Browsing Masters Degrees (Genetics) by Author "Bester, Rachelle"
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- ItemSequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa(Stellenbosch : Stellenbosch University, 2012-12) Bester, Rachelle; Burger, J. T.; Maree, H. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type member of the genus Ampelovirus in the family Closteroviridae and is considered to be the main contributing agent of grapevine leafroll disease (GLD) worldwide. A metagenomic sequencing study of a grapevine leafroll-diseased vineyard led to the discovery of a new variant of GLRaV-3 in South Africa. This new variant was most related to a New Zealand isolate, NZ-1. In this study, we sequenced two isolates, GH11 and GH30, of the new variant group of GLRaV-3. These isolates have less than 70% nucleotide (nt) identity to other known GLRaV-3 variants, indicating that they should be considered variants of a different strain of GLRaV-3. We propose that the GLRaV-3-like virus identified in this study be grouped together with NZ-1 and some Napa Valley isolates as Group VI of GLRaV-3. This study also provided further evidence that next-generation sequencing is an invaluable approach to identify novel viruses and variants, in that the draft sequence generated with bioinformatic tools in this study was 98% identical to the GH11 sequence generated using Sanger sequencing. The study further confirmed that the industry standard ELISA is still an effective GLRaV-3 diagnostic method and that it is able to detect all known variant groups of GLRaV-3. However, this assay is not able to differentiate between GLRaV-3 variant groups. In the current study therefore, a real-time RT-PCR was designed that is able to detect GLRaV-3 variant groups I, II, III and VI, using a single primer pair targeting the Hsp70h gene of GLRaV-3. If high-resolution melting (HRM) curve analysis is added to the real-time RT-PCR, it is possible to differentiate between variant groups based on three melting point intervals. The RT-PCR HRM assay provides a more sensitive and rapid tool to detect and differentiate between different GLRaV-3 variant groups. Finally, a multiplex RT-PCR was designed to differentiate between the variant groups present in South Africa. This multiplex RT-PCR offers a validation method for the RT-PCR HRM and provides an end-point PCR alternative for variant identification. In order to investigate the spread and impact of different GLRaV-3 variants in vineyards, sensitive diagnostic techniques are a necessity. The abovementioned tools will contribute to the understanding of the pathogenesis of GLD and aid epidemiological studies to investigate how these different GLRaV-3 variant groups are spreading, the association of specific GLRaV-3 variants to disease symptoms and the mealybug vector transmission efficiency for each GLRaV-3 variant.