Comparing the methylomes of two genealogically linked Russian wheat aphid biotypes using whole genome bisulfite sequencing

du Preez, Pieter Herodus (2020-03)

Thesis (MScAgric)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae), commonly known as the Russian wheat aphid (RWA), is an economically important cereal pest. Although it does not spread any plant viruses, the severe symptoms caused by RWA feeding poses a significant threat to world wheat production. Commercial wheat cultivars, resistant to RWA, have been developed and are effective at preventing yield losses. However, new, more virulent RWA biotypes (morphologically similar aphid populations, with the ability to successfully feed on previously resistant cultivars) are continuously emerging, leading to a breakdown in resistance. The molecular mechanisms driving biotypification (the development of new biotypes) have not been identified yet. It has been proposed that an epigenetic modification, such as DNA methylation, might a possible means whereby biotypification might occur. The aim of this study was to explore the possible link between levels of DNA methylation and virulence in the RWA, by performing a whole genome bisulfite sequencing (WGBS) analyses on the South African biotypes SA1 and SAM. Together, SA1 and SAM form a good model for the study of virulence as they are closely related, yet at opposite ends of the virulence scale. The overall trends in RWA DNA methylation, observed in this study, correlates with what has previously been reported in insects: genic bodies, especially exons, are the most methylated regions in the genome, with most of the methylation occurring at CpG sites. The ratio of observed to expected CpG sites in a region has been used to infer levels of methylation, as increased methylation has been correlated to a decrease in CpG abundancy; In this study, however, no correlation was found between CpG abundancy and DNA methylation level. This technique, therefore, is not applicable for insect genes. Using the generated WGBS data, 148 genes were found to be differentially methylated between the two biotypes. The relative expression of five of these genes, which were selected based on gene ontology and the degree of differential methylation, along with that of DNA methyltransferase 3 (DNMT3) and ten-eleven translocation enzyme (TET), was quantified and compared between biotypes at 0, 6, and 48-hours after performing host-shifts from susceptible to resistant host plants. DNMT3 is the enzyme responsible for the establishment of DNA methylation, while the TET enzyme catalyses the first step of the process of demethylation. The time points were selected to correlate with defence responses elicited from the host plants. While no clear pattern could be observed in the differences in relative expression of differentially methylated genes between biotypes, or within a biotype between time points, a major increase in expression of both DNMT3 and TET 6-hours after performing a host shift to the resistant cultivar Tugela Dn5 was observed in SAM. This seems to indicate an increased rate of methylation, demethylation, or both methylation and demethylation in SAM, while under stress, which might contribute to its increased ability to respond to, and overcome, plant defence responses.

AFRIKAANSE OPSOMMING: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae), algemeen bekend as die Russiese koringluis (RKL), is ‘n ekonomies relevante graanpes. Alhoewel dit geen plantvirusse versprei nie, bedreig die ernstige simptome van RKL-voeding steeds wêreld graanproduksie. Kommersiële koring kultivars met RKL-weerstand bestaan en is effektief in die verhoeding van opbrengsverliese. Nuwe, meer virulente RKL-biotipes (populasies wat morfologies identies is, maar wat suksesvol kan voed op voorheen weerstandige kultivars) word egter ewig aangetref end dit lei tot ‘n afbreek in weerstand. Die molekulêre meganismes wat biotipifisering (die ontwikkeling van nuwe biotipes) dryf, is nog nie geïdentifiseer nie. Daar is voorgestel dat ‘n epigenetiese modifisering, soos DNA-metilering, ‘n moontlike fasiliteerder van biotipifisering mag wees. Die doel van hierdie studie was om die moontlike verhouding tussen die vlakke van DNA-metilering en virulensie in die RKL te ondersoek, deur heel genoom bisulfiet volgorde bepaling op die Suid-Afrikaanse biotipes SA1 en SAM toe te pas.SA1 en SAM vorm saam ‘n goeie model vir die bestudering van virulensie, aangesien hulle naby verwant is, maar tog op teenoorgestelde ente van die virulensieskaal val. Die algehele eienskappe van DNA-metilering in die RKL stem ooreen met wat tot dusver in insekte gerapporteer is: die hoogste vlakke van metilering word in geniese streke, veral in eksone, aangetref en meeste van die metilering verskyn by CpG setels. Die verhouding tussen die verwagte en waargenome hoeveelheid CpG setels in ‘n streek word soms gebruik vir ‘n beraming van metilering vlakke, aangesien ‘n toename in metilering al gekorreleer is met ‘n afname in CpG-rykheid; in hierdie studie is geen korrelasie egter tussen die vlakke van DNA-metilering en CpG-rykheid waargeneem nie. Dus blyk dit nie of die hierdie tegniek bruikbaar is vir insekgene nie. Deur gebruik te maak van die gegenereerde volgorde data is 148 gene geïdentifiseer as differensieel gemetileer tussen die twee biotipes. Die relatiewe ekspressie van vyf van hierdie gene, geselekteer gebaseer op ontologie en die graad van metilering differensiasie, sowel as die van ‘DNA methyltransferase 3’ (DNMT3) en ‘ten-eleven translocation enzyme (TET) is gekwantifiseer 0, 6, en 48-uur na ‘n gasheerwisseling van ‘n vatbare, tot ‘n weerstandige plant. DNMT3 is die ensiem wat verantwoordelik is vir die stigting van DNA-metileringspatrone, terwyl die TET ensiem die eerste stap in die proses van demetilering kataliseer. Die tydstippe is gekies om ooreen te stem met die verdedigingsresponse van die gasheerplant. Geen patroon is waargeneem in die verskil in relatiewe ekspressie van differensieel gemetileerde gene tussen biotipes, of vir een biotipe tussen tydstippe nie. Daar is egter ‘n drastiese verhoging in die expressive van beide DNMT3 en TET waargeneem in biotipe SAM 6-uur na ‘n verskuiwing na Tugela Dn5. Dit dui moontlik op ‘n verhoogde tempo van metilering, demetilering, of beide metilering en demetilering in SAM gedurende stres, wat moontlik bydra tot SAM se verhoogde vermoë om te reageer op plantverdedigings en dit te oorkom.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/108353
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