Searching for plant viral diversity by using 454-pyrosequencing

Genetic variability in populations of RNA viruses are a very important trait, which can result on the emergence of new more powerful and destructive strains for many plant viruses, e.g. Potato virus Y. Genomic sequencing has been greatly improved due to the generation of new sequencing technologies, such as the 4S4-GS-FLX Titanium series. Different methods of viral RNA extraction and sample preparation were evaluated for their capacity to generate the whole genome of a plant virus. Partial virus purification as a method of viral RNA extraction combined with a double-stranded cDNA sample preparation protocol was determined as the best methodology to obtain the whole genome of a plant virus in this study, generating a good number of virus reads and a reasonable coverage. This method together with amplicon sequencing was used for the study of genetic diversity in populations of Potato virus Y under different transmission bottlenecks. In order to determine the effect different types of transmission bottlenecks, via aphids and mechanical inoculation after a number of passages and infected tubers, have on the genetic diversity of viral population the number of different variants and their mutation frequency was determined from infected potato plants. Overall this study showed that PVY populations do exist as quasispecies and that mechanical transmission bottleneck was responsible for the maintenance or maybe an increase of the genetic diversity existent in the population of two isolates of this virus.EThOS - Electronic Theses Online ServiceGBUnited Kingdo