conference schedule and program with abstracts - Horticulture ...

O-24
Metagenomic sequencing as a tool to elucidate etiology - the case of the virome of a
vineyard
B. Coetzee 1 , M-J. Freeborough 1 , H. J. Maree 1 , J-M. Celton 2 , D.J.G. Rees 2 , J.T. Burger* 1
1
Department of Genetics, Stellenbosch University, Private Bag X1 Matieland, South Africa; 2
Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville,
South Africa
*Corresponding author: jtb@sun.ac.za
Next generation high throughput sequencing is fast gaining credibility and popularity as a tool to
determine the genetic composition of environmental samples, mainly because of the all inclusive
and unbiased nature of analyzing completely unknown samples. Applications vary from studying
microbe populations of the oceans to diagnoses of bacterial infections in humans and even
elucidating the etiological complexity of virus diseases of grapevine, as described in this study.
Double stranded RNA was isolated from a sample consisting of 44 pooled, randomly selected
vines from a leafroll-diseased vineyard in South Africa, and used in a deep sequencing analysis
to build a census of the viral population. The dsRNA was sequenced in an unbiased manner
using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II, and
yielded 837 megabases of metagenomic sequence data. Multiple de novo assemblies were
performed with the assembler Velvet using different parameter settings. The scaffolds generated
by all these de novo assemblies were then combined into contigs using CAP3 contig assembler.
Contigs and scaffolds, from some of the de novo assemblies, were subjected to BLAST searches
against the NCBI databases and the top hit scores used for virus identification. Based on the
BLAST results, full-length genome sequences were selected from the NCBI database and used as
reference sequences in MAQ re-assembly analysis. Four known grapevine viral pathogens were
identified; as expected Grapevine leafroll-associated virus 3 was found to be the most prevalent,
followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine
virus E, a virus not previously reported in South African vineyards, was also identified in the
study. Moreover, viruses not previously identified in grapevine were detected. The second most
prevalent virus detected in the environmental sample was a member of the Chrysoviridae family,
similar to Penicillium chrysogenum virus. Sequences aligning to two other mycoviruses were
also detected. The identification of all the viruses present in the leafroll-diseased vineyard will
assist in the elucidation of the viral disease etiology. This methodology is not restricted to viral
diseases and can be applied to investigate the etiology of diseases caused by other pathogens.
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