Theses and Dissertations (Genetics)

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Die agamiese kompleks Themeda triandra forsk
(University of Pretoria, 1968-11) Geerthsen, Peter J.M.; Liebenberg, Heinrich
Afrikaans: 'n Si togenetiese studie is onderneem op 53 versamelings, oorspronklik vanaf verskillende lokaliteite, van Themeda triandra Forsk. verkry vanuit die rooigrasversameling op die Rietondale Navorsingstasiete Pretoria. Die ondersoek is veral onderneem ten einde vas te stel of die sitogenetika van Themeda triandra nie kan bydra tot 'n beter begrip van die aard en grondliggende oorsake van die komplekse variasiepatroon wat daar by die gras voorkom nie. Die studie is veral toegespits op chromosoomtelling, kiemsakontwikkeling en meiose in die mikrospoormoederselle van al die versamelings. Van die 53 versamelings is 35.8% diploiede, 41.5% tetraploiede, 5.7% pentaploiede en 16.9% heksaploiede. Hierby is ingesluit 7 aneuploiede op die verskillende ploidie-vlakke. B-chromosome kom by vier versamelings voor. Die meiotiese gedrag van die verskillende versamelings varieer baie. By die diploiede is dit baie normaal, asook oor die algemeen by die tetraploiede en heksaploiede. 'n Aantal van die poliploiede versamelings is egter blykbaar heterosigoties vir resiproke translokasies, en drie versamelings vertoon parasentriese inversie-brue. Die kiemsakontwikkeling van die diploiede is geslagtelik en wel van die Polygonu.m tipe, maar 'n paar van die diploiede versamelings bevat 'n hoe persentasie saadknoppe met veelvoudige geslagtelike kiemsakke. Die poliploiede is oor die algemeen fakultatiewe aposporiese apomikte, maar baie variasie kom voor. Twee van die tetraploiede is volkome geslagtelik. Themeda triandra is blykbaar 'n jong9 aktief ontwikkelende agamiese kompleks. Dit verklaar waarskynlik die diskontinue variasie wat daar in die natuur voorkom, sowel as die taksonomiese probleme wat daar by die spesie bestaan. Voorstelle word gemaak vir die verdere bestudering van die kompleks, veral met die oog op die taksonomie van die groep. Die identifisering en afbakening van die oorsprongsbevolkings van die kompleks sal baie daartoe bydra. Hierdie oorsprongsbevolkings is hoofsaaklik diploiede, maar sluit waarskynlik 1n paar geslagtelike tetraploiede in. Hipoteses ter verklaring van die filogenie van die agamiese kompleks, Themeda triandra, in SuidAfrika, word tentatief voorgestel.
Precision phenotyping of pines for resistance against Fusarium circinatum
(University of Pretoria, 2023) Naidoo, Sanushka; Visser, Erik; ruby.ebbeling@up.ac.za; Ebbeling, Ruby May
Pine trees are globally important agricultural and ecological species, with Pinus patula being a key species in South Africa’s economically valuable forestry industry. Natural and commercial pine forests are significantly threatened by the pitch canker fungus, Fusarium circinatum. In the nursery, F. circinatum infection manifests as a wilt, thereby hindering pine production. Since Pinus spp. exhibit a wide range of inter- and intra-specific variation in resistance, insights into the pine resistance phenotype and the pine-F. circinatum interaction may aid in producing resistant planting stock and mitigating F. circinatum damage. Currently, this pine variation is quantified by visually measuring lesion lengths, which is predictive of the pine response phenotype. Visual estimates, however, are limited by subjectivity, inaccuracy, and low reproducibility. This highlights the requirement for a more reliable, precise disease phenotyping method. Quantifying fungal load in infected plants holds promise for greater sensitivity, specificity, timeliness, and high-throughput. To the best of our knowledge, this phenotyping method has not been explored for quantifying F. circinatum resistance in pines. Therefore, our study aimed to develop and optimise a quantitative real-time PCR (qPCR) precision phenotyping tool to quantify the fungal load of F. circinatum in different Pinus patula (susceptible) and Pinus tecunumanii high (intermediately resistant) and low elevation (resistant) families. We also assessed the application of this tool on F1 hybrid pine genotypes of unknown resistance. As an initial approach to estimate resistance, lesion lengths of plants artificially inoculated with F. circinatum were measured weekly for 8 weeks to calculate percentage live stem. The visible lesion length measurements showed the expected differences in resistance for the pine species as well as variation within and between each pine species and family. Next, the fungal load from artificially inoculated pine samples was quantified at 1, 2, 3, 7, 14, 21, and 28 days post-inoculation (dpi) to calculate percentage fungal load, which was compared to percentage live stem. To assess the efficacy of our precision phenotyping tool, we identified correlations between the median percentage live stem and median percentage fungal load. Strong correlations were observed between the median percentage fungal load and median percentage live stem at 3 and 49 dpi, respectively, (0.66, p<0.01) as well as at 28 and 21 dpi, respectively (−0.76, p<0.01). We also observed variation between the F1 genotypes, which allowed them to be classified into three phenotypic groups: susceptible, intermediately resistant, and resistant. We concluded that this phenotyping tool has the potential for phenotypic classes to be developed on a continuum from highly susceptible to highly resistant, to timely and accurately class different pine individuals. This study represents the first step in developing an optimal qPCR precision phenotyping tool to capture the subtle variation in pine responses to F. circinatum. Future studies would involve optimising this tool further and using it to help characterise the genetic architecture of the pine defence response and identify quantitative trait loci governing resistance against F. circinatum.
Pilot study into the molecular mechanisms of canine distemper virus infection
(University of Pretoria, 2019) Bloomer, Paulette; Leisewitz, Andrew L.; simplytessa1@gmail.com; Rossi Schenone, Tessa
In order to understand how a disease should be prevented, treated and managed, one must understand both the host and the pathogen, and how they interact with and influence one another. Canine distemper virus (CDV) causes canine distemper, a multisystemic disease that can spread to infect the central nervous system resulting in profound nervous system clinical signs. A myriad of different host species are affected by this virus, with significant variation to be seen in how severely different hosts are affected and how rapidly the disease progresses, even within different individuals of the same host species. Although multiple studies have looked at the virus itself, fewer studies have focused on the host, and particularly the molecular mechanisms of the host response that may underlie the variation in host response to the same virus. In this project I looked at DNA polymorphisms in the SLAM and CD46 host receptors in wild canid and felid species and how this could result in amino acid and ultimately protein differences in these receptors crucial for viral entry into the cell. I found that the DNA and amino acid sequences of canid species grouped separately to those of felid species in terms of sequence similarity, with small DNA sequence differences resulting in different amino acids between these species. These amino acid differences in turn may partially contribute to different host affinities for CDV at the receptor level by affecting the binding affinity between the virus and the host receptor. The V-domain of the signal lymphocyte activation molecule (SLAM) showed more sequence variability than the selected CD46 exons. Secondly, I compared the gene expression in the brain tissue of healthy dogs to that of dogs infected with CDV. Using RNA sequencing (RNA-Seq) a total of 768 differentially expressed genes were identified between healthy and infected dog brain tissues. Of these, 326 genes were not previously identified by microarray studies that evaluated gene expression associated with CDV infection. It is also worth mentioning that the gene expression differed between different lesion types (as defined histologically) of CDV infection, with certain genes differentially expressed only in each of the lesion types. The variation between lesion types was however smaller than the variation seen between the control versus infected dogs. By looking at both the host differences on a molecular level and studying the differential gene expression in two phases of canine distemper encephalitis, the host-specific differences and variable host affinity observed in CDV infections may be partially explained. This study contributes to improving our understanding of CDV, and the molecular mechanisms in different host species that underlie this disease and its variable manifestations.
Characterization of Phytophthora cinnamomi from avocado
(University of Pretoria, 2014-04) Van den Berg, Noelani; Crampton, Bridget Genevieve; elrea.appelgryn@fabi.up.ac.za; Appelgryn, Elrea
Species in the genus Phytophthora are widely recognized as some of the most devastating plant pathogens, and are responsible for major losses in crops such as potato, tomato, soybean, pepper and alfalfa, and fruit-and forestry trees. The Irish potato famine of 1845 caused by Phytophthora infestans (Mont.) de Bary is a historic example of the devastation this group of pathogens can cause (DE BARY 1876). More than 100 species of Phytophthora have been identified and described to date, and species of this genus are divided into ten clades (BLAIR et al. 2008; KROON et al. 2011; MARTIN et al. 2014). Research on this group of pathogens is focussed on the identification of host factors interacting with effectors, the impact of each effector on pathogenic fitness and their sub-cellular localization in order to identify proposed roles during infection (HUITEMA et al. 2011). This could lead to the application of knowledge on pathogenicity factors to develop novel control strategies.
Population genetic diversity in Spirocerca lupi
(University of Pretoria, 2014-06) De Waal, Pamela Jean; Greeff, Jacobus Maree; Reid, Kerry; janishtha@gmail.com; Mitha, Janishtha R.
Spirocerca lupi is a nematode that parasitises canid species across the world. Infested hosts show symptoms associated with the disease called spirocercosis. The parasite is known to cause significant damage to its final host and often leads to death. Treatment of the disease has been a challenge for veterinarians for many years since symptoms of the disease only become apparent at advanced stages of the disease, by which time, the available treatment is considered ineffective. Many studies to date have given insight into the characterisation and description of spirocercosis, however very few studies have been conducted on the molecular biology, biochemistry, genetics, epidemiology, ecology and host-parasite interactions. This study utilises molecular tools to perform genetic analyses to better understand the parasite’s population structure which will contribute to improved strategies for the treatment, prevention and control of spirocercosis. By integrating a population genetic approach with molecular marker data, it is possible to decipher the transmission dynamics of a parasite. For this purpose, microsatellite markers were developed using the FIASCO (fast isolation by AFLPs of sequences containing repeats) protocol and 454 pyrosequencing. Nine polymorphic microsatellite loci were developed to conduct population genetic analyses on S. lupi nematodes sampled across three geographical locations in South Africa, namely KwaZulu-Natal (Durban), Eastern Cape (Grahamstown) and Gauteng (Tshwane Metropole). Some of these loci proved to be effective in cross-species amplification testing. The loci were also used to provide molecular evidence that S. lupi is in fact found in jackal. Microsatellites proved to be effective markers in detecting subtle levels of population structuring between the three geographical locations, however allelic frequencies indicated that high amounts of gene flow was occurring. High levels of heterozygosity were found in individual hosts as well as between different hosts, suggesting that little or no inbreeding occurs between S. lupi nematodes within the final host. This could be due to the life cycle of the parasite. Spirocerca lupi uses a dung beetle intermediate host as well as several paratenic hosts, which allows for significant mixing of parasite genotypes before reaching the final host. In this way, the final host acquires a highly diverse genetic mixture of parasites, which influences mating patterns and results in outbreeding. This is the first study on S. lupi that uses co-dominant markers to study genetic variation and epidemiology across a wide geographical range. The implications of this study are that since S. lupi populations have high levels of genetic diversity, they have the genetic potential to adapt to changing environmental conditions as well as the ability to withstand possible treatments that are administered to final hosts. This is critical to consider when control mechanisms are implemented aimed to reduce the risk of infestation in canids. Additional research is required on the impact that different life cycle stages of the nematode have in the different hosts and its implication for effective prevention and control of spirocercosis.
Characterisation of the self-assembly and particle formation of major core protein VP7 of AHSV
(University of Pretoria, 2014-07) Van Staden, Vida; Huismans, H. (Henk), 1942-; gaylevwall@gmail.com; Wall, Gayle Victoria
The highly infectious vector-borne disease African horse sickness (AHS) is caused by African horse sickness virus (AHSV), a non-enveloped dsRNA virus of major economic importance in South Africa. The AHSV virion has a double-layered protein capsid comprised of a diffuse outer layer housing an inner icosahedral core. Viral protein (VP) 7 is the major core particle surface protein and forms trimers that can either be incorporated into the core or can self-assemble into flat hexagonal crystalline-like particles, a characteristic unique to AHSV VP7. Little is known about the self-assembly and particle formation of this highly hydrophobic and insoluble protein, and in particular what drives the stable layering of VP7 into these particles. In this study, the effect of various minor and major top domain modifications on the self-assembly and particle formation was investigated by comparing the previously constructed AHSV VP7 vector (VP7-144, VP7-177 and VP7-200) and VP7-eGFP fusion proteins (VP7-144-eGFP, VP7-177-eGFP and VP7-200-eGFP), known to differ with regard to solubility and trimerisation, to wild type (WT) AHSV VP7. The dual confocal and transmission electron microscopy approach showed that self-assembly was unaffected by all of the modifications. Particle formation was however affected, as the morphology of the protein structures formed by all six proteins differed from the hexagonal particles formed by WT VP7. Sucrose gradient sedimentation analysis was used to investigate the kinetics of the VP7-eGFP aggregation observed in previous studies. These results showed that VP7-144-eGFP is an inherently soluble and fluorescing protein, the spontaneous aggregation of which is L-arginine reversible. VP7-200-eGFP, however, is a largely misfolded protein with no evidence of protein aggregation. Immunofluorescence and confocal microscopy was used to investigate the localisation of misfolded, non-fluorescing VP7-eGFP fusion proteins in relation to their correctly folded, fluorescing versions. No such proteins were differentially detected using this approach thereby suggesting that both versions are located in the same area, either due to both versions being transported in a similar manner or due to correctly folded proteins becoming trapped in misfolded protein. Alternatively, the approach itself may be unsuccessful in detecting misfolded VP7-eGFP fusion proteins. Although the role of AHSV VP7 particles in the replication cycle and disease pathogenesis remains to be investigated, this study provides insights into the self-assembly and particle formation thereof. In particular, it is the top domain in combination with a group of associated factors that strongly influences the stable layering of VP7 to form the characteristic particles.
Functional genomics and systems genetics of cellulose biosynthesis in Eucalyptus
(University of Pretoria, 2013-09) Myburg, Alexander Andrew; Mansfield, Shawn D.; Berger, David Kenneth; zander.myburg@up.ac.za; Mizrachi, Eshchar
The globally emerging bioeconomy demands rapid advancement in the sustainable production and utilization of bio-based raw materials for a multitude of downstream applications, particularly in the areas of food, health and bioenergy and biomaterials. These needs, particularly pertaining to plant productivity, quality and stress tolerance, will need to be addressed with advanced biotechnology strategies, which accelerate progress beyond what has been achieved with traditional breeding and cultivation methods. Woody biomass is a readily available source of renewable carbon, and trees from the genus Eucalyptus, displaying superior growth and wood properties and established agricultural practices worldwide, are attractive candidates as short-rotation (5-9 years) feedstocks for biofuels and biomaterials. Guiding advanced strategies in biotechnology in Eucalyptus and other biomass feedstocks requires a sophisticated understanding of the molecular underpinnings of carbon allocation and cell wall biology. In the work presented here, we aimed to characterize the molecular biology of cellulose biosynthesis in Eucalyptus xylem (developing wood) and identify genes, processes and pathways that are linked to and possibly influence this process. We achieved this by detailed characterization of field-grown Eucalyptus hybrid trees, utilizing RNA-sequencing technology and metabolomics of xylem as well as measuring wood properties that are thought to impact the efficiency of industrial processing. Given the lack of information with regards to gene expression in Eucalyptus trees, a major aim was to characterize transcriptomes from various tissues and organs, including a cellulose-enriched form of xylem called tension wood. This involved challenging bioinformatics, which resulted in a high quality assembly and publication of a comprehensive gene catalogue for Eucalyptus, which was one of the first short-read RNA-sequencing based de novo assembly from a eukaryotic organism. We also characterized and modelled the properties of cellulose and xylan biosynthetic pathways as a biological system, the parts of which are segregating in Eucalyptus hybrid tree populations, which has generated novel insights into the allocation and partitioning of sequestered carbon between cellulose, xylan and lignin during active secondary cell wall deposition in woody stem tissues. This research has made important contributions to the field of Eucalyptus biology, but also to the broader field of secondary cell wall biosynthesis in plants, specifically providing (i) resources for transcriptome analysis in a large woody perennial (ii) new biological insight into carbon allocation for polysaccharide biosynthesis in wood, and (iii) annotation and discovery of candidate genes and pathways that may influence wood chemical composition and structures. Importantly, we find that cellulose and xylan biosynthetic genes are transcriptionally hardwired in their co-regulation (along with other important processes for cellulose and xylan transport and deposition), likely due to the fact that they utilize a common source of sucrose-derived carbon for cell wall biosynthesis and the production of sufficient energy to do so. This co-regulation appears to be distinct from the regulation of other cell wall biopolymers. Furthermore, evidence from xylem gene expression and metabolite availability in xylem, as research has made important contributions to the field of Eucalyptus biology, but also to the broader field of secondary cell wall biosynthesis in plants, specifically providing (i) resources for transcriptome analysis in a large woody perennial (ii) new biological insight into carbon allocation for polysaccharide biosynthesis in wood, and (iii) annotation and discovery of candidate genes and pathways that may influence wood chemical composition and structures. Importantly, we find that cellulose and xylan biosynthetic genes are transcriptionally hardwired in their co-regulation (along with other important processes for cellulose and xylan transport and deposition), likely due to the fact that they utilize a common source of sucrose-derived carbon for cell wall biosynthesis and the production of sufficient energy to do so. This co-regulation appears to be distinct from the regulation of other cell wall biopolymers. Furthermore, evidence from xylem gene expression and metabolite availability in xylem, as well as from wood properties of field-grown trees, supports a model in which sucrose-derived cytosolic fructose is shunted to the production of lignin precursors during cellulose and xylan biosynthesis. This model parsimoniously explains a mechanism for trees to partition carbon between polysaccharide and lignin synthesis, and provides exciting new questions and potential strategies to influence carbon allocation in the secondary cell walls of woody plants.
Allele frequencies and evolutionary genetics of acaricide resistance-associated SNPs in Rhipicephalus microplus ticks from South Africa
(University of Pretoria, 2013) Maritz-Olivier, Christine; Van der Merwe, N.A.; samreinecke2@gmail.com; Reinecke, Samantha
No abstract
Qualitative GUS and targeted yeast one-hybrid analyses of the Eucalyptus grandis SND2 promoter region
(University of Pretoria, 2013) Myburg, Alexander Andrew; Maritz-Olivier, Christine; Mizrachi, Eshchar; jonathan.botha1987@gmail.com; Botha, Jonathan
The secondary cell walls of woody plant species are a major source of cellulose. This biopolymer is among the most abundant on the planet and is very useful to industry, having many applications, such as in the paper industry and as a possible source of fermentable sugar for biofuel production. Therefore, much emphasis is placed on understanding the formation of woody biomass (mainly comprising of secondary cell walls) as a possible feedstock for cellulose extraction. Wood fibres are of particular interest as they are most useful for the paper industry and have thick, cellulose rich secondary cell walls. Transcriptional regulation of secondary cell wall biosynthesis is facilitated through a complex network of proteins and genes operating in a semi-hierarchical manner. In xylem, each cell type is controlled by a "master switch", a transcription factor (TF) which is able to activate the entire secondary cell wall biosynthetic program for that particular cell type. In Arabidopsis xylem fibres, SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is the master regulator for secondary cell wall deposition. Overexpression of SND1 results in the upregulation of a number TFs important for secondary cell wall biosynthesis. Three of the upregulated TFs were able to induce expression of a reporter gene under the control of a cellulose synthase (AtCesA8) promoter, indicating these TFs may be involved in cellulose biosynthesis. Of these, only SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN2 (SND2) is not a direct target of SND1. While much work has been done on the regulatory network in the model plant Arabidopsis, there are still major gaps in our understanding. Comparatively little work has been done to study this network in hardwood crop tree species such as Eucalyptus grandis. Properly characterising and understanding this network will allow us to manipulate hardwood crops to obtain trees with commercially desirable traits and will also provide a platform for future studies in the field of transcriptional regulation of wood formation.
Transcriptional responses of Eucalyptus clones to the gall wasp, Leptocybe invasa
(University of Pretoria, 2013) Naidoo, Sanushka; Myburg, Alexander Andrew; Slippers, Bernard; Caryn.Oates@fabi.up.ac.za; Oates, Caryn Nicole
Eucalyptus species constitute some of the most widely grown and economically important hardwood trees in global plantation forestry. This is due primarily to their remarkable growth and adaptability. Much of the initial success of exotic Eucalyptus plantations was attributed to the separation from natural enemies. However, there has been a recent increase in the number of introductions of Eucalyptus pests and pathogens in these exotic plantations. One such scenario is the spread of Leptocybe invasa that is currently described as one of the most devastating pests of global Eucalyptus plantations. Leptocybe invasa (Hymenoptera:Eulophidae) is an Australian gall-inducing wasp that oviposits along immature midribs, petioles and stems. The larvae are endophytic herbivores that cause the development of coalescing galls (abnormal plant growths) and lead to a wide range of symptoms such as stunted growth, die-back and death. In their native environment, populations of L. invasa are maintained to almost below observational level; however, once removed from this environment, the pest causes extensive damage in young, susceptible trees. Pesticides are ineffectual against the gall wasp and biological control is considered the key tool in controlling this pest. The molecular interaction between Eucalyptus and L. invasa is poorly understood and limits the design of biotechnological control measures aimed at reducing losses. Plants have evolved a complex, multi-layered system of constitutive and inducible defences that protect against pests and pathogens. Results from numerous studies have shown that there is extensive overlap in the response of plants to a wide variety of stresses. This means that it is possible to develop a hypothetical model of the response of Eucalyptus to L. invasa by incorporating results from studies investigating the response of other plant species to insect pests. This model can then be refined as evidence for the target system is obtained. Transcriptomic analyses are commonly used to investigate the plant response to biotic stress and allow for the identification of genes that may be manipulated to improve plant resistance through genetic engineering.
Hereditary paraganglioma in South Africa : An investigation of the Succinate-ubiquinone Oxidoreductase subunit genes, SDHB, SDHC and SDHD
(University of Pretoria, 2014) Van Rensburg, Elizabeth J.; nadja.louw@gmail.com; Louw, Nadja
Paraganglioma (PGL) are tumours occurring in the head-and-neck -, intra-abdominal - and thoracic paraganglia. Germ-line mutations in genes encoding the subunits of mitochondrial succinate dehydrogenase complex II (SDHB, SDHC, SDHD) and the SDHAF2 gene are involved in hereditary paraganglioma. Our aim was to identify mutations within these genes in ten South African PGL families. Individuals were screened for mutations in SDHAF2 using Sanger sequencing and Multiplex Ligationdependent Probe Amplification was utilised to investigate large rearrangements in these genes. A 7905bp SDHB exon 3 deletion [c.201-4429_287-933del], was identified in all SA families. The same deletion is reported as a founder mutation in Dutch PGL families. Genotype analysis revealed a common haplotype at the SDHB locus between SA and Dutch patients, indicating common ancestry. This is the first Afrikaner SDHB founder mutation. These results now enable predictive testing of other family members and allow better clinical management of the families.
Two-hybrid analysis and functional annotation of Bm86 and ATAQ from Rhipicephalus microplus
(University of Pretoria, 2013) Maritz-Olivier, Christine; ilkadim.kiper@gmail.com; Kiper, Ilkadim
Vaccination with recombinant Bm86 was found to protect against tick infestation, however, the efficacy of the vaccine varies against different tick species and geographical strains. An improvement of current vaccines could be achieved by identifying homologs of the current Bm86 antigen. A novel protein ATAQ was previously identified during a study that aimed to identify Bm86 homologs. It was shown that these two proteins do not share identical antigenic and immunogenic regions and that they also have different expression patterns. This indicates that ATAQ is not a Bm86 homolog, but rather an entirely different protein. Hence it is reasonable to assume that Bm86 antibodies would not cross-react with ATAQ antigens. Determination of the function of Bm86 and its structurally related protein ATAQ would provide invaluable insight into an unexplored biological system in ticks and potentially lead to the development of an improved vaccine formulation. Therefore, this study sets out to analyze protein-protein interactions by means of the yeast two-hybrid screening system using Bm86 and ATAQ from R. microplus as bait proteins. We were able to obtain that Bm86 and ATAQ have two common (aldehyde and retinol dehydrogenases), as well as a unique (Kunitz-like protein) interacting partners, respectively, via yeast two-hybrid screening. Further confirmation was achieved by immunoprecipitation, western blot and LC/MS analysis. The aldehyde and retinol dehydrogenases, as well as the Kunitz-like protein are involved in retinol metabolism. Therefore, as Bm86 and ATAQ share common binding partners, we hypothesized that they both are involved in the same metabolic pathway in the ticks. Future studies, would involve further confirmation of this pathway in R. microplus and evaluation of a mixture of both Bm86 and its interacting partners in animal vaccination trials.
Phylogeography of the snapper kob Otolithes ruber (Bloch & Schneider 1801) from the South West Indian Ocean
(University of Pretoria, 2014) Bloomer, Paulette; Fennessy, S.T.; Ilkser.Kiper@gmail.com; Kiper, Ilkser Erdem
Studying the distribution and demography of marine species is challenging due to the open nature of the oceans. This latter was historically believed to facilitate extensive gene flow among populations when clear barriers to gene flow were not apparent. Gene flow between populations could be mediated directly by adult migration across large distances for reproduction purposes, feeding and habitat preference or via larval dispersal with the aid of ocean currents, in order to recruit to optimal areas for development and to increase survival. Hence, gene flow among localized populations across large geographic distances most likely results in weak or no genetic differentiation. This recognized model has changed in recent years, as limited gene flow has been demonstrated for many species even in the absence of physical barriers to genetic mixing. To understand fish population dynamics and to manage marine resources sustainably, assessing the degree of population connectivity has become an important focus. It has been reported globally that many marine species are exposed to intensive fishing activities. One such species from the South West Indian Ocean (SWIO) region is the widespread fish species, Otolither ruber. Based on our knowledge, there are no genetic investigations conducted for this species to date. Hence, I studied the evolutionary and ecological processes influencing genetic diversity and the population genetic structure of O. ruber throughout the region based on mitochondrial and nuclear data analyses. Before attempting large scale data generation, critical consideration should be given to marker selection. I was able to find the most suitable mitochondrial gene from three evaluated genes. The cytochrome b gene gave consistently good amplification and showed the most variation within and among O. ruber sampling locations. The advantageous cross-species amplification approach yielded seven microsatellites, previously developed in other sciaenid fishes, with relatively high levels of polymorphism in O. ruber. Two distinct O. ruber populations in the north and the south of the SWIO region were identified based on statistical analyses of cytochrome b sequences and microsatellite genotypes. Low, unidirectional, female gene flow (based on mtDNA) and overall asymmetrical gene flow (based on nuclear DNA markers) were inferred from north to south between these evolutionary units. The isolation with migration model demonstrated a recent past population divergence and a low level of ongoing gene flow. The observed genetic differentiation in this shallow-water demersal sciaenid is mostly likely determined by the oceanography of the region, historical processes and life history traits, such as male-biased dispersal. The biology of the species in terms of reproductive and migratory behaviour should be further investigated to substantiate these findings. The results obtained from this study contribute to improved biological knowledge of O. ruber and its genetic status in the SWIO region, and will be used for future studies undertaking a management assessment of O. ruber. The observed variation among these populations should be maintained by management interventions. The study will also add to comparative phylogeograpic studies of co-distributed species, particularly other sciaenids.
Functional genomics of NAC transcription factor SND2 regulating secondary cell wall biosynthesis in Arabidopsis and Eucalyptus
(University of Pretoria, 2014) Myburg, Alexander Andrew; Berger, David Kenneth; Mizrachi, Eshchar; sghussey@gmail.com; Hussey, Steven Grant
Wood formation is heavily exploited for the manufacturing of pulp, paper, sustainable biomaterials and, potentially, biofuels. Eucalyptus is a favourable fast-growing, short rotation plantation crop grown over millions of hectares globally for its superior fiber properties. Understanding the molecular biology of secondary cell wall (SCW) formation in trees, and in particular how it is transcriptionally and epigenetically regulated, lays the foundation for enhanced woody trait improvement strategies in tree biotechnology. Transcriptional networks regulating SCW biosynthesis have been discovered in the herbaceous model plant Arabidopsis thaliana, in which NAC domain transcription factors (TFs) play a prominent role. The functions of many NAC domain TFs remain to be resolved, and their regulatory roles and evolution in Eucalyptus is unknown. Functional genomics studies of Eucalyptus TFs are currently challenged by a lack of established high-throughput genomics techniques commonly applied to model organisms. In this study, we aimed to better understand NAC family evolution and the epigenetic regulation of xylogenesis in E. grandis, and characterize the role of NAC domain TF SND2 in transcriptional regulation of SCW biosynthesis in A. thaliana and E. grandis. Comparative genomics and bioinformatics analyses of 189 curated gene models of the E. grandis NAC family, one of the largest described to date, revealed extensive tandem duplication in stress response-associated subfamilies, while SCW-associated subfamilies were generally conserved among angiosperms. Novel candidates for wood and tension wood formation as well as cold-stress tolerance were identified from transcriptional profiling in E. globulus and E. grandis. We identified the phenotypic effects and putative targets of the NAC domain TF SND2 in A. thaliana using microarray, microscopy and cell wall chemistry analyses. Moderate SND2 overexpression upregulated genes involved in cellulose, xylan, mannan, signaling and lignin polymerization processes and affected mannose, rhamnose and lignin components of stem cell walls, while strong overexpression resulted in reduced interfascicular fiber SCW deposition. SND2 overexpression in Eucalyptus somatic xylem sectors increased cross-sectional fiber cell area. We optimized a chromatin immunoprecipitation sequencing (ChIP-seq) approach and applied it to developing secondary xylem of mature E. grandis trees to identify the targets of the E. grandis ortholog of SND2, EgrNAC170. In validating the approach, we addressed the regulatory role of the epigenetic mark trimethylated lysine 4 of histone H3 (H3K4me3) in this tissue, showing a strong association with expressed loci, occupation of regions close to transcriptional start sites and tight correlation with transcript abundance, especially that of broadly expressed genes but also genes associated with SCW formation. A pilot study of EgrNAC170 targets was performed using the high-throughput ChIP-seq approach, identifying over 3,000 putative targets in E. grandis developing secondary xylem, but showing evidence that further ChIP-seq data are required for reliable target identification. The results of this thesis contribute to science an understanding of the unique evolution of NAC proteins in Eucalyptus, knowledge of the function of SND2/EgrNAC170 as possible candidates for tree biotechnology, the first genomic profile of a histone modification in developing wood and a high-throughput ChIP-seq protocol for the study of native protein-DNA interactions in developing xylem.
Taxonomy, phylogeny and species diversity in the Ophiostoma quercus complex
(University of Pretoria, 2009) Bloomer, Paulette; Wingfield, Michael J.; Wingfield, Brenda D.; joha.Grobbelaar@fabi.up.ac.za; Grobbelaar, Joha
Polymorphic sequence-characterised marker assays from a recent diversity study on the Ascomycete fungus Ophiostoma quercus from Africa showed that some isolates collected from hardwoods were genetically distinct from O. quercus. In the present study we compared these African isolates with authentic O. quercus isolates, including the neo-type of the species from oak in France, by evaluating morphological characters, growth in culture, mating compatibility, and DNA sequence data. The isolates from Africa exhibited substantial variability in culture and were morphologically similar to O. quercus. Phylogenetic analyses of the ITS, β-tubulin and Translation Elongation Factor 1-α gene regions confirmed that the African group represents a distinct species in the hardwood lineage of the O. piceae-complex, closely related to O. ulmi and O. himal-ulmi. Mating studies between O. quercus and the African isolates showed that isolates predominantly mated with those of their own group, although there were rare cases of fertile crosses between the groups. Isolates residing in the African lineage is described here as a new species, O. tsotsi sp. nov., and is characterized based on morphological characteristics, growth in culture and sequence comparisons.
Mating locus structure in Ceratocystis moniliformis and a gene duplication in Ceratocystis species
(University of Pretoria, 2014) Wingfield, Brenda D.; Wilken,P. Markus; Wingfield, Michael J.; trachyhall887@gmail.com; Godlonton, Tracy Alison
No abstract
Ecology and genetic diversity of Coryphodema tristis on Eucalyptus nitens in South Africa
(University of Pretoria, 2014) Slippers, Bernard; Wingfield, Michael J.; Hurley, Brett P.; Garnas, Jeff; Dawit.Degefu@fabi.up.ac.za; Degefu, Dawit Tesfaye
Forests are complex ecosystems that provide a variety of valuable products, such as timber, fuel wood, fiber and non-wood forest products and they also contribute to socio economic development. Pest problems have a considerable influence on the health of planted and natural woodlands of this industry. Members of the family Cossidae are often of serious concern to forestry and horticulture due to their aggressive, often gregarious wood-boring behaviour. Some species of this family (e.g Coryphodema tristis and Chilecomadia valdiviana) is known to be highly polyphagous and members occupy diverse ecological zones. Coryphodema tristis and Chilecomadia valdiviana are examples of cossid species that have recently expanded their host range to include non-native species. Coryphodema tristis was first reported infesting the non-native plantation species, Eucalyptus nitens, in South Africa in 2004, and C. valdiviana was first recorded infesting the same species in Chile in 1992. These insects have been reported to cause substantial damage to E. nitens. Both species are associated with a number of native trees in their respective countries, as well as non-native fruit trees and ornamentals. Despite what appears to be extreme polyphagy in C. tristis, many trees, including many Eucalyptus species and even hybrids of E. nitens and E. grandis planted in the province of Mpumalanga, are apparently free of any cossid moth infestation. These novel host range expansions raise interesting questions regarding the factors that influence new host associations. This review examines Cossidae as pests in native and non-native forest systems. Possible factors contributing to success of Cossidae in forestry and control strategies employed to manage the group are discussed.
In silico and functional characterization of the cellulose synthase 8 gene promoter of Eucalyptus trees
(University of Pretoria, 2013) Myburg, Alexander Andrew; Maritz-Olivier, Christine; nicky.creux@fabi.up.ac.za; Creux, Nicole Marie
Cellulose is a highly abundant biopolymer which forms the basis of several industrial applications including paper and textile products. Cellulose is deposited into the plant cell wall by a large membrane bound protein complex which is comprised of different cellulose synthase (CesA) subunits. Plants maintain several different CesA genes which have specific expression patterns depending on the cell wall type, tissue type, developmental stage and environment of the cell. While CesA genes and proteins have been the focus of many studies, the upstream regulatory regions which govern their complex expression patterns have remained largely unexplored. The aim of this study was to use the previously identified CesA promoter regulatory modules and putative cis-elements to identify conserved cis-element clusters in the Eucalyptus CesA and the transcription factors which interact with the regulatory regions of the EgrCesA8 promoter. The promoters of six cellulose synthase genes (CesA1, CesA3, CesA6, CesA4, CesA7 and CesA8) were isolated from 13 Eucalyptus species of different sections in the Symphyomyrtus subgenus of Eucalyptus. Species-level nucleotide diversity was calculated for the promoters of each gene. The promoters each contained a highly conserved region at the transcriptional start site (TSS), possibly marking the core promoter. The Eucalyptus promoters appeared to be TATA-less and cis-elements which resembled alternate core plant promoter elements were found clustered close to the TSS. Other localised regions of low species-level nucleotide diversity were identified upstream of the TSS in each promoter set and could indicate the location of cis-regulatory modules (CRMs). The conserved promoter regions and cis-element maps of the SCW-associated EgrCesA8 promoter were used to direct promoter truncation for reporter gene analysis in Arabidopsis, Eucalyptus and Populus. Comparative analysis of the cis-element maps and GUS expression data revealed that two main conserved regions of the CesA8 promoter harboured clusters of cis-elements and modulated GUS expression. The CT(11)-microsatellite in the conserved TSS-associated cis-element cluster produced strong non-specific GUS expression in Eucalyptus and Arabidopsis when appended to the 5’UTR which suggests a role in the EgrCesA8 core promoter. Further upstream in the promoter a second conserved promoter region coincided with a cluster of SCW-associated cis-elements and caused a loss of expression in leaf vasculature, suggesting a role for this CRM in modulating tissue-specific expression of EgrCesA8. The conserved EgrCesA8 promoter regions which coincided with cis-element clusters and GUS regulatory modules were used as baits in a yeast-1-hybrid screen against the a panel of 14 Eucalyptus SCW transcription factors. EgrMYB31 (AtMYB46) and EgrZincFinger-A were found to interact with the EgrCesA8 5’UTR. The interaction of these transcription factors with the 5’UTR were blocked by the presence of the CT(11)-microsatellite and could explain the loss of tissue-specific expression. EgrNAC170 (AtSND2) directly interacted with the CRM containing a cluster of SCW-related cis-elements. A dual Y1H assay revealed that EgrKNAT7 in the presence of EgrMYB80 (AtMYB52) or EgrMYB87 (AtMYB54) could also interact with the ErgCesA8 CRM. Together the results of the dissertation indicate that EgrCesA8 regulation is modulated by different protein-DNA and protein-protein interactions acting at highly conserved regions of the promoter.
Sex determination and symbiont transmission in the Sirex-Amylostereum mutualism
(University of Pretoria, 2014) Slippers, Bernard; Wingfield, Michael J.; Greeff, Jacobus Maree; Hurley, Brett P.; Garnas, Jeff; Amy.Wooding@fabi.up.ac.za; Wooding, Amy Lorraine
Interactions among living organisms fall along the mutualism-parasitism continuum; where mutualistic interactions benefit one or both organisms and parasitic interactions harm them. Mutualisms are a particularly interesting form of interaction as their evolutionary stability is constantly at risk of destabilisation by cheaters which take greater advantage of their partners than other symbionts in the population. This has resulted in organisms involved in mutualistic interactions have evolved many mechanisms to prevent destabilisation by cheaters. Insects are involved in mutualistic interactions with a myriad of organisms, in particular micro-organisms. Some of the most well documented insect–micro–organism interactions are those of the fungus–farming insects; Attine ants, termites and ambrosia beetles, and the obligate mutualistic fungi they cultivate. These mutualisms have remained stable over millions of years. Another, less well studied, apparently stable, obligate insect-fungus mutualism is the interaction between Sirex woodwasps and Amylostereum fungi. In this review we examine the evolution of mutualisms from initial interaction, through to maintenance of a stable obligate interaction, and explore the mechanisms that act to stabilise them. We explore the evolutionary and ecological factors necessary for the maintenance of the Sirex–Amylostereum mutualism in the context of work that has been done on the evolution of other more extensively studied insect-fungus mutualisms.
Establishment of a compatible pathosystem between Eucalyptus nitens and Phytophthora cinnamomi to determine host transcriptional responses
(University of Pretoria, 2013) Naidoo, Sanushka; Van den Berg, Noelani; Myburg, Alexander Andrew; Febéelizabethmeyer@gmail.com; Wilken, Febé Elizabeth
No abstract

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