| Geminiviruses are single-stranded DNA plant viruses with a geminate particle morphology. They have caused serious damages worldwide in recent years. Some monopartite geminiviruses have been shown to be associated with single-stranded DNA satellite, termed DNAβ, most of which are shown to be an essential pathogenicity determinant for inducing typical symptoms in hosts. Satellite DNAβand geminivirus form a geminivirus/satellite DNAβdisease complex and intensifies damages. Populations of plant RNA viruses are intrinsically genetically heterogeneous, which are designated as "quasispecies". Some geminiviruses are also proved to have a high level of population variation. DNAβhas been shown to be co-evoluted with their cognate viral DNA-A. To identify the genetic structure and population variation of satellite DNAβ, Nicotiana glutinosa and Nicotiana benthamianaplants were inoculated with Tobacco curly shoot virus (TbCSV) isolate Y35(Y35) and Tomato yellow leaf curl China virus (TYLCCNV) isolate Y10 associated with their cognate satellite DNAβ(Y35βor Y10β), respectively, by Agrobacterium tumefaciens mediation technique. Based on the molecular clone of DNAβ, the genetic structures and population variation of Y35βand Y10βwere analyzed.Three Y35P populations and eight Y10βpopulations were obtained from N. glutinosa and N. benthamiana at 60 or 120 days post inoculation (dpi). Genetic structures and population variation of these populations were analyzed. The results demonstrated that, both the populations of Y35βand Y10βwere heterogeneous, consisting of a series of sequences that were not identical but closely related to the consensus sequence. About 56.3 to 90 percent clones of Y35βpopulations mutated, and the mutation frequencies, were from 6.00×10-4 to 1.27×10-3.Nearly all clones of Y10βpopulations mutated and the mutation frequencicsof Y10βpopulations were from 2.37×10-3 to 3.20×10-3. These results indicated that the variation level of Y35βpopulations was lower than that of Y10βpopulations. The mutation frequencies of Y35βpopulations in N. glutinosa at 60 and 120 dpi were much varied, but the mutation frequencies of other populations for the same DNAβcollected from different hosts or at different period in the same host have no obvious differences.Mutations were distributed throughout the whole genome sequence of DNAβ. Among the SCR, A-rich andβC1 regions of DNAβ, most mutation sites were found at A-rich region, especially the section from 888 to 901 nucleotide site in Y10βgenome. Mutations occurred atβC1 were obviously less frequent than that at A-rich. Both Y35βand Y10βhad the least mutation sites at SCR, proving this region was most conservative in the genome of DNAβ.Substitution, deletion and insertion mutations were detected in DNAβpopulations. The proportion of base transition was significantly higher than that of base transversion in all of the DNAβpopulations. The predominant mutation types of DNAβvaried with different hosts and times. This indicated that the mutation types could be different although the variation level of DNAβin the same host was maintained at a constant level. The base transitions from G to A, from T to C or from C to T were rather frequent in the mutation types of Y35βpopulations while deletion and insertion mutations occurred much less frequent. On the other hand, the dominant mutation types in Y10βpopulations included base transitions from A to G, from T to C, and deletion of A and G. More base deletion and insertion mutations were detected in Y10βthan in Y35βpopulations, and most of the deletion or insertion of A or G occurred at the 888-901 nucleotide site in A-rich region. The same mutation type was observed at the same nucleotide position of several DNAβclones.βC1 region was the only functional gene identified for DNAβ. Analysis of base mutations and aminoacid mutations inβC1 indicated that the proportion of non-synonymous mutations was higher than that of synonymous mutations both for Y35βand Y10β, and all synonymous mutations occurred at the third site of start codon Mutations were uniformly distributed among the three sites of codon in Y10βpopulations but they occurred mostly at the third site of codon in Y35βpopulations.To conclude, both the populations of Y35βand Y10βhad a high level of population variation and they were consistent with the natures of "quasispecies". The population variation level of DNAβwas much higher than its assistant viruses, and the high mutation frequencies are probably correlated to the frequent mutations in A-rich region. The rapid mutation of DNAβcould be thought of a factor in improving the host adaptability of the geminivirus-DNAP disease complex and intensifying damages. These findings on the genetic structures and population variation of DNAβin different hosts may have important implications for understanding the evolution models and epidemics of geminiviruses and satellite DNAβs. |
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