Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Metagenomic studies that make use of next-generation sequencing (NGS) generate large amounts
of sequence data, representing the genomes of multiple organisms of which no prior knowledge is
necessarily available. In this study, a metagenomic NGS approach was used to detect multiple
novel mycoviral sequences in grapevine phloem tissue. Individual sequencing libraries of doublestranded
RNA (dsRNA) from two grapevine leafroll diseased (GLD) and three shiraz diseased (SD)
vines were sequenced using an Illumina HiScanSQ instrument. Over 3.2 million reads were
generated from each of the samples and these reads were trimmed and filtered for quality before
being de novo assembled into longer contigs. The assembled contigs were subjected to BLAST
(Basic Local Alignment Search Tool) analyses against the NCBI (National Centre for Biotechnology
Information) database and classified according to database sequences with which they had the
highest identity. Twenty-six putative mycovirus species were identified, belonging to the families
Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae and Totiviridae. Two of the identified
mycoviruses, namely grapevine-associated chrysovirus (GaCV) and grapevine-associated
mycovirus 1 (GaMV-1) have previously been identified in grapevine while the rest appeared to be
novel mycoviruses not present in the NCBI database. Primers were designed from the de novo
assembled mycoviral sequences and used to screen the grapevine dsRNA used for sequencing as
well as endophytic fungi isolated from the five sample vines. Only two mycoviruses, related to
sclerotinia sclerotiorum partitivirus S and chalara elegans endornavirus 1 (CeEV-1), could be
detected in grapevine dsRNA and in fungus isolates. In order to validate the presence of
mycoviruses in grapevine phloem tissue, two additional sequencing runs, using an Illumina
HiScanSQ and an Applied Biosystems (ABI) SOLiD 5500xl instrument respectively, were
performed. These runs generated more and higher quality sequence data than the first sequencing
run. Twenty-two of the putative mycoviral sequences initially detected were detected in the
subsequent sequence datasets, as well as an additional 29 species not identified in the first
HiScanSQ sequence datasets. The samples harboured diverse mycovirus populations, with as
many as 19 putative species identified in a single vine. This indicates that the complete virome of
diseased grapevines will include a high number of mycoviruses. Additionally, the complete genome
of a novel endornavirus, for which we propose the name grapevine endophyte endornavirus
(GEEV), was assembled from one of the second HiScanSQ sequence datasets. This is the first
complete genome of a mycovirus detected in grapevine. Grapevine endophyte endornavirus has
the highest sequence similarity to CeEV-1 and is the same virus that was previously detected in
fungus isolates using the mycovirus primers. The virus was detected in two fungus isolates,
namely Stemphylium sp. and Aureobasidium pullulans, which is of interest since mycoviruses are
not known to be naturally associated with two distinctly different fungus genera. Mycoviral
sequence data generated in this study can be used to further investigate the diversity and the
effect of mycoviruses in grapevine. / AFRIKAANSE OPSOMMING: Metagenomiese studies, wat gebruik maak van volgende-generasie volgordebepalingstegnologie,
het die vermoë om die genetiese samestelling van veelvoudige onbekende organismes te bepaal
deurdat dit groot hoeveelhede data genereer. Die bogenoemde tegniek was in hierdie studie
aangewend om aantal nuwe mikovirusse in die floëem weefsel van wingerd te identifiseer.
Dubbelstring-RNS was gesuiwer vanuit twee druiwestokke met rolbladsiekte en drie met shirazsiekte
en Illumina HiScanSQ instrument is gebruik om meer as 3.2 miljoen volgorde fragmente te
genereer van elk van die monsters. Lae-kwaliteit volgordes was verwyder en die oorblywende kort
volgorde fragmente was saamgestel om langer konstrukte te vorm wat met behulp van BLAST
soektogte teen die NCBI databasis geïdentifiseer kon word. Ses-en-twintig mikovirus spesies, wat
aan die families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae en Totiviridae behoort,
was geïdentifiseer. Twee van die geïdentifiseerde mikovirusse, naamlik grapevine-associated
chrysovirus (GaCV) en grapevine-associated mycovirus 1 (GaMV-1), was voorheen al in wingerd
gekry terwyl die res nuwe mikovirusse is wat tans nie in die NCBI databasis voorkom nie. Inleiers
was ontwerp vanaf die saamgestelde mikovirus basisvolgordes en gebruik om wingerd
dubbelstring-RNS sowel as swamme wat vanuit die wingerd geïsoleer is te toets vir die
teenwoordigheid van hierdie mikovirusse. Slegs twee mikovirusse, wat onderskeidelik verwant is
aan sclerotinia sclerotiorum partitivirus S en chalara elegans endornavirus 1 (CeEV-1), kon deur
middel van die inleiers in wingerd en swam isolate geïdentifiseer word. Twee addisionele
volgordebepalingsreaksies, wat gebruik gemaak het van die Illumina HiScanSQ en ABI SOLiD
5500xl volgordebepalingsplatforms, was gebruik om die teenwoordigheid van mikovirusse in
wingerd te bevestig. Groter hoeveelheid volgorde fragmente was geprodusser wat ook van
hoër gehalte was as dié van die eerste volgordebepalingsreaksie. Twee-en-twintig mikovirus
spesies kon weer geïdentifiseer word, sowel as 29 spesies wat nie in die eerste HiScanSQ
basisvolgorde datastelle gevind was nie. Die wingerdstokke wat in hierdie studie ondersoek was,
het hoë diversiteit van mikovirusse bevat aangesien daar tot 19 mikovirus spesies in enkele
wingerdstok geïdentifiseer was. Dit is aanduiding dat volledige virus profiele van siek
wingerdstokke aantal mikovirusse sal insluit. Die vollengte genoomvolgorde van voorheen
onbekende endornavirus was saamgestel vanuit een van die tweede HiScanSQ volgorde
datastelle. Dit is die eerste mikovirus wat in wingerd gevind word waarvan die volledige
genoomvolgorde bepaal is en ons stel die naam grapevine endophyte endornavirus (GEEV) voor
vir hierdie virus. Grapevine endophyte endornavirus is die naaste verwant aan CeEV-1 en is
dieselfde virus wat voorheen in wingerd dubbelstring-RNS en swam isolate gevind was deur
middel van die mikovirus inleiers. Swam isolate waarin GEEV gevind is, was geïdentifiseer as
Stemphylium sp. en Aureobasidium pullulans. Dit is van belang dat GEEV in twee swam isolate
gevind is wat aan verskillende genusse behoort aangesien hierdie verskynsel nog nie voorheen in
die natuur gevind is nie. Mikovirus nukleiensuurvolgordes wat in hierdie studie bepaal was kan gebruik word in toekomstige studies om die verskeidenheid en impak van mikovirusse in wingerd
verder te ondersoek. / National Research Foundation (NRF) / Stellenbosch University
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/80025 |
| Date | 03 1900 |
| Creators | Espach, Yolandi |
| Contributors | Burger, J. T., Maree, H. J., Mostert, L., 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 | xv, 75 p. : col. ill. |
| Rights | Stellenbosch University |
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