Doctoral Degrees (Genetics)

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    Characterisation of EMS mutagenic bread wheat (Triticum aestivum l.) lines to investigate their water deficit stress tolerance and adaptability
    (Stellenbosch : Stellenbosch University, 2023-03) Kayaga, Helen Ninsiima; Botha-Oberholster, Anna-Maria; Van der Vyver, Christell; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Wheat is a staple food for 2.5 billion people worldwide and is the second most important cereal grain grown in South Africa. Water deficit stress has adverse effects on wheat productivity in the world. This is exacerbated under current unpredictable climatic patterns due to global warming. In 2017, most of the wheat production areas in South Africa were lost to alternate crops due to prevailing drought conditions. This created a need to develop water deficit stress tolerant wheat lines using ethyl methanesulfonate (EMS) because currently, no officially released varieties in the country carry such a trait. This study aimed to characterise newly developed mutagenic water stress-resilient bread wheat lines. The agro-morphological traits of three wild types and ten M3 lines were determined based on wheat descriptors in a randomised experiment with three replicates in a greenhouse. The chemical properties of flour were measured using the Inframatic 9500 NIR Grain Analyser machine, and the sedimentation tests of flour were quantified using the AACC International Method 56-60.02. The M3 lines were screened for tolerance to water deficit stress in a split-plot (3x5 factorial) experimental design that induced stress at the following growth stages: stress at seedling growth (emergence), tillering (forty days after planting), anthesis (flowering), milk development stage (seed set), and control (well-watered), and 13 subplots (bread wheat lines). Results from the analysis indicated a variation in the agro- morphological traits (18 traits) of mutants from the wild types (WTs) aside from ten traits that were similar across all bread wheat lines. Baking quality analysis showed that mutant Big 8.3 had the most desirable moisture content percentage of flour, a high protein, and wet gluten. Hence its dough has good elasticity and extensibility. The mutants performed better than WTs under water deficit stress. Big 395.1 was the most tolerant at emergence and forty days after planting, while Big 8.1 and Big 8.3 were most tolerant to water deficit stress at flowering and seed set, respectively. Previously identified drought-related genes, Sal1 (an inositol polyphosphate 1- phosphatase encoding gene) and Era1 (enhanced response to Abscisic acid 1), were analysed for single nucleotide polymorphism (SNPs) variants induced by chemical mutagenesis through sequencing complementary DNA (cDNA) of the genes and aligning them to the reference wheat genome of “Chinese Spring” available on the National Centre for Biotechnology Information (NCBI) web-based platform. I could not attain results from the Era1 sequences, and alignment results showed no differences between the Sal1 sequences of the mutant lines to that of reference genome “Chinese Spring”. In conclusion, chemical mutagenesis increased the genetic diversity of the parental bread wheat lines through induction of point mutations resulting in desirable new mutant lines like Big 8.3 with a desirable agrotype, baking quality characteristics, and high tolerance to water deficit stress at seed set.
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    Analysis of interactions between glucan, water dikinase and either isoamylase or starch branching enzymes in detrmination of starch structure
    (Stellenbosch : Stellenbosch University, 2022-12) Adegbaju, Muyiwa Seyi; Lloyd, James Richard; Van der Vyver, Christell; Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: Starch is the main form in which carbon is stored in plants and it is used in a wide array of applications, from its role as a source of calories in human diets, to livestock feed and as an industrial feedstock. It is composed of two types of glucan polymer, amylose which is com- posed mainly of α-1,4 linked chains and highly branched amylopectin which contains α1,4 chains which are connected together with α1,6 branchpoints. Synthesis of the starch polymer involves co-ordinated activities of starch synthases, starch branching enzymes (SBE) and de- branching enzymes (DBE). In addition, the enzyme glucan, water dikinase (GWD) can intro- duce phosphate into amylopectin. The critical roles these enzymes play during starch metabo- lism have been investigated by knockout or silencing of genes encoding them in some plants, mainly Arabidopsis thaliana. Interactions between some of these enzymes have also been demonstrated, but there are still many questions about how these interactions occur and how they influence starch metabolism. In this study two sets of experiments were performed. The first involved analyses of potato (Solanum tuberosum L.) plants where the expression of SBEI, SBEII and/or Glucan, Water Dikinase 1 (GWD1) were repressed using RNAi technology. Individual or joint repression of the two SBE isoforms resulted in a significant increase in starch phosphate, whereas repression of GWD1 led to synthesis of low phosphate starch. Starch phosphate decreased in SBEI/GWD1 and SBEII/GWD1 lines but not to the levels found in lines where only GWD1 was repressed. The apparent amylose content increased in starch from SBEI/GWD1 lines was greater than either the SBEI or GWD1 lines. These alterations in starch composition influenced its granule morphology, swelling power and freeze-thaw stability. Silencing of GWD1 reduced starch deg- radation in cold-stored tubers, but this was not the case in lines repressed in either starch branching enzyme. The second set of experiments examined polyglucan metabolism in potato lines repressed in isoamylase 2 (ISA2) and/or GWD1. Transgenic potato lines were produced by RNAi gene silencing and tuber starch metabolism analysed. The water-soluble glucan content in ISA2 and ISA2/GWD1 lines tuber was higher than GWD1 lines. Analysis of starch structure indicated that there was an increase in the apparent amylose content of starches from GWD1 lines whereas it was unchanged ISA2/GWD1 lines. There was a reduction in the starch phosphate in both GWD1 and ISA2/GWD1 lines, and this reduction was greatest in the GWD1 lines. The ISA2 lines contained a proliferation of small starch granules, but this was not observed in ISA2/GWD1 lines which indicates that ISA2 and GWD1 interact during granule initiation. The effect of both enzymes on cold-induced sweetening (CIS) was also investigated. As expected, CIS was reduced in lines where GWD1 was silenced. Interestingly CIS was also inhibited in the ISA2 lines indicating that the pathway of starch degradation may differ between cold-stored potato tubers and Arabidopsis leaves.
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    Defining the QTL for chill requirement during dormancy and dormancy release in apple (Malus x domestica Borkh.).
    (Stellenbosch : Stellenbosch University, 2021-12) Cornelissen, Stephanie; Burger, Johan T.; Rees, Jasper; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Dormancy is a physiological stage that all deciduous fruit trees experience during winter. It consists of three sub stages, para-, endo- and ecodormancy. During the endodormic stage, apple trees need to be exposed to cold temperatures for their chill requirement to be fulfilled. Most apple varieties have a unique chill requirement, for instance ‘Anna’ requires less than 300 hours. After this requirement is fulfilled, the plant will break dormancy if the environmental conditions are optimal, i.e. the less the chill requirement the earlier the plant will bud. This study looks at the genetic component of chill requirement by firstly determining the region of the apple genome that is primarily associated with budbreak and thereafter the genes within this region and their expression patterns. A mapping population was created by crossing ‘Lady Williams’ with ‘Anna’. ‘Lady Williams’ is a medium to high chill variety and ‘Anna’ is a low chill variety that requires less than 300 chill units. This population was used to generate a genetic map and subsequent QTL analysis, as well as association mapping, were used to determine the region of the apple genome that is associated with budbreak. A major QTL in the first 10Mb distal region of Linkage Group/chromosome 9 was discovered. This region consists of 741 genes, of which 27 were differentially expressed over time when exposed to cold. An RNA time series analysis was performed by periodically extracting RNA from the meristems of cuttings from ‘Anna’, incubated at 4°C for up to 800 hours. The expression patterns of the 27 genes grouped into seven distinct clusters. The major observation that could be made is that there is a difference between the expression patterns before and after the theoretical time of budbreak (300 hours) in ‘Anna’. The 27 genes were differentially expressed before budbreak but there was little to no differential expression after budbreak. Another observation that was made, is that the biggest differences in gene expression were between 0-hours and a 100-hours. This indicated that the plant could have experienced cold shock by transferring it to low temperature incubation and subsequently the onset of endodormancy. Known dormancy-associated DAM genes, were not differentially expressed over time. However, an ortholog of the predicted transcription factor ICE1-like, bHLH, and the gene RING domain ligase 2, that have been shown to be associated with the regulation of ICE1-like, was differentially expressed over time when first introduced to cold. Furthermore, genes regulating the expression of the plant hormones ethylene and auxin were differentially expressed. Other notable genes include ones that encode Tubulin alpha-2 chain and xyloglucan endotransglucosylase/hydrolase 9 proteins, both known to regulate transport through cell wall/membrane, and 5'-adenylyl phosphosulfate reductase 2 that is associated with oxidation-reduction homeostasis. This study serves as a starting point for further investigations of the complex nature of the onset of dormancy, chill accumulation during dormancy and the eventual budbreak, by identifying genes that are associated with dormancy.
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    Analysis of aspects of starch metabolism in Physcomitrella patens
    (Stellenbosch : Stellenbosch University, 2021-12) Mdodana, Ntombizanele Thobela; Lloyd, James Richard; Kossmann, Jens; Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: Starch is an important polysaccharide produced by plants and is widely used in industry mainly as a food thickener, but also in other important processes, such as the development of textiles and p aper manufacture. This polyglucan consists of two glucose polymers, amylose and amylopectin. As the major storage carbohydrate, starch is synthesised during the day before being catabolised at night to sustain plant growth and metabolism. Starch metabolism is well studied in vascular plants such as Arabidopsis thaliana and Solanum tuberosum, however, information about these processes are less well understood in non-vascular plants. The bryophyte Physcomitrella patens is an excellent plant model system for many reasons; for example its fully sequenced genome and the ability to produce knockout mutants using homologous recombination. Attempts to gain a better understanding of the function and regulation of some of the key metabolic enzymes involved in starch metabolism in non-vascular plants have recently emerged (Stander, 2015; Jacobs, 2018; Mdodana et., al 2019). This project focused on two aspects of starch metabolism to determine whether some of the pathways and mechanisms involved during these processes are conserved between Physcomitrella patens and vascular plants. The first part of this dissertation examines the roles of glucan water dikinase enzymes (GWD) in P. patens. In angiosperms these polypeptides are involved in starch degradation through catalysing starch phosphorylation. Five isoforms, PpGWDa-e, were identified in and phylogenetic analysis demonstrated the two (PpGWDa and PpGWDb) were most similar to Arabidopsis GWD1 or GWD2, while another two (PpGWDd and PpGWDe) were most similar to GWD3/PWD. The final isoform (PpGWDc) was likely to be inactive as it lacks the essential catalytic histidine. Both PpGWDa and PpGWDb targeted to chloroplasts. Using homologous recombination, knockout mutant lines were successfully generated for PpGWDa and PpGWDb each isoform both as individually and together in double mutants. Inserts in either gene resulted in reduced amounts of starch phosphate compared to the control, with Ppgwda mutant lines interestingly containing less glucose 6-phosphate in starch than Ppgwdb lines. Double mutant (Ppgwda/Ppgwdb; DM) lines contained even less glucose 6-phosphate in starch than Ppgwda single mutants. When plants were grown over a diurnal cycle it was shown that, like vascular plants, starch accumulated in the light period and was degraded at night. Both Ppgwd1a and DM lines accumulated significantly higher amounts of starch compared to Ppgwd1b and the control lines at almost all time points. Soluble sugars on the other hand were significantly reduced in Ppgwd1a and the DM lines compared with Ppgwd1b and the control lines. The Ppgwd1a and DM mutant lines also demonstrated a n observable morphological phenotypic alteration characterized by lack of gametophore development which could be reversed by growing the plant on media supplemented with glucose. In the second experimental chapter, two isoamylase (ISA) like starch debranching enzymes, ISA1 and ISA2, were examined. Mutations in these genes in vascular plants can result in the production of the water-soluble polysaccharide (WSP), phytoglycogen at the expense of starch. Both single and double mutants showed loss of a debranching enzyme activity band in activity gels indicating that ISA1 and ISA2 exist as a heterocomplex in Physcomitrella patens. Analysis of purified recombinant protein demonstrated, surprisingly that both PpISA1 and PpISA2 peptides were catalytically active. Analysis of single and double mutant plants demonstrated that all contained increased amounts of WSP.
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    Genetic studies for sustainable aquaculture of the sea urchin, Tripneustes gratilla
    (Stellenbosch University, 2020-12) Brink, Marissa; Roodt-Wilding, Rouvay; Rhode, Clint; Macey, Brett Marc; Cyrus, Mark Digby; Stellenbosch University. Faculty of AgriSciences. Department of Genetics.
    ENGLISH ABSTRACT: The sea urchin, Tripneustes gratilla, has been identified as a species with potential for aquaculture production in South Africa, as these animals are distributed along the eastern coast, produce bright orange roe and have a fast growth rate. This study aimed to assess various aspects of T. gratilla that could contribute to successful future aquaculture practices, through population genetics, pedigree reconstruction, qualitative assessments, quantitative genetics and metagenomic approaches. Chapter 2 evaluated the genetic diversity and population structure of T. gratilla populations along the South African coast, through the application of 22 species-specific microsatellite markers. Geographically representative sampled populations formed a single, interbreeding population, with a moderate degree of genetic diversity. In chapter 3, the markers were applied in two T. gratilla cultured cohorts to assess parental contributions, as well as changes in genetic diversity from the progenitor natural population. In these cohorts, the parental skew often associated with broadcast spawning animals was observed, where a single female and male dominated the respective spawning events. This resulted in a decline in genetic diversity, which could have implications for the genetic management of future commercial production. These results suggested that other factors, such as diet, breeding design, gonad and gamete quality could affect reproductive success. Consequently, chapter 4 aimed to assess biological and genetic aspects in T. gratilla that could influence reproductive competition, larval growth and juvenile performance. Results illustrated that a factorial breeding design is an effective approach for retaining genetic diversity in cultured populations. Broodstock conditioned on a mixed feeding regime outperformed animals fed the other diets included in this study (formulated feed, Ecklonia maxima and Ulva rigida). These animals could have had a higher ingestion efficiency, may have been exposed to a broader array of nutrients, displayed improved maternal provisioning or had an improved digestibility promoted by the bacteria introduced through natural feeds. The bacterial communities associated with sea urchin systems play an important role in animal health. In the studied aquaculture environment, bald sea urchin disease has been observed. Chapter 5 explored this disease using a 16S rDNA metagenomics approach, where samples included healthy animals from natural locations along the eastern coast of South Africa, as well as different cultured cohorts: healthy-, diseased- and stressed animals. Results showed that this disease is more likely caused by complex interactions between opportunistic bacteria, rather than by a specific pathogenic agent. Overall, this study showed that the preservation of genetic diversity in cultured T. gratilla populations is possible through factorial breeding designs and broodstock conditioning, where precautionary measures and effective animal husbandry practices can contribute to the prevention of diseases associated with opportunistic bacteria. Therefore, an integrated approach should be implemented to maintain genetic diversity, promote reproductive success and manage disease outbreaks in this emerging echinoculture industry.