Browsing by Author "Maree, Hans J."

Now showing 1 - 9 of 9
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    Citrus tristeza virus genotype detection using high-throughput sequencing
    (MDPI, 2021-01-23) Bester, Rachelle; Cook, Glynnis; Maree, Hans J.
    The application of high-throughput sequencing (HTS) has successfully been used for virus discovery to resolve disease etiology in many agricultural crops. The greatest advantage of HTS is that it can provide a complete viral status of a plant, including information on mixed infections of viral species or virus variants. This provides insight into the virus population structure, ecology, or evolution and can be used to differentiate among virus variants that may contribute differently toward disease etiology. In this study, the use of HTS for citrus tristeza virus (CTV) genotype detection was evaluated. A bioinformatic pipeline for CTV genotype detection was constructed and evaluated using simulated and real data sets to determine the parameters to discriminate between false positive read mappings and true genotype-specific genome coverage. A 50% genome coverage cut-off was identified for non-target read mappings. HTS with the associated bioinformatic pipeline was validated and proposed as a CTV genotyping assay.
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    Draft genome sequence of a “candidatus phytoplasma asteris” - related strain (aster yellows, subgroup 16SrI-B) from South Africa
    (American Society for Microbiology, 2019-04-25) Coetzee, Beatrix; Douglas-Smit, Nicoleen; Maree, Hans J.; Burger, Johan T.; Kruger, Kerstin; Pietersen, Gerhard
    Here, we report the draft genome sequence of a phytoplasma discovered in grapevine. The genome size is 600,116 nucleotides (nt), with 597 predicted open reading frames. It is most similar to a maize bushy stunt phytoplasma of group 16SrI-B (aster yellows). The possible presence of a 3,833-nt plasmid was also noted.
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    Extending the sRNAome of Apple by next-generation sequencing
    (Public Library of Science, 2014-04) Visser, Marike; Van der Walt, Anelda P.; Maree, Hans J.; Rees, D. Jasper G.; Burger, Johan T.
    The global importance of apple as a fruit crop necessitates investigations into molecular aspects of the processes that influence fruit quality and yield, including plant development, fruit ripening and disease resistance. In order to study and understand biological processes it is essential to recognise the range of molecules, which influence these processes. Small non-coding RNAs are regulatory agents involved in diverse plant activities, ranging from development to stress response. The occurrence of these molecules in apple leaves was studied by means of next-generation sequencing. 85 novel microRNA (miRNA) gene loci were predicted and characterized along with known miRNA loci. Both cis- and trans-natural antisense transcript pairs were identified. Although the trans-overlapping regions were enriched in small RNA (sRNA) production, cis-overlaps did not seem to agree. More than 150 phased regions were also identified, and for a small subset of these, potential miRNAs that could initiate phasing, were revealed. Repeat-associated siRNAs, which are generated from repetitive genomic regions such as transposons, were also analysed. For this group almost all available repeat sequences, associated with the apple genome and present in Repbase, were found to produce siRNAs. Results from this study extend our current knowledge on apple sRNAs and their precursors significantly. A rich molecular resource has been created and is available to the research community to serve as a baseline for future studies.
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    High-throughput sequencing reveals small RNAs involved in ASGV infection
    (BioMed Central, 2014) Visser, Marike; Maree, Hans J.; Rees, D. Jasper G.; Burger, Johan T.
    Background : Plant small RNAs (sRNAs) associated with virulent virus infections have been reported by previous studies, while the involvement of sRNAs in latent virus infection remains largely uncharacterised. Apple trees show a high degree of resistance and tolerance to viral infections. We analysed two sRNA deep sequencing datasets, prepared from different RNA size fractions, to identify sRNAs involved in Apple stem grooving virus (ASGV) infection. Results sRNA analysis revealed virus-derived siRNAs (vsiRNAs) originating from two ASGV genetic variants. A vsiRNA profile for one of the ASGV variants was also generated showing an increase in siRNA production towards the 3′ end of the virus genome. Virus-derived sRNAs longer than those previously analysed were also observed in the sequencing data. Additionally, tRNA-derived sRNAs were identified and characterised. These sRNAs covered a broad size-range and originated from both ends of the mature tRNAs as well as from their central regions. Several tRNA-derived sRNAs showed differential regulation due to ASGV infection. No changes in microRNA, natural-antisense transcript siRNA, phased-siRNA and repeat-associated siRNA levels were observed. Conclusions This study is the first report on the apple sRNA-response to virus infection. The results revealed the vsiRNAs profile of an ASGV variant, as well as the alteration of the tRNA-derived sRNA profile in response to latent virus infection. It also highlights the importance of library preparation in the interpretation of high-throughput sequencing data.
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    In silico analysis of the grapefruit sRNAome, transcriptome and gene regulation in response to CTV-CDVd co-infection
    (BioMed Central, 2017) Visser, Marike; Cook, Glynnis; Burger, Johan T.; Maree, Hans J.
    Background: Small RNA (sRNA) associated gene regulation has been shown to play a significant role during plant-pathogen interaction. In commercial citrus orchards co-infection of Citrus tristeza virus (CTV) and viroids occur naturally. Methods: A next-generation sequencing-based approach was used to study the sRNA and transcriptional response in grapefruit to the co-infection of CTV and Citrus dwarfing viroid. Results: The co-infection resulted in a difference in the expression of a number of sRNA species when comparing healthy and infected plants; the majority of these were derived from transcripts processed in a phased manner. Several RNA transcripts were also differentially expressed, including transcripts derived from two genes, predicted to be under the regulation of sRNAs. These genes are involved in plant hormone systems; one in the abscisic acid, and the other in the cytokinin regulatory pathway. Additional analysis of virus- and viroid-derived small-interfering RNAs (siRNAs) showed areas on the pathogen genomes associated with increased siRNA synthesis. Most interestingly, the starting position of the p23 silencing suppressor’s sub-genomic RNA generated a siRNA hotspot on the CTV genome. Conclusions: This study showed the involvement of various genes, as well as endogenous and exogenous RNA-derived sRNA species in the plant-defence response. The results highlighted the role of sRNA-directed plant hormone regulation during biotic stress, as well as a counter-response of plants to virus suppressors of RNA-silencing.
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    Next-generation sequencing for virus detection : covering all the bases
    (BioMed Central, 2016-06) Visser, Marike; Bester, Rachelle; Burger, Johan T.; Maree, Hans J.
    Background: The use of next-generation sequencing has become an established method for virus detection. Efficient study design for accurate detection relies on the optimal amount of data representing a significant portion of a virus genome. Findings: In this study, genome coverage at different sequencing depths was determined for a number of viruses, viroids, hosts and sequencing library types, using both read-mapping and de novo assembly-based approaches. The results highlighted the strength of ribo-depleted RNA and sRNA in obtaining saturated genome coverage with the least amount of data, while even though the poly(A)-selected RNA yielded virus-derived reads, it was insufficient to cover the complete genome of a non-polyadenylated virus. The ribo-depleted RNA data also outperformed the sRNA data in terms of the percentage of coverage that could be obtained particularly with the de novo assembled contigs. Conclusion: Our results suggest the use of ribo-depleted RNA in a de novo assembly-based approach for the detection of single-stranded RNA viruses. Furthermore, we suggest that sequencing one million reads will provide sufficient genome coverage specifically for closterovirus detection.
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    Phylogenomic analysis reveals deep divergence and recombination in an economically important grapevine virus.
    (Public Library of Science, 2015) Maree, Hans J.; Pirie, Michael D.; Oosthuizen, Kristin; Bester, Rachelle; Rees, D. Jasper G.; Burger, Johan T.
    The evolutionary history of the exclusively grapevine (Vitis spp.) infecting, grapevine leafroll-associated virus 3 (GLRaV-3) has not been studied extensively, partly due to limited available sequence data. In this study we trace the evolutionary history of GLRaV-3, focussing on isolate GH24, a newly discovered variant. GH24 was discovered through the use of next-generation sequencing (NGS) and the whole genome sequence determined and validated with Sanger sequencing. We assembled an alignment of all 13 available whole genomes of GLRaV-3 isolates and all other publicly available GLRaV-3 sequence data. Using multiple recombination detection methods we identified a clear signal for recombination in one whole genome sequence and further evidence for recombination in two more, including GH24. We inferred phylogenetic trees and networks and estimated the ages of common ancestors of GLRaV-3 clades by means of relaxed clock models calibrated with asynchronous sampling dates. Our results generally confirm previously identified variant groups as well as two new groups (VII and VIII). Higher order groups were defined as supergroups designated A to D. Supergroup A includes variant groups I-V and supergroup B group VI and its related unclassified isolates. Supergroups C and D are less well known, including the newly identified groups VII (including isolate GH24) and VIII respectively. The inferred node ages suggest that the origins of the major groups of GLRaV-3, including isolate GH24, may have occurred prior to worldwide cultivation of grapevines, whilst the current diversity represents closely related isolates that diverged from common ancestors within the last century.
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    Real-time RT-PCR high resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated associated virus 3 variant groups I, II, III and VI
    (BioMed Central, 2012-09) Bester, Rachelle; Jooste, Anna E. C.; Maree, Hans J.; Burger, Johan T.
    Abstract Background Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. Methods In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. Results A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. Conclusion The real-time RT-PCR HRM provides a sensitive, automated and rapid tool to detect and differentiate different variant groups in order to study the epidemiology of leafroll disease.
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    Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids
    (BioMed Central, 2021-03-22) Bester, Rachelle; Cook, Glynnis; Breytenbach, Johannes H. J.; Steyn, Chanel; De Bruyn, Rochelle; Maree, Hans J.
    Background: High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods: Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results: The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions: This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.