Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The first aim of this study was to reliably and rapidly detect Grapevine rupestris stem pittingassociated
virus (GRSPaV) in grapevine. This was achieved by screening 94 grapevines
using crude plant extracts in both quantitative and conventional reverse transcription
polymerase chain reaction (RT-PCR). The second aim was to establish a technique capable of
differentiating GRSPaV sequence variants. The application of this technique is for the largescale
screening of diseased vines to associate sequence variants of GRSPaV with disease
symptoms. Nested quantitative polymerase chain reaction and high resolution melting assays
(qPCR-HRM) were developed for three regions of the GRSPaV genome (coat protein, RNAdependant
RNA-polymerase and triple gene block movement protein). The qPCR-HRM
technique using the high saturation dye, EvaGreen™, and the Rotor-Gene™ 6000 analyzer
was validated with a panel of sixteen sequence-characterized viral isolates. Diluted RT-PCR
products and cloned cDNA gave the most consistent amplification plots and dissociation
profiles. RT-PCR products generated from total RNA extracts were used as template for
qPCR-HRM assays and for direct sequencing of sixteen samples in the three aforementioned
regions. The average amplification efficiency for qPCR was 1.52±0.04. Auto-calling of userdefine
genotypes was performed at a confidence interval of 70%. Phylogenetic analysis of the
three regions of the GRSPaV genome was performed with published GenBank sequences to
confirm the HRM data. The dominant sequence variants found in the South African sample
set radiated with Group II, reference full-length variant GRSPaV-SG1. GRSPaV-infected
samples can in future be subjected to qPCR-HRM assays developed during this study. This
can be performed to establish similarity to known genotypes and therefore phylogenetic
groups. Mixed infection of sequence variants and quasi-species were a common occurrence.
The assay will be useful in establishing correlation of specific genotypes to different
phenotypical expression of viral disease. This could provide insight into the etiology of
diseases associated with GRSPaV. / AFRIKAANSE OPSOMMING: Die eerste doel van hierdie studie was om die virus wat met Rupestris-stamverpitting
(Grapevine rupestris stem pitting-associated virus of “GRSPaV”) in wingerd verbind is,
vinnig en betroubaar op te spoor. Dit is bereik deur 94 wingerdstokke vir die
teenwoordigheid van die virus te toets met beide kwantitatiewe en konvensionele trutranskripsie
polimerase kettingreaksies (RT - PCR) vanaf ongesuiwerde plant-ekstraksies.
Die tweede doel was die daarstelling van ’n tegniek om onderskeid te tref tussen variante van
GRSPaV met verskillende nukleotiedvolgordes. Hierdie tegniek kan op groot skaal gebruik
word om ge-affekteerde wingerdstokke te toets om sodoende siektesimptome met spesifieke
variante van GRSPaV te verbind. Ge-neste kwantitatiewe polimerase-kettingreaksies (qPCR)
en hoë-resolusie smelt-analises (HRM) is ontwikkel vir drie streke van die GRSPaV-genoom
(mantelproteïen, RNS-afhanklike RNS-polimerase en trippelgeenblok bewegingsproteïen).
Die tegniek van qPCR-HRM met die hoë-versadingingskleurstof EvaGreen™ en die Rotor-
Gene™ 6000 ontleder se geldigheid is bevestig deur vergelyking met ’n paneel van sestien
virus-isolate waarvan die volgorde reeds bepaal is. Verdunde RT-PCR-produkte en
gekloneerde DNS het die mees konsekwente amplifikasie-uitstipping en dissosiasieprofiele
opgelewer. RT-PCR-produkte wat vanuit totale RNS-ekstrakte verkry is, is as templaat vir
qPCR-HRM-analises gebruik. Dieselfde produkte is ook gebruik, om die volgorde van
sestien monsters in drie streke direk te bepaal. Die gemiddelde amplifikasiedoeltreffendheid
van die qPCR was 1.52±0.04. Gebruiker-gedefinieerde genotipes is deur middel van outooproeping
teen ’n vertroue-interval van 70% uitgevoer. Filogenetiese analises vir drie streke
van die GRSPaV-genoom is uitgevoer met gepubliseerde GenBank-volgordes om die HRMdata
te bevestig. Die dominante volgorde-variante in die stel Suid-Afrikaanse monsters het
ooreengestem met Groep II, vollengte-verwysingsvariant GRSPaV-SG1. Monsters wat met
GRSPaV besmet is kan in die toekoms onderwerp word aan die qPCR-HRM-analises wat in
hierdie studie ontwikkel is. Dit kan uitgevoer word om ooreenkomste met bekende genotipes
te bepaal, en dus ook met filogenetiese groepe. Die besmetting van plante met meer as een
volgorde-variant het algemeen voorgekom. Die kwasi-spesies populasie-struktuur van die
virus het ook gedurig na vore gekom. Die toets sal nuttig wees in die bepaling van korrelasies
tussen spesifieke genotipes en verskillende fenotipiese voorkomste van virussiektes. Dit kan
insig verleen in die etiologie van siektes wat met GRSPaV verbind word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/5252 |
| Date | 12 1900 |
| Creators | Noach, Liesl Christine |
| Contributors | Burger, J. T., University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 113 p. : ill. |
| Rights | University of Stellenbosch |
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