Graduate Thesis Or Dissertation

Identification of Endogenous and Exogenous Pararetroviruses in Red Raspberry (Rubus idaeus L.) and Blueberry (Vaccinium corymbosum L.)

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  • The Pacific Northwest (Oregon and Washington in the United States and British Columbia in Canada) is one of the major producers of red raspberry (Rubus idaeus L.) and blueberry (Vaccinium corymbosum L.) in the world. The expansion of growing area with these crops has resulted in the emergence of new virus diseases that cause serious economic losses. The majority of viruses affecting plants (including blueberry and red raspberry) contain RNA genomes. In contrast, plant viruses with DNA genomes are relatively rare and most of the time ignored in virus surveys. The family Caulimoviridae is a group of plant pararetroviruses (reverse-transcribing viruses) with the ability to integrate their DNA into the host genome, resulting in complex molecular interactions that lead to inconsistencies in terms of detection and disease symptoms. Albeit, few studies have been conducted to determine the nature of plant pararetroviruses and their relationships with the associated host. To investigate the presence of pararetroviruses in blueberry and red raspberry, and their possible integration events, different plant material suspected to be infected with viruses was collected in nurseries, commercial fields and clonal germplasm repositories for a period of four years. For blueberry, using rolling circle amplification (RCA) a new virus was identified and named Blueberry fruit drop-associated virus (BFDaV) because of its association with fruit-drop disorder. Based on the genome organization and phylogenetics, BFDaV may represent a novel genus in the Caulimoviridae, and it has the largest genome known thus far in this virus family. Until recently, Rubus yellow net virus (RYNV) has been the only plant pararetrovirus reported to occur naturally in Rubus spp. Over the past few years, it was observed that several plants regarded as negative for RYNV, based on graft transmission onto indicator plants, produced positive results by PCR-based assays. Additionally, these plants tested positive for after virus clean-up using thermal therapy and meristem tip-culture, which has been shown to successfully eliminate RYNV when assays were based on graft-transmission. A series of methods including bioassays, reverse transcription-PCR, RCA and Southern blotting was used to demonstrate that RYNV not only exists as infectious particles, but also as an integrated element into the red raspberry genome. This was confirmed by a further characterization of the plant-virus junctions (virus genome inserts) by next-generation sequencing. Simultaneously, an atypical strain of RYNV was described, which differed in genome organization from the previously described isolate. In addition, it was shown that the new RYNV variant lacked aphid transmissibility. Altogether, this work highlights the importance of plant pararetroviruses and describes a methodology for their identification and discrimination between endogenous and exogenous viruses in red raspberry and other plant species.
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