Browsing Masters Degrees (Genetics) by Author "Aldrich, Dirk Jacobus"
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- ItemCharacterisation of microRNA expression profiles of Vitis vinifera in response to grapevine leafroll-associated virus 3 infection(Stellenbosch : Stellenbosch University, 2017., 2017-03) Aldrich, Dirk Jacobus; Maree, H. J.; Burger, Johan T.; Stellenbosch University. Faculty of AgriScience. Dept. of Genetics.ENGLISH ABSTRACT: Grapevine leafroll disease (GLD) is endemic to all grape-growing regions of the world and is considered the most significant grapevine viral disease. Grapevine leafroll-associated virus 3 (GLRaV-3) is considered the primary cause of GLD and in South African vineyards five genetic variant groups (I, II, III, VI and VII) have been confirmed. Small RNAs (sRNAs) have been shown to play a significant role in a plant’s response to biotic and abiotic stress. This has led to a growing interest in evaluating sRNAs, such as microRNAs (miRNAs), for their role in mediating gene regulation in response to virus infections. In this study, stem-loop RT-qPCR probe-based assays were utilised for miRNA quantitation in GLRaV-3 positive and negative grapevines. A set of own-rooted Cabernet Sauvignon plants representing GLRaV-3 variant groups I, II, III and VI has been established from cuttings of highly symptomatic GLRaV-3 infections found in commercial vineyards. These plants were sampled and screened to yield the first data set. Additionally, young Cabernet Sauvignon plants were established and graft-inoculated with single infections of the five known variants of GLRaV-3 found in South African vineyards. All these plants were maintained in a climate-controlled greenhouse and sampled twice, six months apart, to yield two data sets. A fourth data set comprised of GLRaV-3 positive and negative Cabernet Sauvignon plants sampled from various vineyards in Stellenbosch. Eleven miRNAs were quantified in both infected and healthy grapevine samples. Putative miRNA targets were predicted and annotated using in silico analyses. These targets were subsequently quantified in both greenhouse and field samples using a SYBR Green RT-qPCR assay. This study validated statistically significant differences in virus concentrations, expressed as virus concentration ratios (VCRs), in plants singly infected with different GLRaV-3 variants. Interestingly, no difference in mean VCRs were observed between data sets, despite notable differences in plant age, duration of GLRaV-3 infection, scion/rootstock combination and growing conditions. Several miRNAs showed statistically significant expression modulation between infected and healthy samples. miRNA expression between data sets varied substantially and a greater miRNA/target response was observed in plants with more established GLRaV-3 infections. The lack of significant differences in mean VCRs between data sets, coupled with the consistent modulation of certain miRNAs in plants that have likely been infected for longer is a promising result. This finding could indicate that successful inhibition of further virus replication by plant defence mechanisms occurred and that these miRNAs and their targets are implicated in this response. The predicted targets for these miRNAs are genes involved in disease resistance, apoplastic processes, oxidation-reduction processes and growth and developmental processes. Additionally, possible variant-specific miRNA responses to infection were observed across all data sets, which could aid in elucidating possible biological differences between variants of GLRaV-3.