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Genetic Variability of Dasheen Mosaic Virus and Consequences for Detection

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Whole thesis(6.56 MB)
DsMV and BCMV seq.geneious(378.19 KB)
DsMV and BCMV seq.xml(672.17 KB)
DsMV consensus sequence alignment(529.76 KB)
FASTQC analysis guide(650.95 KB)

Date

2012

Authors

Supervisor

Higgins, Colleen

Item type

Thesis

Degree name

Master of Applied Science

Journal Title

Journal ISSN

Volume Title

Publisher

Auckland University of Technology

Abstract

The genus Potyvirus is one of the largest groups of plant viruses having RNA genomes. RNA viruses show high genetic diversity which can influence virus infectivity and host selection. In this study, deep sequencing profiles of a potyvirus, namely Dasheen mosaic virus (DsMV) were analysed to determine the level of genetic variation occurring in two different DsMV strains (DsMV-NZ1 and DsMV-B) and isolates (DsMV-NZ1.1, NZ1.2, B1.1 and B1.2). Potyviruses are one of the most important groups of plant viruses that cause severe damage to agricultural, horticultural and ornamental crops. Within this genus, DsMV is an important viral pathogen of cultivated aroids worldwide, causing a 40-60% reduction in total crop yield. Therefore, the precise and accurate identification of DsMV infection is important for developing appropriate control mechanisms. The first objectives of this study was to test the effectiveness of universal potyvirus primers in detection and identification of DsMV infection in taro. Three pairs of universal primers, namely HPFor-HPRev, CIFor-CIRev and NIb2F-NIb3R were tested in this study using reverse-transcriptase polymerase reaction (RT-PCR). After testing each of these primer pairs, it was concluded that these primers can be used for detection and identification of the DsMV infection of taro, but only under the experimental conditions used in this study. The second objective was to determine the level of variation within each strain/isolate of the DsMV. For that Illumina HiSeq 2000 platform was used for creating the deep sequencing profiles of each sample. The bioinformatics analysis revealed that the level of variation across all the samples was consistent and an average 20% of genetic variation was observed within each strain/isolate from the DsMV reference genome. It was also found that the level of variation within the isolates of a same strain was lower than variation between the strains. A phylogenetic tree constructed based on the whole genome nucleotide sequences of the DsMV showed that DsMV belongs to the Bean common mosaic virus (BCMV) group. The phylogenetic analysis also revealed that DsMV is most closely related to Vanilla mosaic virus (VaMV) than to any other virus of the BCMV group. The third objective of this study was to determine the level of variation within each region of the DsMV genome. The bioinformatics analysis of the Illumina sequencing data showed that the variation within the 5’ and 3’ untranslated regions was ~28% and 30%, respectively. With the protein coding genes, the P1 gene showed the highest level of genetic variation (~30%) while NIa-VPg gene showed the lowest (~16%). The low level of variation in the NIa-VPg gene suggested that this gene may be functionally very important for the DsMV while the high variation of the P1 may provide a mechanism for altering the host range. Each region needs to be investigated in more detail, which may help in identification of the regions of conservation within the potyviral genome. The results showed that genetic variability can interfere with the efficient detection and identification of potyviral infection in plants. The findings of this study, helped in understanding the pattern of genetic variability among potyviruses. and in developing a better understanding of the functionality of the potyviral genome. The findings of this study can also be used for identifying the conserved regions within the potyviral genome, which would be expected to assist designing better universal primers for precise and accurate detection of potyvirus infection in plants, as well as deepening or understanding of viral processes such as translation replication.

Description

Keywords

Molecular genetics, Potyvirus, Dasheen mosaic virus, Genetic variability, Potyvirus detection

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Permanent link

https://hdl.handle.net/10292/5170

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Masters Theses