Masters Degrees (Genetics)

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Assessing the cyto-genotoxic impacts of un-neutralised and pH-neutralised acid mine drainage on the human breast cancer cell line, MCF-7
(Stellenbosch : Stellenbosch University, 2015-12) Botha, Shirmone; Botha-Oberholster, Anna-Maria; Genthe, B.; Oberholster, P.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: The use of toxicity tests to evaluate the quality of streams affected by mixtures such as acid mine drainage (AMD), adds value to assessments whereby site-specific toxicological data may identify toxicants that pose a threat to humans. To successfully evaluate the risk of combined mixtures, an improved understanding of the individual components, their uptake, metabolism, excretion and mode of action is required. This study aimed to identify the extent of AMD toxicity in a dose dependant manner on the MCF-7 cell line. The first study site associated with gold mining was chosen as the Tweelopies Stream situated in the Gauteng province of South Africa. The AMD effluent (un-neutralised) contaminating the Tweelopies Stream had undergone pH-neutralisation using a reactor-bed limestone technology incorporating the use of both calcium carbonate (CaCO3) powder and limestone beds. The second study site, the Kromdraai River, is situated in the eMalahleni region of South Africa where a predominance of coal mining exists. The pH -neutralisation of the AMD (un-neutralised) contaminated Kromdraai River was performed using a caustic soda (NaOH) precipitation technique. This study demonstrated the rapid and effective application of the comet assay as a screening tool for AMD-associated DNA breakages in the human cell line, MCF-7. Moreover, the study analysed parameters of cellular survival, DNA fragmentation and variations in morphologies indicative of cellular death. Collectively, the cyto-genetic aberrations observed in the MCF-7 cells as a result of exposure to gold and coal mining associated AMD, confirms the urgency of incorporating high-throughput screening in ecological toxicity assessment to evaluate cellular damage at genetic levels in low dose exposures where detection might be missed.
Characterization of the mitochondrial genomes of Diuraphis noxia biotypes
(Stellenbosch : Stellenbosch University, 2014-12) De Jager, Laura-Ellen; Botha-Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) commonly known as the Russian wheat aphid (RWA), is a small phloem-feeding pest of wheat (Triticum aestivum L). Virulent D. noxia biotypes that are able to feed on previously resistant wheat cultivars continue to develop and therefor the identification of factors contributing to virulence is vital. Since energy metabolism plays a key role in the survival of organisms, genes and processes involved in the production and regulation of energy may be key contributors to virulence: such as mitochondria and the NAD+/NADH that reflects the health and metabolic activities of a cell. The involvement of carotenoids in the generation of energy through a photosynthesis-like process may be an important factor, as well as its contribution to aphid immunity through mediation of oxidative stress. The complete mitochondrial genome of global Diuraphis noxia populations was characterised using Next Generation sequencing, and was found to be 15 721bp in size and consisting of 38 genes typically found within most insects. Single nucleotide polymorphism (SNP) analyses of the genomes of nine populations revealed 125 SNPs in the protein coding genes with the majority of the SNPs occurring in the ND genes, and the least in the ND4L gene. Low SNP variant frequency was found for the atp6 and atp8 genes, which differed from other reports in the Hemiptera. Variable ND5 expression levels were observed among the biotypes, although no correlation was apparent between ND5 expression and the virulence associated with each biotype. Whereas atp6 transcription was higher in the highly virulent biotype (SAM) under normal and stressful conditions in comparison to the least virulent biotype (SA1). A significantly higher NAD+/NADH ratio was also observed for the SAM biotype under stressful conditions in comparison to the lesser virulent biotypes. UPLC-MS analysis did not reveal any lycopene or β-carotene due to low compound concentrations in the extracted samples but various hydrophobic compounds were present in different concentrations among the biotypes. The carotene desaturase expression profile revealed that SA1 had the lowest relative expression of the gene involved in carotenoid products, while SAM had the highest, under normal and stressful conditions. The results indicate that sequence conservation in mitochondrial genes are associated with key energy processes to maintain a state of homeostasis under variable conditions and that the generation of energy is a contributing factor to the virulence development of D. noxia. The results also show that carotenoids may possibly contribute to fitness of D. noxia through reactive oxygen species scavenging or the production of additional energy, but further investigation is needed for confirmation.
Comparing different siRNA delivery systems to target Diuraphis noxia
(Stellenbosch : Stellenbosch University, 2018-12) Swiegers, Hendrik Willem; Botha-Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Diuraphis noxia, also known as the Russian wheat aphid, is a major pest of wheat. Breeding for resistance against D. noxia has been relatively successful in wheat as there has been many resistance genes incorporated into wheat in the past. However, this resistance has more often than not been counteracted by D. noxia through the development of a new biotype. The mechanism with which D. noxia is able to do this is not well understood. Previously, a highly virulent, laboratory generated biotype, known as SAM (South African Mutant), was compared to its avirulent progenitor, SA1, through proteome analysis of the salivary glands and complete genome sequence analysis. It was found that, among other differences, the cuticle protein, Dncprr1-8, containing a Rebers and Riddiford consensus was present in the salivary gland of SAM but not SA1. The gene also contained single nucleotide polymorphisms (SNPs) between the biotypes. In this study the function of Dncprr1-8 was investigated through RNA interference (RNAi). As RNAi has never been performed in D. noxia, several methods of siRNA delivery to this organism were compared. Injection of siRNA into the aphid haemolymph and ingestion of siRNA through artificial feeding medium was not successful. Allowing D. noxia to feed on wheat inoculated with a virus-induced gene silencing (VIGS) vector modified to contain D. noxia transcript sequence was partly effective, but overall had variable results. Finally, siRNA delivery through injection into wheat and allowing D. noxia to feed around the injection site, proved to be the most effective. Delivery of Dncprr1-8-siRNA using this method resulted in reduced survival and fecundity of biotype SAM while feeding on resistant wheat. The phenotypic responses were then compared to that of another aphid species, Myzus persicae, feeding on Arabidopsis thaliana injected siRNA targeting the same gene. M. persicae did not display reduced survival, but did produce fewer nymphs. Collectively, the results were then used to draw conclusions on the putative function of Dncprr1-8 in the plant-aphid interaction.
Comparing the methylomes of two genealogically linked Russian wheat aphid biotypes using whole genome bisulfite sequencing
(Stellenbosch : Stellenbosch University, 2020-03) du Preez, Pieter Herodus; Botha-Oberholster, Anna-Maria; Burger, Nicolaas Francois Visser; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae), commonly known as the Russian wheat aphid (RWA), is an economically important cereal pest. Although it does not spread any plant viruses, the severe symptoms caused by RWA feeding poses a significant threat to world wheat production. Commercial wheat cultivars, resistant to RWA, have been developed and are effective at preventing yield losses. However, new, more virulent RWA biotypes (morphologically similar aphid populations, with the ability to successfully feed on previously resistant cultivars) are continuously emerging, leading to a breakdown in resistance. The molecular mechanisms driving biotypification (the development of new biotypes) have not been identified yet. It has been proposed that an epigenetic modification, such as DNA methylation, might a possible means whereby biotypification might occur. The aim of this study was to explore the possible link between levels of DNA methylation and virulence in the RWA, by performing a whole genome bisulfite sequencing (WGBS) analyses on the South African biotypes SA1 and SAM. Together, SA1 and SAM form a good model for the study of virulence as they are closely related, yet at opposite ends of the virulence scale. The overall trends in RWA DNA methylation, observed in this study, correlates with what has previously been reported in insects: genic bodies, especially exons, are the most methylated regions in the genome, with most of the methylation occurring at CpG sites. The ratio of observed to expected CpG sites in a region has been used to infer levels of methylation, as increased methylation has been correlated to a decrease in CpG abundancy; In this study, however, no correlation was found between CpG abundancy and DNA methylation level. This technique, therefore, is not applicable for insect genes. Using the generated WGBS data, 148 genes were found to be differentially methylated between the two biotypes. The relative expression of five of these genes, which were selected based on gene ontology and the degree of differential methylation, along with that of DNA methyltransferase 3 (DNMT3) and ten-eleven translocation enzyme (TET), was quantified and compared between biotypes at 0, 6, and 48-hours after performing host-shifts from susceptible to resistant host plants. DNMT3 is the enzyme responsible for the establishment of DNA methylation, while the TET enzyme catalyses the first step of the process of demethylation. The time points were selected to correlate with defence responses elicited from the host plants. While no clear pattern could be observed in the differences in relative expression of differentially methylated genes between biotypes, or within a biotype between time points, a major increase in expression of both DNMT3 and TET 6-hours after performing a host shift to the resistant cultivar Tugela Dn5 was observed in SAM. This seems to indicate an increased rate of methylation, demethylation, or both methylation and demethylation in SAM, while under stress, which might contribute to its increased ability to respond to, and overcome, plant defence responses.
A comprehensive comparison of the methylomes between three Russian wheat aphid biotypes differing in virulence and associated bacteria
(Stellenbosch : Stellenbosch University, 2021-12) De Jager, Nicholas Stratos; Botha-Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae), commonly known as Russian wheat aphid (RWA), is a phloem-feeding pest capable of causing severe damage to wheat crop yields. In the United States and South Africa, rapid biotypification of this aphid has been documented, allowing it to overcome previously resistant commercial cultivars. It is speculated that differences in DNA methylation patterning allow genetically similar RWAs to become more virulent. Additionally, aphid-associated bacteria have been shown to facilitate aphid virulence in wheat (Triticum aestivum L). To establish whether these bacteria affect DNA methylation patterning in the US RWA biotypes [US1 (co-fed); US2 (co-fed); US2 (isolated)], and by extent the virulence of these biotypes, the methylation profiles and global DNA methylation and hydroxymethylation (5mC + 5hmC) levels were investigated using the methylation sensitive amplified polymorphism (MSAP) and enzyme- linked immunosorbent assay (ELISA) methods, respectively. Furthermore, whole genome bisulfite sequencing (WGBS) of these biotypes was performed and using a modified Bismark pipeline, the context of DNA methylation (CpG, CHH, CGH) was established, while differentially methylated genes (DMGs) were analysed using the Bioconductor DSS (Dispersion Shrinkage for Sequencing data) package. The results from the MSAP and ELISA methods suggested that there were differences in the DNA methylation profiles of the biotypes, suggesting indicating that aphid-associated bacteria might influence methylation in the RWA. The WGBS data revealed that generally, throughout each context and region, the less virulent biotypes [US1 (co-fed); US2 (isolated)] were more methylated (and less hemimethylated) than the more virulent biotype [US2 (co-fed)], corroborating previous findings on RWAs. More DMGs were found between the isolated and co-fed US2 populations than US1 (co-fed) and US2 (co-fed), indicating that aphid-associated bacteria had a major effect on gene methylation. Between both comparisons, several DMGs were involved in lipid metabolism and energy regulation, and some were identified to be involved in reactive oxygen species (ROS) detoxification, defence signalling during microbial invasion and sterol conversion, hinting at interactions between the aphid and its associated bacteria. Lastly, five potential effectors were predicted using a custom effector prediction pipeline.
Engineering wheat (Triticum aestivum L.) for abiotic resilience by manipulating small ubiquitin-like modifiers
(Stellenbosch : Stellenbosch University, 2016-03) Le Roux, Marlon Luke; Botha-Oberholster, Anna-Maria; Van der Vyver, Christell; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Research is becoming increasingly more focused on innovative strategies to improve wheat (Triticum aestivum L.) to meet current and future consumer demands. With the number of countries facing extreme climate variability increasing, drastic steps need to be taken to ensure local food security. Although genetic engineering of staple crops has been explored widely, they require challenging platforms with highly efficient explants with enhanced regenerative abilities for callus formation and somatic embryogenesis to consistently yield plantlets with altered attributes. Thus, this study describes the necessary steps to obtain transgenic wheat with relative ease, with the goal of improved abiotic stress tolerance. The developed in vitro regeneration and cryopreservation system was then used to introduce genes into bread wheat, namely Overly Tolerant to Salt 1 (OTS1) and OTS2 (both are Small Ubiquitin-like Modifier (SUMO) protease genes) and transcription factor known as Inducer of CBF expression 1 (ICE1). The class of molecules have emerged as an influential mechanism for targeted protein management, as SUMO proteases play a vital role in regulating pathway flux and are therefore ideal targets for manipulating stress-responsive SUMOylation. This study thus describes the isolation and cloning of three genes and its manipulation into constitutive and drought inducible vectors. The latter makes use of a unique promoter which was characterized in silico, after which it was then applied. Finally this study also demonstrated the compatibility of the vectors within wheat as it was confirmed that genetic modification of wheat was achieved by particle bombardment.
Gene silencing in bread wheat (Triticum aestivum L.) following a biolistics approach
(Stellenbosch : Stellenbosch University, 2014-04) Fisher, Nadia Mitilda; Botha-Oberholster, Anna-Maria; Van der Vyver, Christell; Van Eck, L.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Global food security is hampered by a variety of insects/pest and plant diseases. In wheat, the Russian wheat aphid (RWA) is a significant pest problem in many areas of the world. Wheat has developed defensive mechanisms against the RWA over time which are activated upon feeding. One such mechanism is the hypersensitive response (HR) which is effective against phloem-feeding insects i.e. D. noxia (Diuraphis noxia, Kurdjumov, RWA). In this study, two genes associated with the hypersensitive response i.e. ascorbate peroxidase (APX) and glutathione S transferase (GSTF6b) were investigated to elucidate their function in the defensive mechanism of wheat using a reverse genetic approach i.e. particle bombardment. This study has succeeded in the established of a tissue culture and transformation system which generated three genetically modified wheat plants with decreased resistance to RWA feeding due to gene silencing. The establishment of this system enabled to test the association of defensive related genes in wheat to RWA resistance. Expression analysis performed on obtained transgenics before and after RWA infestation reavealed that the silenced plants were more susceptible to RWA feeding. Chlorosis was observed in the Gamtoos-S-APX transgenic plant which is an indicator of oxidative damage to the photosynthetic machinery of the plant. Decreased GSTF6b transcripts was found in the transgenic Gamtoos-S-GSTF6b and transgenic Gamtoos-R-GSTF6b transgenic plants but no visible symptoms of infestation was observed in these two plants. Resistance breeding could be strengthened by developing broad spectrum resistance plants by incorporating wheat defensive related genes with known function into the breeding programs. The use of this transformation system will allow rapid identification and introduction of agronomically important genes by upregulating these genes to enhance bread wheat against aphid infestation.
Inducing mutations in bread wheat (Triticum aestivum L.) using chemical treatments
(Stellenbosch : Stellenbosch University, 2014-12) Mbwanji, Kenneth; Botha-Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Bread wheat (Triticum aestivum L.) production is hindered by a variety of abiotic stresses with drought being the most devastating. Mutation breeding through induced mutagenesis is one way for wheat breeders to adapt to the challenges posed by climate change. Especially, chemically induced mutagenesis showed promise in improving drought tolerance using different recommended mutagens at optimum concentrations and treatment durations. The aim of the study was to improve drought tolerance in wheat by means of chemical induced mutagenesis. In the study, the mutagenic properties of four chemicals, namely Sodium azide (SA, 0.1 mM, 1.0 mM and 10 mM), Ethyl methanesulfonate (EMS, 0.1%, 0.5%, 1.0% v/v), Maleic hydrazide (MH, 0.5 mM, 1.0 mM, 2.0 mM), and N-methyl-N-nitrosourea (MNU, 0.5 mM, 1.0 mM, 2.0 mM) at different concentrations and treatment durations (2h, 4h and 8h) were compared. To select for mutants that express drought tolerance, the M1 plants were exposed to water stress. NMU and MH treated M1 plants demonstrated zero survival rates, while a few of the plants treated with SA and EMS survived. In the study, treatments with 0.5% (EMS) 4h, 1% (EMS) 2h, 1 mM (SA) 2h and 1 mM (SA) 8h were considered optimum, since these treatments resulted in fertile plants. However, the chemically derived mutant wheat lines displayed a lower germination rate, delayed maturity, stunted growth and lower than average seed mass when compared to the control. The latter traits were also verified in the M2 and M3 generations. The M2 and M3 generations also displayed a shorter growth form and delayed maturity phenotype, but had higher germination rates and produced more seeds. Screening for drought tolerance conducted on the M3 plants confirmed the tolerant phenotype found in the M1 generation plants. Amplified fragment length polymorphism (AFLP) profiling was also conducted on the mutants using three primer combinations (MTT/ECG, MTG/ECT and MTG/EGC) in order to assess the extent and significance of the induced mutations. From the obtained data, it was revealed that SA 1 mM (2h) 16 had the highest number of induced total character differences (109) relative to the control of all the SA and EMS derived mutants, suggesting that a treatment with 1 mM SA for 2h induced more mutations than any other SA or any of the EMS treatments. Of the sequenced clones, only one revealed similarity to a T. aestivum isolate AAC/CTG7 scab resistance-linked AFLP fragment gene sequence, an important disease of wheat, but due to time constrains this finding was not investigated further.
RNAi of selected insect genes
(Stellenbosch : Stellenbosch University, 2016-12) Visser, Ilze; Botha-Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae), commonly known as the Russian wheat aphid (RWA), is regarded as one of the most destructive and widely distributed insect species in the world. Nonetheless, the currently available control strategies, including chemical pesticides, biological control agents, and RWA resistant wheat cultivars, are still very limited and rather ineffective. The process of double-stranded RNA (dsRNA)-mediated interference (RNAi) displays high specificity and the prospect of developing into a new specific method for managing agricultural pests. Plants can potentially be genetically engineered to express dsRNA to down-regulate vital gene functions present in pest insects, resulting in the protection of plants. In order to survive and reproduce, aphids require close interaction with their host plants, during which effectors are transported inside the plant to modify host cell processes. Four previously identified RWA salivary secretion proteins were investigated in the present study. However, cloning and sequencing results indicated that only two of the aforementioned proteins – C002 and 14-3-3 epsilon (ɛ) – could be potential protein elicitors in RWA. Thus, these two transcripts were subjected to RNAi experiments via artificial diet feeding and feeding on siRNA injected wheat leaf trials in order to investigate their role in RWA-host interactions and their importance in the survival and reproduction of the RWA. The relative expression levels of C002 and 14-3-3 ɛ at 0h were compared between SAM, the most virulent RWA biotype, and SA1, the least virulent RWA biotype in South Africa, and the results indicated that both transcripts had a higher relative expression in SAM than in SA1. Therefore, suggesting that C002 and 14-3-3 ɛ might play an important part in RWA virulence. From the RT-qPCR results it was evident that successful silencing of both C002 and 14-3-3 ɛ were achieved at 24h after initial siRNA exposure and that the transient silencing effect subsided thereafter. The expression data pertaining to the wheat leaf injection experiments, however, displayed high standard deviations that are not ideal and suggested that the expression of the transcripts differs greatly between the aphids within each group. This is likely due to the custom-made aphid cages and injection procedure of the siRNA into wheat leaves that appears to hinder the accuracy of the results. The fecundity data produced quite inconclusive results due to previously mentioned inadequacies and therefore an accurate and decisive conclusion cannot be drawn as to how the C002 and 14-3-3 ɛ silencing effects the survival and reproduction of the RWA. Both methods used for RNAi – the artificial diet trial and the injection of wheat leaves trial – have their drawback. After considering the RT-qPCR data, it appears as though the artificial diet trial produced more accurate and feasible results. Even so, the injection method establishes a more natural mode of feeding for the aphids and consequently more optimal cages need to be designed and tested to produce precise results.
A targeted investigation of Diuraphis noxia (Hemiptera: Aphididae) methylation
(Stellenbosch : Stellenbosch University, 2017-12) Breeds, Kelly; Botha-Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae – or Russian wheat aphid, RWA) is an economically important phloem-feeding pest of wheat and barley. The most effective method for controlling RWA infestation of wheat is the deployment of resistant cultivars. However, new biotypes – aphid populations expressing virulence towards these cultivars – continue to develop. Consequently, a dire need exists to understand the molecular mechanism underlying increases in aphid virulence. The epigenetic modification of methylation has been proposed as one such mechanism, yet its effect on virulence remains largely unexplored. The aim of the study was thus to determine if methylation plays a role in biotypification and the associated increase in aphid virulence. To this end, two methods, namely methylation-sensitive amplification polymorphism (MSAP or MS-AFLP) and restriction site-specific fluorescent labelling (RSSFL), were tested for their ability to detect and quantify RWA methylation. The former was successful on both counts, specifically in the CG and CC dinucleotide contexts. Use of this methodology also revealed 22 polymorphic loci between the least and most virulent South African biotypes, SA1 and SAM, with 18 resulting from an increase in methylation during SAM‟s biotypification from SA1. Restriction site-specific fluorescent labelling is a novel technique that makes use of a fluorescently labelled adaptor, which binds to the sticky ends produced after the restriction of DNA using the isoschizomers HpaII and MspI. Although unable to detect or quantify methylation, RSSFL was able to detect trends in methylation. Various aspects of the DNA methyltransferases (DNMTs), which catalyse methylation, were also investigated. A homology search identified four putative RWA DNMT genes, namely DNMT1, DNMT2, DNMT3A and DNMT3B. Sequencing of these genes detected only one single nucleotide polymorphism between biotypes SA1 and SAM. Baseline DNMT expression, quantified using RT-qPCR, revealed significant differences in DNMT3A expression, which could be explained by the virulence of the respective biotypes. An antibody specific to 5-methylcytosine (5mC) was used to quantify both the DNMT protein activity (by detecting the relative number of methyl groups transferred by the DNMTs to a universal substrate) and the global 5mC levels, both of which did not differ significantly between the biotypes. The 5mC levels ranged from 0.1% to 0.16% and were in line with levels reported for numerous insects. Global hydroxymethylation levels were quantified using an antibody specific to 5-hydroxymethylcytosine (5hmC, a demethylation intermediate). Biotype SAM‟s 5hmC level was significantly higher than that of biotypes SA1, SA2 and SA3. Based on the results obtained, it is recommended that future studies of RWA methylation first perform RSSFL, followed by either MSAP or antibody-mediated methylation quantification (or both), depending on the needs of the specific study. The results also made clear the fact that methylation, and the removal thereof is related to differences in RWA virulence. Although many aspects of methylation were similar between the biotypes, local increases in methylation proved beneficial to the development of the highly virulent biotype SAM. During biotypification SAM also attained an increased ability to demethylate its genome, which affords this biotype greater flexibility to adapt to changing environments, by means of alterations in gene regulation. An increased demethylation capacity might therefore be a key contributory factor to increases in aphid virulence and hence biotypification.

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Browsing Masters Degrees (Genetics) by browse.metadata.advisor "Botha-Oberholster, Anna-Maria"

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