Browsing Doctoral Degrees (Genetics) by browse.metadata.advisor "Botha-Oberholster, Anna-Maria"
Now showing 1 - 3 of 3
Results Per Page
- ItemCharacterisation 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.
- ItemElucidating functional interactions between the Russian wheat aphid (D. noxia Kurjumov) and bread wheat (Triticum aestivum L.)(Stellenbosch : Stellenbosch University, 2014-12) Schultz, Thia; Botha-Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: The Russian wheat aphid (Diuraphis noxia, Kurdj., Hemipetra, Aphididae, RWA) is an important pest of wheat, causing large-scale damage and yield losses. Various studies have been done at a transcriptomics level, including complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), suppressive subtractive hybridization (SSH) and micro-array, which have identified genes putatively involved in RWA resistance. Even though these candidate genes have been identified, their role in host defence still needs to be verified using a functional genetics approach. In this study virus induced gene silencing (VIGS) using a barley stripe mosaic virus (BSMV) vector, has been utilized to knock-down candidate genes of interest in a wheat cultivar with the Dn1-resistance gene (TugelaDN). In this study it was hypothesized that genes involved in the hypersensitive response (HR) may contribute towards resistance and were thus targeted for silencing. These include glutathione-S-transferase (GST), superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX). However, since aphid feeding also results in wounding, the genes were also analyzed under wounding only. Aphid fecundity is considered an indicator of involvement in RWA resistance, as susceptible plants result in higher aphid fertility. Findings in the study suggest that with wounding only, that Dn1 containing plants produce a greater hypersensitive response than susceptible controls. Ascorbate peroxidase was found to be important for wounding-induced resistance in Dn1 wheat plants. Under infestation conditions, silencing of superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX) was found not to have an effect on aphid fertility and thus are not directly involved in resistance signaling. Knock-down of a phi-class glutathione-S-transferase F6 (TaGSTF6) transcripts however, had a large effect on aphid nymph numbers and thus may contribute to Dn1-resistance. Putative resistance genes silenced under aphid infestation conditions were a nucleotide binding protein (NBP) and resistance gene analogue 2 (RGA2). Analysis of NBP revealed its identity as a part of the iron homeostasis machinery in the cytosol, responsible for Fe-cluster assembly. Silencing of both NBP and RGA2 resulted in the expression of a susceptible phenotype. T10rga2-1A is an NBS-LRR protein known to be required for rust resistance in concert with resistance gene Lr10. T10rga2-1D silenced treatments resulted in susceptibility and plant death after aphid infestation, suggesting that T10rga2-1D may be a good up-stream candidate in Dn1-resistance.
- 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.