Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) is globally associated with latent infection in commercial apple trees. Little
is known about this plant-‐virus interaction. This study made use of
next-‐generation sequencing to investigate the effect of virus-‐infection on the expression of the different small RNA (sRNA) species
namely, miRNAs, nat-‐siRNAs, phasiRNAs, rasiRNAs, tRNA-‐derived
sRNAs and vsiRNAs. Broad and narrow size-‐range datasets were generated using sRNA libraries prepared from total and size-‐selected
RNA, respectively. Through bioinformatic data analyses, 130 genomic
loci were predicted to give rise to miRNAs, 85 of which were novel
MIR genes. Targets were predicted for the majority of miRNAs, a
few of which could be validated with a publicly available degradome
dataset. Cis-‐ and trans-‐natural antisense transcripts (NATs) were
identified, of which only the latter were highly enriched for sRNAs
in their overlapping regions. Transcript as well as genomic regions
were also identified that can give rise to phasiRNAs. For 25 of these
loci an in-‐phase miRNA target site was identified, half of which could
be validated with the degradome dataset. Nearly all apple repeat sequences in Repbase were associated with sRNA synthesis. sRNAs
derived from both ends of mature tRNAs were identified. These
sRNAs corresponded to tRFs and tRNA-‐halves. Reads associated
with tRNA-‐halves were prominent in the broad range datasets.
sRNAs, originating from the central regions of tRNAs, were also
observed. Analysis of the vsiRNAs suggested the presence of two
ASGV genetic variants in two of the samples, while the third sample
was infected with only one variant. Comparison of the vsiRNA profiles
generated from the two datasets highlighted the influence of library
preparation on the interpretation of results. Differential expression
analysis of the identified apple sRNA species showed no variation
between healthy and infected plants, except for the tRNA-‐derived
sRNAs, which did show altered expression levels. Taken together,
the various sRNA species characterised in this study significantly
extended the existing knowledge of apple sRNAs and provide a
broad platform for future functional studies in apple. This study
also presents the first and most comprehensive report on sRNAs
involved in ASGV infection in apple. / AFRIKAANSE OPSOMMING: Appel gleufstam virus (ASGV) word wêreldwyd geassosieer met latente infeksie in kommersiële appelbome. Min inligting oor hierdie plant-‐virus interaksie is beskikbaar. Hierdie studie het van volgende-‐generasie volgordebepaling gebruik gemaak om die effek van virusinfeksie
op die uitdrukking van verskillende klein RNA (sRNA) spesies, nl.
miRNAs, nat-‐siRNAs, phasiRNAs, rasiRNAs, tRNA-‐afkomstige sRNAs
en vsiRNAs, te ondersoek. Datastelle met breë en smal grootte-‐verspreiding is gegenereer m.b.v. sRNA biblioteke wat onderskeidelik
voorberei is vanaf totale RNA en RNA van ‘n bepaalde grootte.
Deur middel van bioinformatiese data-‐ontleding is 130 genomiese
loci voorspel wat aanleiding kan gee tot miRNAs, waarvan 85 nuwe
MIR gene is. Teikens is voorspel vir die meerderheid van die miRNAs
en 'n aantal daarvan kon bevestig word m.b.v. 'n publiek-‐beskikbare
degradoom datastel. Cis-‐ en trans-‐natuurlike antisense transkripte
(NATs) is geïdentifiseer, waarvan slegs die laasgenoemde verryk
was vir sRNAs in hul oorvleuelende areas. Transkrip sowel as
genomiese areas, wat aanleiding kan gee tot phasiRNAs, is ook
geïdentifiseer. Vir 25 van hierdie loci is 'n in-‐fase miRNA teiken
geïdentifiseer, waarvan die helfte bevestig kon word met die degradoom datastel. Byna al die appel herhalende volgordes in
Repbase was geassosieer met sRNA sintese. sRNAs afkomstig van
beide kante van volwasse tRNAs is geïdentifiseer. Hierdie sRNAs
het ooreengestem met tRFs en tRNA-‐helftes. Volgordes geassosieer
met tRNA-‐helftes was prominent in die breë grootte-‐verspreiding
datastelle. sRNAs, afkomstig van die sentrale dele van tRNAs, is
ook waargeneem. Ontleding van die vsiRNAs het die teenwoordigheid
van twee ASGV genetiese variante in twee van die monsters aangetoon, terwyl die derde monster met slegs een variant geïnfekteer was. Die vergelyking van vsiRNA profiele, gegenereer
vanaf die twee datasteltipes, beklemtoon die invloed van biblioteek
voorbereiding op die interpretasie van resultate. Ontleding van die
differensiële uitdrukking van die geïdentifiseerde appel sRNA spesies
het geen verskil tussen gesonde en geïnfekteerde plante getoon
nie, behalwe vir die tRNA-‐afkomstige sRNAs, wat wel verandering die
vlak van uitdrukking getoon het. Die verskillende sRNA spesies wat
in hierdie studie geïdentifiseer is, het die bestaande kennis van appel
sRNAs aansienlik uitgebrei en bied 'n breë platform vir toekomstige
funksionele studies in appel. Hierdie studie bied ook die eerste, en
mees omvattende verslag oor sRNAs betrokke in ASGV infeksie in
appel.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/95828 |
| Date | 12 1900 |
| Creators | Visser, Marike |
| Contributors | Burger, Johan T., Maree, Hans J., Rees, D. Jasper G., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xii, 93 p. : ill. |
| Rights | Stellenbosch University |
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