Browsing Masters Degrees (Genetics) by browse.metadata.advisor "Cook, Glynnis"
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- ItemDetermining the distribution and genetic diversity of Coguvirus eburi in South African citrus and development of a citrus-infecting coguvirus detection assay(Stellenbosch : Stellenbosch University, 2022-04) De Bruyn, Rochelle; Maree, H. J.; Bester, Rachelle; Cook, Glynnis; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.ENGLISH ABSTRACT: Citrus virus A (CiVA) is a novel negative-sense single-stranded RNA virus discovered with high- throughput sequencing (HTS). CiVA is classified as a member of the species Coguvirus eburi and is closely related to a member of the species Citrus coguvirus, named citrus concave gum-associated virus (CCGaV). CCGaV and CiVA are members of the genus Coguvirus in the family Phenuiviridae in the order Bunyavirales. CiVA has a bipartite genome encoding an RNA-dependant RNA polymerase (RdRp) on RNA 1 and a nucleocapsid protein (NP) and a putative movement protein (MP) on the ambisense RNA 2. The confirmed presence of CiVA in cultivars of grapefruit (Citrus paradisi Macf.), sweet orange (C. sinensis (L.) Osb.) and clementine (C. reticulata Blanco) in South Africa initiated a study to determine the distribution, genetic diversity and symptom association of CiVA in three provinces and seven citrus production regions. CiVA was detected in six citrus production regions in symptomatic and asymptomatic sweet orange trees. In three citrus production regions, CiVA was detected in sweet orange trees displaying a fruit rind symptom similar to citrus impietratura. CiVA was also detected in grapefruit trees with typical citrus impietratura symptoms and in symptomless clementine trees. The three encoded gene regions of CiVA were Sanger sequenced to investigate the genetic diversity between isolates from the six citrus production regions and three citrus species. Phylogenetic analysis of the nucleotide sequences (nt) of each encoded gene region was performed through the construction of Maximum-likelihood (ML) phylogenetic trees and nucleotide identity matrices. Phylogenetic analysis and nt identity matrices indicated a higher genetic diversity within the NP than the MP and RdRp. More genetic diversity was observed between isolates from the three citrus species than between isolates from the different citrus production regions. A real-time RT-PCR detection assay targeting the RdRp was also developed to simultaneously detect CiVA and CCGaV. Two cDNA synthesis methods for reverse transcription (RT) were compared and a degenerate, dual priming oligo (DPO) reverse primer was designed to improve the specificity of the detection assay. Two PCR assays that utilised the DPO reverse primer with two different forward primers were compared. The cDNA synthesis method and choice of primer pair had an influence on the amplification efficiency, specificity and sensitivity of the real-time detection assay. A tissue specificity assay was also performed and CiVA was detected throughout the plant in leaf midribs, leaf lamina, green bark and roots. Lower Ct values were consistently associated with the green bark and leaf midribs. The detection assay was implemented for pathogen screening within the Citrus Improvement Scheme (CIS) of South Africa, ensuring the release of CiVA free budwood to the citrus industry.