Browsing by Author "Coetzee, Beatrix"

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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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    Genome and transcriptome sequencing of vitis vinifera cv pinotage
    (Stellenbosch : Stellenbosch University, 2018-03) Coetzee, Beatrix; Burger, Johan T.; Maree, H. J.; Stellenbosch University. Faculty of AgriScience. Dept. of Genetics.
    ENGLISH ABSTRACT: Examining the genetic basis of natural phenotypic variation, and the transfer of this knowledge to a breeding program for improved crop cultivars or livestock races, is a major goal for biological sciences. As grapevine (Vitis vinifera) is one of the most important crop plants in the world, research into its genetics is imperatave, both in terms of sustainable food production and the vast economic impact of the wine industry. Grapevine displays a great level of intraspecies phenotypic diversity in viticultural and oenological traits, between cultivars. Understanding this genetic diversity is an important step towards developing improved grapevine cultivars, but also the conservation of the important traditional cultivars. Vitis vinifera cv Pinotage is an artificial Pinot noir/ Cinsaut cross, created with the South African climate and growing conditions in mind. Today it is a commercial cultivar, used for the production of premium wines, deeply rooted in the South African wine culture and history. This study focused on the next-generation sequencing and bioinformatic analysis of the Pinotage genome and transcriptome. A de novo assembly strategy was followed to produce the first Pinotage draft genome sequence. Sequencing read data were also aligned to the available reference Pinot noir genome, and from this alignment the Pinotage/ Pinot noir variant density, determined. This was followed by a more in-depth focus on a number of functional gene clusters with more than 50% of their genes influenced by these variants. Furthermore, this is the first research to lend scientific support to the current wine trend of exclusive, superior wines produced from old vineyards. These old-vine wines are assumed to have a deeper character and more flavour. To explore the role of genetics and differential gene expression in this phenomenon, RNA-seq data were used to survey and compare the leaf and berry transcriptomes of young and old Pinotage vines, at harvest. Differential gene expression between young and old vines was studied, and the involvement of these genes in fruit ripening, discussed. A general trend towards delayed ripening in older vines was observed. This suggests that the berries remain attached to the vine for a longer period, thereby allowing more time for flavour compounds to accumulate. In the final part of the study, the Pinotage genome and transcriptome data were combined to identify Pinotage genes present in neither the reference Pinot noir PN40024 nor ENTAV115. These genes were classified as both structural and regulatory genes and it was shown that genes involved in the stress response network are a major gene class contributing to the genetic differences between Pinotage and Pinot noir. A plant species is constantly challenged by various biotic and abiotic stresses and it is an evolutionary investment to diversify genes responsible for stress response, to be able to efficiently overcome these stresses. The information generated in this study will aid in grapevine breeding programs for sustainable production of high quality wine in a changing environment.
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    A metagenomic approach using next-generation sequencing for viral profiling of a vineyard and genetic characterization of grapevine virus E
    (Stellenbosch : University of Stellenbosch, 2010-12) Coetzee, Beatrix; Burger, J. T.; Freeborough, M-J.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Next-generation sequencing technologies are increasingly used in metagenomic studies, largely due to the high sequence data throughput capacity and unbiased approach in determining the genetic composition of an unknown environmental sample. This study investigated the applicability of the Illumina next-generation sequencing platform for metagenomic sequencing of grapevine viruses to provide the first complete viral profile, or virome, of a diseased vineyard. Leaf material was harvested from 44 randomly selected vines in a leafroll-diseased vineyard in South Africa. Sample material was pooled and double-stranded RNA extracted. The dsRNA was sequenced as a paired-end sequencing run using the Illumina sequencing-by-synthesis technique, and more than 19 million sequence reads, equivalent to approximately 837 megabases of metagenomic sequence data, were obtained. Of these data, approximately 400 megabases could be assembled into 449 scaffolds, using the de novo assembler Velvet. These scaffolds were subjected to BLAST searches against the NCBI databases and top hit scores were used for virus identification. Based on the BLAST results, suitable sequences were selected from the NCBI database and used as reference sequence in MAQ mapping assemblies. The bioinformatic analyses allowed for the determination of the virus species present, the most prominent variants, and the relative abundance of each. Four known grapevine viral pathogens were identified. Grapevine leafroll-associated virus 3, representing 59% of the analyzed short read sequence data, was identified as the most prominent virus species. Three variants of this virus were detected: GP18 was the most abundant, followed by a minor Cl766/NY1 variant and a potential novel grapevine leafroll-associated ampelovirus. A single Grapevine rupestris stem pitting ]associated virus variant, similar to SG1, and a Grapevine virus A variant, a member of molecular group III, were identified. This study is also the first to report the presence of Grapevine virus E (GVE) in South African vineyards. Grapevine virus E was further genetically characterized and the genome sequence of GVE isolate SA94 determined. The GVE SA94 genome sequence, 7568 nucleotides in length, is the first complete genome sequence for the virus species. The genome organization of GVE SA94 is typical of vitiviruses, but in contrast to other RNA viruses, the AlkB domain is located within the helicase domain in open reading frame 1 (ORF 1). Grapevine virus E SA94 shares nearly 100% nucleotide identity with the Japanese TvP15 isolate and GVE 3404, a de novo scaffold generated from the metagenomic sequence data. Bioinformatic analysis of metagenomic sequence data further revealed the presence of three fungus-infecting viral families, Chrysoviridae, Totiviridae and the unclassified dsRNA virus, Fusarium graminearum dsRNA mycovirus 4. A virus from the family Chrysoviridae, similar to Penicillium chrysogenum virus, was the second most abundant virus detected. We demonstrated the successful application of a short read sequencing technology, such as the Illumina platform, for viral profiling of an infected vineyard. To our knowledge this is the first application of the Illumina technology for this purpose.
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    Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits
    (BioMed Central, 2012-08) Meiring, Tracy L; Salimo, Anna T; Coetzee, Beatrix; Maree, Hans J; Moodley, Jennifer; Hitzeroth, Inga I; Freeborough, Michael-John; Rybicki, Ed P.; Williamson, Anna-Lise
    Abstract Background Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women. Methods Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR. Results Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence’s of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6%, 12.8%, 4.6% and 8.3% respectively. Conclusions Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.