Browsing Masters Degrees (Genetics) by Author "Andrews, Kelsey Jayne"
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- ItemDevelopment of a molecular detection assay for accurate identification of five economically important tephritid species of commercial fruit in South Africa(Stellenbosch : Stellenbosch University, 2022-12) Andrews, Kelsey Jayne; Maree, H. J.; Bester, Rachelle; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: South African fresh fruit production and export are plagued by five major fruit fly pests (Diptera: Tephritidae). These fruit flies: Ceratitis capitata (Wiedemann) Mediterranean fly, Ceratitis cosyra (Walker) Marula fly, Ceratitis quilicii (de Meyer, Mwatawala & Virgilio) Cape fly, Ceratitis rosa (Karsch) Natal fly, and Bactrocera dorsalis (Hendel) the Oriental fly, are of quarantine significance in certain export markets. Apart from economic loss incurred due to limitations in export markets, these flies also cause physical damage to fresh fruit through oviposition. Accurate identification of these fruit flies can be tricky as this fruit fly family consists of multiple morphologically cryptic species and species complexes. Morphological identification of these fruit flies is unreliable when larvae or closely related species are intercepted. This has highlighted a need for accurate methods to distinguish between these species that do not rely on morphological traits. Therefore, this study aimed to develop a molecular identification assay that can differentiate between the five fruit flies of concern to South Africa accurately and timeously. To achieve this, two colony-reared insects from each species underwent DNA extraction and high throughput sequencing (HTS). HTS data were subjected to de novo assembly and used to construct ten complete mitochondrial genomes using a combination of de novo and reference-based assembly methods. From this, two identification assays were developed: a sequencing- based assay targeting a mitochondrial intergenic region and a multiplex PCR assay targeting the gene opsin Rh4. Regarding the sequencing-based assay, a single primer set was designed to amplify a mitochondrial region between tRNAⁱˡᵉ and tRNAᵐᵉᵗ. The intergenic region between tRNAⁱˡᵉ and tRNAᵍˡⁿ (designated intergenic region I) within the amplicon is species-specific in size and proposed as a potential tool for species differentiation of the five species of interest in this study. In the multiplex PCR assay, five sets of species- specific primers with varying sizes were designed and optimised for use in a multiplex format. The resulting species-specific amplicons can be separated using a 2% agarose-TAE gel, providing accurate species identification. Both assays were validated using larval stages and wild, trap-collected specimens. The assays developed in this study can be applied in pest surveillance and monitoring activities and during fruit inspection at packhouses and ports of entry (PoE).