Browsing by Author "Breeds, Kelly"

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    DNA methylation and demethylation are regulated by functional DNA methyltransferases and DnTET enzymes in diuraphis noxia
    (Frontiers Media, 2020) Du Preez, Pieter H.; Breeds, Kelly; Burger, N. Francois V.; Swiegers, Hendrik W.; Truter, J. Christoff; Botha, Anna-Maria
    Aphids are economically important insect pests of crops worldwide. Despite resistant varieties being available, resistance is continuously challenged and eventually broken down, posing a threat to food security. In the current study, the epigenome of two related Russian wheat aphid (Diuraphis noxia, Kurdjumov) biotypes (i.e., SA1 and SAM) that differ in virulence was investigated to elucidate its role in virulence in this species. Whole genome bisulfite sequencing covered a total of 6,846,597,083 cytosine bases for SA1 and 7,397,965,699 cytosine bases for SAM, respectively, of which a total of 70,861,462 bases (SA1) and 74, 073,939 bases (SAM) were methylated, representing 1.126 ± 0.321% (SA1) and 1.105 ± 0.295% (SAM) methylation in their genomes. The sequence reads were analyzed for contexts of DNA methylation and the results revealed that RWA has methylation in all contexts (CpG, CHG and CHH), with the majority of methylation within the CpG context (± 5.19%), while the other contexts show much lower levels of methylation (CHG − ± 0.27%; CHH − ± 0.34%). The top strand was slightly (0.02%) more methylated than the bottom strand. Of the 35,493 genes that mapped, we also analyzed the contexts of methylation of each of these and found that the CpG methylation was much higher in genic regions than in intergenic regions. The CHG and CHH levels did not differ between genic and intergenic regions. The exonic regions of genes were more methylated (±0.56%) than the intronic regions. We also measured the 5mC and 5hmC levels between the aphid biotypes, and found little difference in 5mC levels between the biotypes, but much higher levels of 5hmC in the virulent SAM. RWA had two homologs of each of the DNA methyltransferases 1 (DNMT1a and DNMT1b) and DNMT3s (DNMT3a and DNMT3b), but only a single DNMT2, with only the expression of DNMT3 that differed significantly between the two RWA biotypes. RWA has a single ortholog of Ten eleven translocase (DnTET) in the genome. Feeding studies show that the more virulent RWA biotype SAM upregulate DnDNMT3 and DnTET in response to wheat expressing antibiosis and antixenosis.
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    A targeted investigation of Diuraphis noxia (Hemiptera: Aphididae) methylation
    (Stellenbosch : Stellenbosch University, 2017-12) Breeds, Kelly; Botha-Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae – or Russian wheat aphid, RWA) is an economically important phloem-feeding pest of wheat and barley. The most effective method for controlling RWA infestation of wheat is the deployment of resistant cultivars. However, new biotypes – aphid populations expressing virulence towards these cultivars – continue to develop. Consequently, a dire need exists to understand the molecular mechanism underlying increases in aphid virulence. The epigenetic modification of methylation has been proposed as one such mechanism, yet its effect on virulence remains largely unexplored. The aim of the study was thus to determine if methylation plays a role in biotypification and the associated increase in aphid virulence. To this end, two methods, namely methylation-sensitive amplification polymorphism (MSAP or MS-AFLP) and restriction site-specific fluorescent labelling (RSSFL), were tested for their ability to detect and quantify RWA methylation. The former was successful on both counts, specifically in the CG and CC dinucleotide contexts. Use of this methodology also revealed 22 polymorphic loci between the least and most virulent South African biotypes, SA1 and SAM, with 18 resulting from an increase in methylation during SAM‟s biotypification from SA1. Restriction site-specific fluorescent labelling is a novel technique that makes use of a fluorescently labelled adaptor, which binds to the sticky ends produced after the restriction of DNA using the isoschizomers HpaII and MspI. Although unable to detect or quantify methylation, RSSFL was able to detect trends in methylation. Various aspects of the DNA methyltransferases (DNMTs), which catalyse methylation, were also investigated. A homology search identified four putative RWA DNMT genes, namely DNMT1, DNMT2, DNMT3A and DNMT3B. Sequencing of these genes detected only one single nucleotide polymorphism between biotypes SA1 and SAM. Baseline DNMT expression, quantified using RT-qPCR, revealed significant differences in DNMT3A expression, which could be explained by the virulence of the respective biotypes. An antibody specific to 5-methylcytosine (5mC) was used to quantify both the DNMT protein activity (by detecting the relative number of methyl groups transferred by the DNMTs to a universal substrate) and the global 5mC levels, both of which did not differ significantly between the biotypes. The 5mC levels ranged from 0.1% to 0.16% and were in line with levels reported for numerous insects. Global hydroxymethylation levels were quantified using an antibody specific to 5-hydroxymethylcytosine (5hmC, a demethylation intermediate). Biotype SAM‟s 5hmC level was significantly higher than that of biotypes SA1, SA2 and SA3. Based on the results obtained, it is recommended that future studies of RWA methylation first perform RSSFL, followed by either MSAP or antibody-mediated methylation quantification (or both), depending on the needs of the specific study. The results also made clear the fact that methylation, and the removal thereof is related to differences in RWA virulence. Although many aspects of methylation were similar between the biotypes, local increases in methylation proved beneficial to the development of the highly virulent biotype SAM. During biotypification SAM also attained an increased ability to demethylate its genome, which affords this biotype greater flexibility to adapt to changing environments, by means of alterations in gene regulation. An increased demethylation capacity might therefore be a key contributory factor to increases in aphid virulence and hence biotypification.