Doctoral Degrees (Genetics)
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- ItemGenetic diversity of edible Lepidoptera in Southern Africa and implications for sustainable use of wild populations(Stellenbosch : Stellenbosch University, 2024-03) Nethavhani, Zwannda; Van Asch, Barbara; Veldtman, Ruan; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: The African Saturniidae encompass 400 species, some of which contribute significantly to the nutrition and income of many rural livelihoods across the continent. Due to their significance, species such Gonimbrasia belina are subjected to unregulated harvesting and hostplant destruction which threaten their populations. The taxonomic classification of Saturniidae is challenging, impeding accurate documentation of the biodiversity in this family. Additionally, genetic data that could guide conservation and management efforts of Saturniidae in the wild is largely unavailable. Our study aimed to address these knowledge gaps by pursuing four primary objectives. Firstly, we generated DNA barcode (COX1) data for 17 species to contribute to cataloguing the biodiversity of African Saturniidae. The genetic diversity of 170 specimens representing 17 species across 12 genera was assessed in the context of 1586 DNA barcodes publicly available for those genera. Results show that the current taxonomy of African Saturniidae for the 12 genera is affected by taxonomic inconsistencies and clerical errors resulting in 56% of cases of non-monophyly. Integrating alpha taxonomy and genetic data resolved 87% of these cases, thus demonstrating the importance of combining the two disciplines. Secondly, in the context of the mitochondrial phylogeny of the family, Africa was represented by only two species (Gonimbrasia belina and Gynanisa maja), a small number relative to the Asian counterparts. We generated complete mitochondrial genomes for 12 African Saturniidae from five tribes, including the first representatives of the tribes Eochroini and Micragonini. We performed comparative mitogenomics and phylogenetic reconstruction of the family, which strongly supported the monophyletic tribal structure in Attacini, Bunaeini, Micragonini and Saturniini, the sister relationship between Saturniini and Attacini, and the placement of Eochroa trimenii in the tribe Eochroini. Thirdly, despite the extensive harvesting of edible caterpillars of Saturniidae across southern Africa, genetic data for informing conservation and management of these species is largely lacking. We assessed the phylogeography, genetic diversity and historical demography of Gynanisa maja in northwestern Namibia based on sequence data for the standard DNA barcoding region (COX1) and two mitochondrial polymorphic (ND6 and Cytb). This baseline data showed that Gy. maja in northwestern Namibia is genetically diverse and panmictic, indicating healthy levels of genetic diversity and demographic status. Lastly, we evaluated the phylogeography, genetic diversity and demographics of Gonimbrasia belina using a multi-marker approach (mitochondrial sequences and nuclear RADseq data). Gonimbrasia belina exhibited high overall genetic diversity and showed large scale phylogeographical structure between Namibia and the South Africa/Botswana region. Some level phylogeographic structure was also apparent at the fine scale, with a large proportion of haplotypes exclusive to Botswana or South Africa, and shared haplotypes present at the border between the two countries. Furthermore, G. belina showed signs of strong population structure, low genetic diversity, small effective population size and recent bottlenecks across individual sampling sites in Botswana and South Africa, raising conservation concerns. Overall, our study offers important baseline contributions for species identification, genetic characterization, and conservation strategies of African Saturniidae, particular the edible species G. belina and Gy. maja.
- ItemThe physiological and genetic underpinnings that influence the xylem properties of Eucalyptus grandis, when subjected to different levels of water availability(Stellenbosch : Stellenbosch University, 2024-03) Keret, Rafael; Hills, Paul Norman; Drew, David Michael; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: Prospects for climate change forecast increases in temperature, with unpredictable rainfall. These future environmental scenarios are not promising for commercial forestry, especially since the main culprit of tree dieback is runaway cavitation, caused by drought. Consequently, foresters have opted to propagate more adaptable, fast-growing tree species, of which the genus Eucalyptus offers some promising candidates and, as such, has become an extensively propagated hardwood, carrying global significance. Given the wide native range of Eucalyptus, particularly E. grandis, these trees often display diverse physiological responses to abiotic stress that typically manifest in phenotypic plasticity in the xylem to prevent hydraulic failure. However, the broad range of physiological responses in Eucalyptus, coupled with the complexities associated with xylogenesis, have caused great difficulty in identifying the core mechanisms that govern the commercially valuable wood properties. To uncover how the environmental inputs are translated into anatomical outputs in the xylem of E. grandis, highly controlled drought experiments were conducted, with careful consideration of the physiology, anatomy, and transcriptome. As an early adaptive strategy, isohydric stomatal responses were elicited in the leaves of water-stressed plants to mitigate water loss through transpiration and maintain a stable water potential. Gaseous exchange was preserved at lower levels under drought, which likely provided the photosynthates necessary for the development of hydraulically-safer xylem. A novel method for procuring quantitative wood anatomy data from transverse stem microsections was developed in QuPath (v0.4.4) to assess the adaptive xylem anatomy stimulated under drought. Predominantly, smaller vessels and fibers were produced at a higher density, which enhanced the hydraulic- safety through improved implosion resistance and functional redundancy. Although these features reduced the theoretical hydraulic conductance, the associated decrease in water flow to the leaves may have reduced the transpiration rate to further mitigate water loss. Likewise, smaller ray parenchyma cells were elicited, which manifested in an increased number of rays and ray-vessel contacts. This arrangement increased the ray network reach within the xylem and may have facilitated a more efficient distribution of solutes for embolism prevention and repair. To assess the underlying mechanisms that govern cellular plasticity in E. grandis during water deficit, a comparative transcriptomics approach was conducted on the mRNA transcripts of the xylem. Genes associated with responses to reactive oxygen species and hypoxia were shown to exist at the core of the stress response, triggering a molecular cascade of events that ultimately led to the droughted xylem phenotype. For instance, cell cycle processes including DNA replication, cellular expansion, and division, were severely downregulated in water-stressed plants, which reduced vegetative growth. Similarly, genes associated with cell wall remodelling and component biosynthesis were expressed to a considerably lower degree compared to the controls. These gene expression patterns strongly suggest putative candidates for controlling cell size in E. grandis. Lastly, the expression of genes associated with the metabolism and transport of lipid-surfactants and osmotica suggest the capacity of E. grandis for embolism prevention or repair. This study has thus identified numerous regulatory mechanisms that influence the wood structure and physiology, to improve survivability under drought.
- ItemInvestigating the interactome of grapevine leafroll-associated virus 3 and Vitis vinifera(Stellenbosch : Stellenbosch University, 2023-12) Mostert, Ilani; Maree, Hano; Bester, Rachelle; Burger, Johan; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: Grapevine leafroll disease (GLD) is a globally important grapevine disease that affects the yield and fruit quality of affected vines. Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus, family Closteroviridae) has been identified as the main causal agent of GLD due to its consistent association with the symptoms of GLD. GLRaV-3 has not been successfully eliminated from mature vines, and no natural source of resistance to GLRaV-3 has been reported. Although the impact of GLRaV-3 infection on the transcriptome and metabolome of infected vines has been investigated, little is known about the exact mechanisms by which these effects occur. Furthermore, the roles of proteins encoded by GLRaV-3 open reading frames (ORFs) have largely been inferred by sequence homology or analogy to related viruses, and functional studies to determine their involvement in virion assembly and movement have not been performed. The aim of this study was to investigate the GLRaV-3 interactome to identify virus or host genes that play a key role in the proliferation and pathogenesis of GLRaV-3. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were employed to screen GLRaV-3 ORFs for pairwise interactions. The majority of interactions between structural proteins suggest that GLRaV-3 shares a common mechanism of assembly with members of the genus Closterovirus, family Closteroviridae, although some unexpected interactions were also found. Interaction of p20B, a silencing suppressor, with structural proteins has not been reported for other members of the family, indicating its possible involvement in other aspects of the viral replication cycle. The GLRaV-3 transmembrane protein self-interacted; however, the mechanism by which this interaction occurs remains unknown as it lacks a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. To identify virus-host interactions, a Vitis vinifera Y2H prey library was constructed and screened against GLRaV-3 ORFs encoding proteins involved in virion assembly, intracellular movement, and suppression of host silencing. BiFC was then used to demonstrate these interactions in planta. Two interactions identified using Y2H could not be demonstrated in planta and involved GLRaV-3 p20A, a protein of unknown function proposed to play a role in suppression of host defence responses and long-distance transport. In yeast, p20A was found to interact with a V. vinifera chlorophyll a-b binding protein and a V. vinifera SMAX1-LIKE 6 protein. Y2H and BiFC assays both demonstrated the interaction of p20A with V. vinifera mitogen-activated protein kinase and a V. vinifera small heat shock protein, as well as the interaction of GLRaV-3 minor coat protein with V. vinifera 3-deoxy-D-arabino- heptulosonate 7-phosphate synthase 02. All five of these host proteins are associated with host defence responses against pathogens. Furthermore, these interactions demonstrate that the symptoms of GLD may be caused by interference with a variety of pathways. This study contributes to our knowledge on the roles of GLRaV-3-encoded proteins in its replication and spread and provides information on cellular responses by grapevine against GLRaV-3. Understanding the proteins involved in the pathogenesis of GLRaV-3 can lead to the development of novel approaches to manage GLD.
- ItemCharacterisation of citrus tristeza virus-induced stem pitting in citrus(Stellenbosch : Stellenbosch University, 2023-12) Aldrich, Dirk Jacobus; Maree, Hans Jacob; Bester, Rachelle; Burger, Johan Theodorus; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: Citrus tristeza virus (CTV) is the most important viral pathogen of citrus and causes several disease syndromes in different citrus hosts. CTV-induced stem pitting leads to substantial economic losses in sensitive citrus varieties, including grapefruit. The exact mechanisms of stem pitting development in CTV-infected citrus remain unclear. This study aimed to utilise CTV infectious clone mutants in a reverse genetics approach to study stem pitting induction. A panel of recombinant CTV clones was generated focussing on the open reading frames (ORFs) implicated in stem pitting induction and pathogenicity, namely p33, p18, p13 and p23. ORF replacements from severe- and mild-pitting South African CTV isolates were introduced into the mild-pitting infectious clone (CTV-fl6 - genotype T36) to determine if severe stem pitting could be induced. Stem pitting assessments were complemented with the determination of virus concentration ratios in ‘Mexican’ lime and ‘Duncan’ grapefruit to relate stem pitting outcomes to virus concentration. The various infectious clone and wild type CTV infections yielded a broad range of stem pitting outcomes and specific ORF replacements associated with enhanced stem pitting were identified. Plant responses to different stem pitting pressures were further assessed by untargeted metabolite profiling and the quantitation of the stress-responsive phytohormones, abscisic acid, jasmonic acid and salicylic acid. In both citrus hosts, the metabolite profiling yielded fourteen statistically significant compounds that differed between stem pitting groups. These compounds were mainly phenolic acids and phenolic glycosides and are known to function as antioxidant and stress signalling molecules. Significant differences in phytohormone content were also found between test groups, particularly in plants that were severely stunted. Stem pits were also characterised at the molecular anatomical level using a combination of known and novel techniques to better understand the nature of the xylem and phloem tissues impacted by severe pitting. Established methods such as biological staining and fluorescence microscopy were used to visualise CTV-induced stem pitting and virus localisation. The utility of two novel technologies that have not previously been used to study CTV-induced stem pitting, namely high-resolution CT scanning and serial block face scanning electron microscopy was also evaluated. This study contributed to our understanding of CTV-induced stem pitting in citrus. Importantly, the use of CTV infectious clones for South African CTV research was established, which can allow for targeted follow-up experiments to further characterise the local citrus-CTV pathosystem. This can also provide novel opportunities for potentially employing CTV infectious clones as a crop protection tool against other important citrus diseases on the African continent.
- ItemAn investigation into the wheat (Triticum aestivum L.) host response to Russian wheat aphid (Diuraphis noxia Kurd.) feeding(Stellenbosch : Stellenbosch University, 2023-12) Fisher-Smith, Nadia; Botha-Oberholster, Anna-Maria; Van der Vyver, Christell; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjomov), is a major pest of wheat, causing damage and high yield losses worldwide. One of the undesirable effects of aphid feeding is leaf rolling, which serves as a shelter for the aphids protecting them from their natural predators and from insecticide spraying. Leaf rolling reduces the plant’s ability to photosynthesise and grow effectively, in addition, leaf rolling can also increase the aphid fitness, as it provides an ideal environment for growth. Therefore, identifying natural sources of resistance and introducing them into susceptible cultivars seems to be the most efficient strategy against RWA feeding. The use of genetic resistance is an efficient and environmentally safe method for controlling the RWA. This study aimed to ascertain if enhanced tolerance to biotic stress can be achieved by modifying plants either through genetic manipulation or chemical mutagenesis. Various studies have been done at a transcriptome level, allowing the identification of genes likely involved in RWA resistance. Utilising proteomics data in this study, allowed for the identification of differentially expressed peptides between resistant and susceptible wheat lines. Among the identified proteins were: glutathione-S-transferase (GST) and peroxidase. Literature suggests that GST forms part of the detoxification system in plants against biotic stress. This protein was uniquely expressed in the W1320-W1278 line that contains the Dn5 resistance gene. Peroxidase is associated with the oxidative burst, usually in response to stress, was identified in this study in the Gamtoos-S (Dn0) susceptible cultivar. Utilising genetic manipulation, a partial gene fragment of glutathione-S-transferase F6 (GSTF6b) was isolated from wheat and sequenced to confirm its identity. The gene fragment was cloned into a plant expression vector in the antisense orientation and bombarded into four- to six-day-old wheat immature embryos. Resulting in a putative transgenic plant, namely Gamtoos-S (Dn0)-pUBI-510:GSTF6b. Quantitative reverse- transcriptase-linked polymerase chain reaction (RT-qPCRs) were conducted to quantify the expression of the GSTF6b gene with/without RWA infestation. A reduction of nearly 50% was observed in GSTF6b expression in the respective transgenic plants when compared with the control. The T₁ was successfully hardened off, and allowed to seed and a T₂ generation was generated, which was functionally analysed through phenotypic screening, aphid fecundity, enzymatic responses and measuring oxidative burst. A decrease GST transcript level was observed post-infestation in the transgenic plants suggesting that plant susceptibility can probably be linked to a decrease in GST transcript promoting aphid growth and increasing the rate of reproduction. The last part of the study involved chemical mutagenesis, whereby drought-tolerant mutagenic M6 lines were screened for aphid resistance. A phenotyping assessment was performed on available mutant lines infested with South African (SA) biotype 1. A total of 33 mutant lines selected for drought tolerance, consisting of 21 ethyl methanesulfonate (EMS) and 12 Sodium azide (NaN₃) mutants, showed variation in aphid tolerance. Furthermore, drought-tolerant mutants were found to be more susceptible to aphid infestation, excluding the M12 (RYNOB8.012) line, shown to be intermediate to aphid feeding. The anti-oxidative enzyme GSTF6b expression was found to be significantly up-regulated in the mutagenic lines before infestation, therefore, contributing to the notion that GSTF6b is present at the basal level. A positive correlation was observed between GSTF6b gene expression and the intrinsic rate of increase (rm) in 25 mutagenic lines.