of the CODIS STR loci cannot be made into smaller amplicons. For this reason non-CODIS miniSTRs have been developed. The present study was undertaken for the population genetic analysis of microsatellite variation in four South African populations / Afrikaner, Xhosa, Mixed Ancestry and Asian Indian using eight non-CODIS miniSTR loci. These miniSTRs loci were characterized within the populations by estimating the levels of diversity of the markers, estimating the population genetic parameters, and studying the inter-population relationships. All of the miniSTRs were amplified successfully and the genetic variability parameters across all loci in Afrikaner, Mixed Ancestry, Asian Indian and Xhosa were estimated to be in the range of 3 (D4S2364) to 12 (D9S2157) alleles, the total number of alleles over all loci ranged from 100 to 204, the allelic richness ranged from 3.612 to 10.307 and the heterozygosity ranged from 0.4360 to 0.8073. Genetic distance was least between Afrikaner and Asian Indian and highest between Xhosa and Mixed Ancestry. Deviations from Hardy-Weinberg equilibrium were not observed for most of the loci. The low mean FIS (-0.027) and FIT (-0.010) and FST (0.017) values across the populations indicated low level of inbreeding within (FIS) and among (FST) the populations. The Asian Indian population showed higher levels of the inbreeding coefficient, indicating less gene exchange between it and other populations. These 8 markers can be used for genetic investigations and assessing population structure. The study contributed to the knowledge and genetic characterization of four South African populations. In addition, these MiniSTRs prove to be useful in cases where more genetic information is needed.</p>

Magister Scientiae - MSc / In many forensic cases, such as mass disasters reconstruction cases, the recovered DNA is highly degraded. In such incidences, typing of STR loci has become one of the most powerful tools for retrieving information from the degraded DNA. However, as DNA degradation proceeds, three phenomena occur consecutively: loci imbalance, allele dropout and no amplification. To solve the problem of degraded DNA, redesigned primer sets have been developed in which the primers were positioned as close as possible to the STR repeat region. These reduced primer sets were called Miniplexes. Unfortunately, a few of the CODIS STR loci cannot be made into smaller amplicons. For this reason non-CODIS miniSTRs have been developed. The present study was undertaken for the population genetic analysis of microsatellite variation in four South African populations; Afrikaner, Xhosa, Mixed Ancestry and Asian Indian using eight non-CODIS miniSTR loci. These miniSTRs loci were characterized within the populations by estimating the levels of diversity of the markers, estimating the population genetic parameters, and studying the inter-population relationships. All of the miniSTRs were amplified successfully and the genetic variability parameters across all loci in Afrikaner, Mixed Ancestry, Asian Indian and Xhosa were estimated to be in the range of 3 (D4S2364) to 12 (D9S2157) alleles, the total number of alleles over all loci ranged from 100 to 204, the allelic richness ranged from 3.612 to 10.307 and the heterozygosity ranged from 0.4360 to 0.8073. Genetic distance was least between Afrikaner and Asian Indian and highest between Xhosa and Mixed Ancestry. Deviations from Hardy-Weinberg equilibrium were not observed for most of the loci. The low mean FIS (-0.027) and FIT (-0.010) and FST (0.017) values across the populations indicated low level of inbreeding within (FIS) and among (FST) the populations. The Asian Indian population showed higher levels of the inbreeding coefficient, indicating less gene exchange between it and other populations. These 8 markers can be used for genetic investigations and assessing population structure. The study contributed to the knowledge and genetic characterization of four South African populations. In addition, these MiniSTRs prove to be useful in cases where more genetic information is needed. / South Africa

Culicoides (Diptera: Ceratopogonidae) are small (<3mm) blood feeding flies. These flies are biological vectors of viruses, protozoa and filarial nematodes affecting birds, humans, and other animals. Among the viruses transmitted those causing bluetongue (BT), African horse sickness (AHS) and epizootic haemorrhagic disease (EHD) are of major veterinary significance. Culicoides (Avaritia) imicola Kieffer, a proven vector of both AHS and BT viruses, is the most abundant and wide spread livestock-associated Culicoides species in South Africa. Field isolations of virus and oral susceptibility studies, however, indicated that a second Avaritia species, C. bolitinos Meiswinkel may be a potential vector of both BT virus (BTV) and AHS virus (AHSV). Differences in oral susceptibility, which are under genetic control, of populations from different geographical areas to viruses may be an indication of genetic differences between these populations, which may be the result of limited contact between these populations. A good knowledge of the distribution, spread and genetic structure of the insect vector is essential in understanding AHS or BT disease epidemiology. In the present study, an effort was made to gather field specimens of both C. imicola and C. bolitinos from different areas within their natural distribution in South Africa. The aim was to partially sequence two mitochondrial genes from these specimens and to analyse the sequence data making use of phylogenetic trees to clarify the genetic relationships between individuals or groups collected from geographically distinct sites. The two species were collected from four geographically separated areas in South Africa viz. Gauteng Province, Eastern Cape Province, Western Cape Province as well as the Free State Province. DNA was extracted from a total of 120 individual midges of the two Culicoides species using DNA extraction kits. Extracted DNA was analysed using PCR, sequencing as well as phylogenetic methods. A total of 117 mitochondrial DNA COI and 104 mitochondrial 16S ribosomal RNA Culidoides</i. sequences were analysed. DNA sequence polymorphism and phylogenetic relationships of various groups of C. imicola and C. bolitinos midges were determined. The results of the phylogenetic analysis of Culicoides populations using mitochondrial COI gene fragment showed that, at least one subpopulation of C. imicola and two distinct genotypes of C. bolitinos species do exist in South Africa, and further analysis is necessary. This study showed that COI has the potential to separate Culicoides midges based on their geography / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted