Study On Seed-borne Viruses Of Raphanus And Brassica With Double-stranded RNA Genomes

In the long-term process of interaction game between viruses and their hosts, two lifestyles of viruses are established, acute infection and persistent infection. Persistent infection of viruses been largely neglected. In recent years, increasing number of studies concerning persistent infection and related viruses has beening arosen, bringing more and more attentions. Thus, a large class of viruses with dsRNA genomes has been found, mainly infecting fungi and plants, with persistent infection and vertical transmission manner. At present, these persistent dsRNA viruses are classified into five different families, Totiviridae, Partitiviridae, Chrysoviridae and the recently established Endornaviridae and Picobirnaviradae. Plants persistent viruses are known as the cryptic viruses. Previously, there is evidence that cryptic viruses may exist in Brassica crops, and there are several partitiviruses were reported in radish. In this study, we have investigated these seed-borne cryptic viruses in Raphanus and Brassica crops using dsRNA diagnostic technique, and established a new method that characterize the population genetic diversity of Raphanus and Brassica crops based on their dsRNA diversity. And we have obtained the genome sequences of the viruses corresponding to these dsRNAs. The taxonomic status and evolutionary meaning were analyzed further. The findings are as follows:1. A cryptic virus survey of26different varieties from7species of Brassica and Raphanus was done by using dsRNA diagnostic technique. DsRNAs in these crops were classified in five different groups, which were named ds1500, ds1600, ds1800, ds1900and ds3500according their size and patterns. These five group putatively correspond to five different cryptic viruses at least. Neither low temperature processing nor tissue culture regeneration of the crops in sterile environment could eliminate these dsRNAs. And no endophytic and symbiotic fungi were observed under light microscope in the leaf after trypan blue staining. These all excluded the possibility that these dsRNAs are related to fungi. The patterns of dsRNA are different among species, and different varieties, as well as with different individuals within the same variety. We conducted a statistical analysis of such patterns in different varieties, and the exhibiting frequency of different dsRNA group. And we found that the diversity of dsRNA in Raphanus cultivars is much higher than that in Brassica; ds1800and ds1900only specificly exist in Raphanus; ds1500exists in almost all Brassica and Raphanus cultivars; and differently, ds3500showed both in some Raphanus cultivars and some Brassica cultivars.2. The polymorphism of dsRNA was used to characterize the genetic distance of the Raphanus and Brassica cultivars based clustering analysis. We found that inter-cultivar dsRNA difference can only differentiate Raphanus and Brassica at the genus level, and both the intra-cultivar polymorphism of dsRNA and the combined inter-cultivar&intra-cultivar polymorphism can distinguish not only the Raphanus and Brassica at the genus level, but also at the cultivar level for the Raphanus and partial Brassica cultivars with a higher degree of reliability. These population genetic results are consistent with the previous hybridizational orgin hypothesis of Raphanus. This new molecular marker method can be useful to distinguish radish germplasm resources for their high dsRNA diversity. This method also can be used in Brassica and the related taxa in Cruciferae. The application in other orgnisms was also disscussed for the widely distribution of the persitent dsRNA virus. It could be expected that this method will provided a new perspective for the study of the genetic structure and evolutionary relationships.3. DsR3500was found to be consisted with three large dsRNAs related to a chrysovirus whoes whole sequence were determined by using the modified single primer amplification technique. Sequence analysis revealed that these three dsRNA molecules with the sizes of3638,3517and3299bp, all shared conserved untranslated terminal regions, and each contained a major open reading frame encoding the chrysoviral replicase, capsid protein and the putative protease respectively. Isometric virus-like particles (VLP) of about45 nm in diameter were isolated from the infected radish plants. Northern blotting indicated that these dsRNAs are encapsidated in the VLPs. Further, no fungi mycelia were observed in the radish leaf tissues stained by trypan blue dye. The phylogenetic analysis based on the alignment between RasCV1genomic sequences and other closely related sequences revealed that RasCV1should be classified as a new species of the family Chrysoviridae and might form a plant chrysovirus taxon with other putative plant chrysovirus. These results all support that RasCV1most likely to be a plant chrysovirus other than a chrysovirus hosting in fungi symbiosis with radish. Exhaustive blast analysis of RasCV1and AmaCV found that chrysovirus-like virus might widely exist in eudicot and monocot plant, and endogenization into the plant genome of chrysovirus segments once happened.4. Selected dsRNAs varying from1500bp to2000bp in Brassica and Raphanus were also cloned using single primer application technique. A1915bp dsRNA molecule related to a novel virus corresponding to ds1900in radish, the coat protein segment of a novel virus related to ds1500in B. rapa cv. Youdongercaitai, the full genome sequences of a novel virus related to ds1500in Sinapis alba cv. Lintao were obtained. These sequences’ encoding capacity was analysis, then NCBI Genbank database searches were done for functional analysis. We found that they correspond to three different partitivirus, named Raphanus sativus partitivirus1, Brassica rapa cryptic virus1and Sinapis alba cryptic virus1respectively. Phylogenetic analysis showed that these three viruses belong to the different subgroups in Partitiviridae. The evolutionary relationship with other reported radish cryptic viruses were also disscussed.5. Typical partitivirus contains two dsRNA molecules, which encodes the viral replicase and coat protein respectively. In recent years, some tri-partite partitiviruses containing three dsRNAs were found. The origin of the third dsRNA was rarely investigated. We selected the second dsRNA (dsRNA2) and the third dsRNA (dsRNA2)(also including the putatively protein encoding by them) of five reported tri-partite partitivirus for further phylogenetic analysis. It showed that each dsRNA2is closely related to the dsRNA3in the same virus, other than clustering into two groups, dsRNA2group and dsRNA3group. This indicated that dsRNA2and dsRNA3in a single tri-partite partitivirus originated from a common ancestor. Finally, we propose a model of the origin of the third dsRNA of tri-partite partitivirus with respect to the co-infecting reassortment view, which reveals a novel evolutionary path of persistent virus.