Browsing by Author "Rees, D. Jasper G."

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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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    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.