Viral metagenomic analysis of sweet potato using high throughput deep sequencing

Nhlapo, Thulile Faith
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Sweet potato is an orphan crop with significant importance in developing countries due to its high β-carotene content and ability to address food insecurity in rural communities. Sweet potatoes also serve as an economically viable crop to resource poor farmers. Production constraints in the form of viral diseases negatively impact crop quality and yield, which often results in catastrophic economic losses. Detailed genomic characterisation of sweet potato viruses in East and Southern Africa is still lacking. The development of virus detection, identification and characterisation techniques is essential for viral disease control. The aim of this study was to identify and characterise the diversity of sweet potato viruses causing sweet potato virus disease (SPVD) in the Eastern and Western Cape provinces of South Africa. Viral metagenomics and high-throughput deep sequencing revealed the presence of six previously detected viruses and two novel badnaviruses. Sweet potato chlorotic stunt virus (SPCSV) was identified as a major role player and causal agent of SPVD. SPCSV was detected in mixed infections with sweet potato feathery mottle virus (SPFMV) and begomoviruses. These viruses are known to act in synergy to exacerbate viral disease symptoms. The reference-guided and de novo assembly of next generation sequencing (NGS) data achieved over 70% genome coverage for all viruses. The use of deep sequencing of nucleic acids is therefore a reliable diagnostic tool for virus detection as well as for differentiating between diverse viral strains. Small RNA profiles in the NASPOT 1 (resistant) and Blesbok (susceptible) leaves were also investigated by analysing the expression patterns of virus derived small interfering RNAs (vsiRNAs) and endogenous micro RNAs (miRNAs) in response to mixed viral infection at 60 days post infection (dpi). MiRNAs are small noncoding RNAs involved in the regulation of important biological processes such as plant development, biotic and abiotic stress response and pathogen defense. Over 55 miRNA families were identified collectively from the two cultivars. Two miRNAs (miR160 and miR6300) were downregulated in both resistant and susceptible cultivars, while miR393, miR398, miR168, miR162 and miR482) were upregulated in both cultivars, after infection with viruses. These miRNAs could play a key role in pathogen defense responses, as they are known to target mRNAs that encode major genes, enzymes and proteins, which are involved in the plant defense mechanisms. This study lays a firm foundation for understanding hostpathogen interactions in sweet potato
A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2019