Doctoral Degrees (Genetics)

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Characterization of citrus tristeza virus variants and their influence on symptom expression in grapefruit
(Stellenbosch : Stellenbosch University, 2019-04) Cook, Glynnis; Maree, H. J.; Burger, Johan T.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: Citrus tristeza virus (CTV), a member of the family Closteroviridae, was responsible for large scale destruction of citrus, especially in the Americas, due to tristeza disease and necessitated a production switch to less sensitive rootstocks. CTV however continues to affect citrus through the stem-pitting disease phenotype which is especially problematic in sweet orange, grapefruit and lime cultivars. In South Africa, the productive lifespan of grapefruit trees was severely affected by stem-pitting, requiring early tree replacement with an associated lag in production. This affect was later mitigated by applying cross-protection, a management strategy using non-stem-pitting sources of CTV, but without prior knowledge of which CTV strains were responsible for stempitting or which strains were present in the cross-protection sources. To understand the disease and unravel mechanisms underlying cross-protection, it is necessary to characterise CTV strains to investigate both virus-host- and strain-interactions. The aim of this study was firstly to identify single-strain isolates belonging to different strains, to characterise them biologically and to determine full-genome sequences. These characterised CTV isolates were further used in a complementation study to investigate possible synergistic interactions affecting stem-pitting. Complete viral genomes of eight single-strain isolates were determined during the study. Two commercial grapefruit cultivars, ‘Star Ruby’ and ‘Marsh’, were used in a glasshouse trial to evaluate the ability of specific strains to induce stem-pitting in single or mixed infections. Evaluation over four years showed that symptom expression of mild strains did not result in altered symptom expression when in combination with each other. Importantly demonstrating that there was no additive effect on stem-pitting expression with multiple isolates. Relative quantitation of the strains in ‘Marsh’ and ‘Star Ruby’ plants indicated that the individual strain concentrations were not significantly altered when in combination with the other strains. A valuable discovery made within this project was the characterisation of two variants of the T68 strain, derived from the same GFMS12 source, but displaying differences in stem-pitting severity in grapefruit. This finding demonstrates the co-existence of severe and mild variants of the same strain in one source and provides an explanation for the presumed strain segregation event observed for the GFMS12 cross-protection source that resulted in the discontinuation of the source for use in cross-protection of grapefruit. The characterisation of these variants will further assist in the identification of the sequence determinants for stem-pitting in grapefruit.
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.
Genetic enhancement of pearl millet
(Stellenbosch : Stellenbosch University, 2004-03) O'Kennedy, Martha Margaretha; Botha, F. C.; Burger, Johan T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).
ENGLISH ABSTRACT: The aim of this study was toe stablish a reliable protocol for the production 0 f transgenic pearl millet as this will open new avenues for augmenting the gene pool of this crop. This was achieved by identifying a highly regenerabie genotype and optimisation of a tissue culture system, and biolistic protocol f or stable integration of selected transgenes. Both a negative, herbicide resistance selectable marker gene, bar, and a positive selectable marker gene, manA, were individually introduced in order to identify and establish a reliable transformation protocol. The optimised transformation protocol was then used to introduce an antifungal gene in the genome of pearl millet to enhance resistance to the biotrophic fungus Sclerospora graminicola. S. graminicola, an obligate oomycetous fungal phytopathogen, is the causal agent of downy mildew in pearl millet plants and a major constraint in the production of pearl millet. A single component of antifungal resistance was introduced into the genome of pearl millet, as preliminary work towards determining its role in the total plant defence system. The approach chosen was to introduce a hydrolytic enzyme, 13-1,3- glucanase, from Trichoderma atroviride (formerly T. harzianum), a soil-borne filamentous fungus, capable of parasitizing several plant pathogenic fungi. It was anticipated that introducing this glucanase gene from T. atroviride which degrades glucan in the fungal cell walls, would significantly contribute to the improvement of resistance against downy mildew. Constructs were prepared containing the gene (gluc78) encoding a 78 kDa beta-1,3- glucanase. The constructs were prepared containing the gluc78 gene driven either by a strong constitutive promoter (ubiquitin promoter, exon and intron) or a wound inducible promoter, the potato proteinase inhibitor ilK gene promoter. The wound inducible promoter includes either an AMV leader' sequence or the rice Act1 intron to obtain higher expression levels in the monocotyledonous plant. The transformation efficiency using the particle inflow gun and the herbicide resistance gene, bar, was improved from 0.02% on a MS based medium, to 0.19 or 0.72% with manA as selectable marker gene on MS or L3 based medium, respectively. However, individual experiments, introducing manA as selectable marker gene, resulted in frequencies of 1.2 and 3%. This translated to one transformation event per plate, which contains on average 31-35 pre-cultured immature zygotic embryos. This is the first report of t he successful introduction and expression of a 13-1,3-glucanase encoding gene from a biocontrol fungus not only under constitutive expression but also under wound inducible expression in a plant. Optimisation of genetic engineering of pearl millet, a cereal crop recalcitrant to transformation, and the introduction of an antifungal transgene, was accomplished in this study. Initial results hint that expression of this transgene enhances resistance to S. graminicola.
Genome and transcriptome sequencing of vitis vinifera cv pinotage
(Stellenbosch : Stellenbosch University, 2018-03) Coetzee, Beatrix; Burger, Johan T.; Maree, H. J.; Stellenbosch University. Faculty of AgriScience. Dept. of Genetics.
ENGLISH ABSTRACT: Examining the genetic basis of natural phenotypic variation, and the transfer of this knowledge to a breeding program for improved crop cultivars or livestock races, is a major goal for biological sciences. As grapevine (Vitis vinifera) is one of the most important crop plants in the world, research into its genetics is imperatave, both in terms of sustainable food production and the vast economic impact of the wine industry. Grapevine displays a great level of intraspecies phenotypic diversity in viticultural and oenological traits, between cultivars. Understanding this genetic diversity is an important step towards developing improved grapevine cultivars, but also the conservation of the important traditional cultivars. Vitis vinifera cv Pinotage is an artificial Pinot noir/ Cinsaut cross, created with the South African climate and growing conditions in mind. Today it is a commercial cultivar, used for the production of premium wines, deeply rooted in the South African wine culture and history. This study focused on the next-generation sequencing and bioinformatic analysis of the Pinotage genome and transcriptome. A de novo assembly strategy was followed to produce the first Pinotage draft genome sequence. Sequencing read data were also aligned to the available reference Pinot noir genome, and from this alignment the Pinotage/ Pinot noir variant density, determined. This was followed by a more in-depth focus on a number of functional gene clusters with more than 50% of their genes influenced by these variants. Furthermore, this is the first research to lend scientific support to the current wine trend of exclusive, superior wines produced from old vineyards. These old-vine wines are assumed to have a deeper character and more flavour. To explore the role of genetics and differential gene expression in this phenomenon, RNA-seq data were used to survey and compare the leaf and berry transcriptomes of young and old Pinotage vines, at harvest. Differential gene expression between young and old vines was studied, and the involvement of these genes in fruit ripening, discussed. A general trend towards delayed ripening in older vines was observed. This suggests that the berries remain attached to the vine for a longer period, thereby allowing more time for flavour compounds to accumulate. In the final part of the study, the Pinotage genome and transcriptome data were combined to identify Pinotage genes present in neither the reference Pinot noir PN40024 nor ENTAV115. These genes were classified as both structural and regulatory genes and it was shown that genes involved in the stress response network are a major gene class contributing to the genetic differences between Pinotage and Pinot noir. A plant species is constantly challenged by various biotic and abiotic stresses and it is an evolutionary investment to diversify genes responsible for stress response, to be able to efficiently overcome these stresses. The information generated in this study will aid in grapevine breeding programs for sustainable production of high quality wine in a changing environment.
Identification and characterisation of grapevine leafroll-associated virus 3 genomic and subgenomic RNAs
(Stellenbosch : University of Stellenbosch, 2010-12) Maree, Hans Jacob; Burger, Johan T.; Freeborough, M-J.
ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus Ampelovirus, family Closteroviridae. There has been only one report that claimed the complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended UTR was found in all other South African isolates of GLRaV-3 that were tested. In two collaborative studies the existence of the extended 5’ UTR was confirmed and further investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended 5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the different genetic variants, however within a variant the 5’ UTR was found to be highly conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’ half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3 mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation of putative sg-promoters is also described.
Identification and molecular characterization of three genetic variants of Grapevine leafroll-associated virus 3 (GLRaV-3) from South African vineyards and their spread in local vineyards
(Stellenbosch : University of Stellenbosch, 2011-03) Jooste, Anna Elizabeth Catharina; Burger, Johan T.; Goszczynski, D. E.; University of Stellenbosch. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Grapevine diseases, in particular virus and virus-like diseases, are threatening grapevine industries worldwide; also in South Africa. Grapevine leafroll (GLR) is one of the most important diseases of grapevines, occurring in all grape-producing countries worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) is known to be closely associated with GLR disease and occurs commonly in South African vineyards. In this study three genetic variants of GLRaV-3 were identified in vineyards of the Western Cape, South Africaby single strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. A specific SSCP profile could be assigned to each variant group and these wereconfirmed by sequencing of the ORF5 regions.These results demonstrated that SSCP analysis on this region in ORF5 provides a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5’ ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5’UTRs indicated significant sequence and length variation in this region, between the three South African variant groups. Nucleotide sequence alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three GLRaV-3 variants in South Africa, and that two or three additional variant groups occurs elsewhere in the world. We further investigated the prevalence of these three GLRaV-3 variants in mother blocksof different cultivars and from different vine growing regions, using SSCP analysis. The majority of the plants studied, were infected with the group II variant, similar to isolates 623 and GP18. The distribution of the three GLRaV-3 variants within a spatio-temporally recorded cluster of diseased plants was studied by means of SSCP profile analysis. We showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection cluster. Results showed that, in some leafroll disease clusters, the variant that was present in the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row and across rows. Some plants in the cluster were also infected with variants not present in the original plant. These infections could have been caused by mealybug vectors feeding on plants from surrounding areas and then infecting these plants. The scientific information generated on GLRaV-3 variants in this project contributed to the advancement of our knowledge of genetic variability and provides a basis of further epidemiology and vector-virus studies. The study showed for the first time that different GLRaV-3 variants were transmitted to adjacent plants in a row and across rows in a GLR disease cluster. The diversity detected in the 5’UTR between variants from the three genetic groups provides a platform for the further study of the biological characteristics of GLRaV-3 variants.
Modulation of the Vitis vinifera cv. ‘Chardonnay’ microRNA and mRNA transcriptomes in response to aster yellows phytoplasma-infection
(Stellenbosch : Stellenbosch University, 2019-04) Snyman, Marius Christian; Burger, Johan T.; Stephan, Dirk; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.
ENGLISH ABSTRACT: Aster yellows (AY) phytoplasmas are part of a group of cell wall-less plant pathogenic bacteria responsible for a detrimental disease known as grapevine yellows (GY). The molecular mechanisms of AY phytoplasma pathogenicity on highly susceptible cultivars, such as Chardonnay, are still largely unknown. This has sparked considerable interest to gain knowledge about the basis of host susceptibility to GY in order to develop control strategies that may mitigate the scale of infection or even prevent spread. Leaf total RNA was extracted from both healthy and AY-infected plants to generate small RNA (sRNA) sequencing libraries, as well as mRNA sequencing libraries. These libraries were subjected to Illumina transcriptome sequencing (small RNA-seq and mRNA-seq, respectively), and comparative transcriptome profiling, to explore the involvement of microRNA (miRNA) and gene expression pathways in AY phytoplasma-infected Chardonnay. Multiple known miRNA sequence variants (isomiRs) were identified, and 13 known miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a previously uncharacterised genomic location, were identified, of which 23 were differentially expressed. Some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of some of these known and novel miRNAs was confirmed with stem-loop RT-qPCR analysis, thereby validating the trend of miRNA expression in the normalised sRNA-seq read count data. miRNA target prediction, using a complementary-based in silico approach, followed by functional annotation, allowed the identification of potential target genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. mRNA-seq results showed that 175 genes were differentially expressed in the AY phytoplasma-infected leaf material. Functional annotation of differentially expressed genes (DEGs) enabled the identification of mRNAs involved in plastid and cell wall metabolism/architecture, signalling, innate immunity, pathogen defence, secondary metabolism and photosynthesis. RT-qPCR analysis was used to validate the trend of expression of significant DEGs. Taken together, this study presents the first report on the modulation of miRNAs and genes associated with AY phytoplasma-infection in Chardonnay. The knowledge generated during this study may be crucial in understanding disease symptom development in AY phytoplasma-infected grapevines. Importantly, the findings of this study may also aid in developing GY disease control strategies and could provide added insight for future plant pathogenesis-related studies.
Small RNA profiling of virus-infected apple plants
(Stellenbosch : Stellenbosch University, 2014-12) Visser, Marike; Burger, Johan T.; Maree, H. J.; Rees, Jasper; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) is globally associated with latent infection in commercial apple trees. Little is known about this plant-‐virus interaction. This study made use of next-‐generation sequencing to investigate the effect of virus-‐infection on the expression of the different small RNA (sRNA) species namely, miRNAs, nat-‐siRNAs, phasiRNAs, rasiRNAs, tRNA-‐derived sRNAs and vsiRNAs. Broad and narrow size-‐range datasets were generated using sRNA libraries prepared from total and size-‐selected RNA, respectively. Through bioinformatic data analyses, 130 genomic loci were predicted to give rise to miRNAs, 85 of which were novel MIR genes. Targets were predicted for the majority of miRNAs, a few of which could be validated with a publicly available degradome dataset. Cis-‐ and trans-‐natural antisense transcripts (NATs) were identified, of which only the latter were highly enriched for sRNAs in their overlapping regions. Transcript as well as genomic regions were also identified that can give rise to phasiRNAs. For 25 of these loci an in-‐phase miRNA target site was identified, half of which could be validated with the degradome dataset. Nearly all apple repeat sequences in Repbase were associated with sRNA synthesis. sRNAs derived from both ends of mature tRNAs were identified. These sRNAs corresponded to tRFs and tRNA-‐halves. Reads associated with tRNA-‐halves were prominent in the broad range datasets. sRNAs, originating from the central regions of tRNAs, were also observed. Analysis of the vsiRNAs suggested the presence of two ASGV genetic variants in two of the samples, while the third sample was infected with only one variant. Comparison of the vsiRNA profiles generated from the two datasets highlighted the influence of library preparation on the interpretation of results. Differential expression analysis of the identified apple sRNA species showed no variation between healthy and infected plants, except for the tRNA-‐derived sRNAs, which did show altered expression levels. Taken together, the various sRNA species characterised in this study significantly extended the existing knowledge of apple sRNAs and provide a broad platform for future functional studies in apple. This study also presents the first and most comprehensive report on sRNAs involved in ASGV infection in apple.

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