Molecular Divergence And Gene Recombination Of Grapevine Leafroll Associated Virus 2

Grapevine leafroll is the most widespread viral diseases of grapevine. This disease occurs in almost all major grapevine growing areas in the world and adversely affects fruit quality and grape yields which lead to enormously economical losses. To date, at least 11 serologically distinct viruses have been reported and designated as Grapevine leafroll associated virus-1 to-9. Besides these, two new Ampelovirus isolates (GLRaV-Pr and-De) was just reported by Maliogka in 2008. All of these viruses belong to the family Closteroviridae, and of the nine GLRaVs, only GLRaV-2 belongs to the genus Closterovirus. GLRaV-1,3,4,5,6,8 and 9 are members of the genus Ampelovirus.In this study, RT-PCR were used to detect 70 Grapevine canes showing leafroll symptoms collected from three Grapevine growing areas:Fruit Research Institute, Chinese Academy of Agriculture Sciences,Institute of Fruit and Tea Science, Hubei Academy of Agricultural Sciences and Grapevine yard of huazhong agricultural university. After RT-PCR detection,11 samples were tested to be infected with GLRaV-2. All the 11 samples were rooted in greenhouse for further study. The obtained results are illustrated as follows:1. The partial HSP70h gene (432 bp) and complete CP gene (597 bp) of GLRaV-2 from grapevine 11 samples were amplified and sequenced. Results showed that both genes of the virus had very low intra-isolate sequence diversity and the value of genetic diversity were 0.000-0.008 for HSP70h and 0.002-0.010 for CP among randomly selected three clones within each isolate. Therefore, each of the 11 isolates could be considered comprising a single variant.2. Sequence analyzing showed that the CP genes among these isolates were relatively more conserved. In pair wise comparisons, the 11 CP sequences showed high identities ranging 97.3-100% at the nt level and 98.5-100% at the aa level. However, the high genetic variation was observed in the HSP70h. The identities of these isolates were between 74.5%-100% at the nt level and between 79.9%-100% at the aa level. Two HSP70h sequences from tr and hn possessing 99.3% identities with each other, showed the highest divergences from other 9 isolates, only 74.5%-75.8% and 79.9%-82.6% identities with other Chinese isolates at the nt and the aa level.3. The obtained sequences were analyzed using the BLAST (NCBI) program. The CP-based tree showed that all Chinese isolates were clustered into the same group represented by GLRaV-2-PN. However, the HSP70h-based tree showed that all these Chinese isolates were clearly clustered into two different groups, in which isolates tr and hn were clustered together with the isolate RG.4. The polymorphic region near 3'-NCR of the viral genome was analyzed for isolates zdq and db from HSP-based group PN and isolate tr from HSP-based group RG using primer GLR2-4 and GLR2-5. The fragment from tr were 99% identical to that from isolate RG and only 76.5-76.8% identical to that from isolate zdq and db.5. The possible recombination was investigated with the suite of programs included in the RDP3 package. Results showed that the sequences of tr and hn were recombination events generated from GLRaV-2-PN and GLRaV-2-RG lineages.