Genetic diversity analysis of grapevine rupestris stem pitting-associated virus from grapevine by colony PCR-SSCP and -RFLP.

We have developed methods for detecting the genetic diversity of grapevine rupestris stem pitting-associated virus (GRSPaV) based on restriction fragment length polymorphism (RFLP) and single stranded conformational polymorphism (SSCP) in the 905 nt 3' sequence. The amplicons were cloned from six grapevine cultivars, and colony polymerase chain reaction (colony PCR) using recombination bacteria was subsequently analyzed by RFLP and SSCP. Four haplotypes of SSCP and six haplotypes of Sac I RFLPs were defined. The two methods had a 40% discrepancy rate in showing the degree of diversity. All clones were sequenced and were used to construct a phylogenetic tree with seven previously reported GRSPaV sequences. In the tree, all the newly acquired sequences were divided into three clusters, I, II, and III, which corresponded to haplotypes I, II, and III of SSCP, respectively. Haplotype IV of SSCP was grouped into cluster II. A recombination analysis showed that haplotype IV has undergone a recombination event. Together, these results indicate that the SSCP assay is useful for the rapid identification of genetic diversity of GRSPaV. This is the first report of an analysis of the large fragment of GRSPaV by colony PCR-SSCP. Keywords: grapevine; grapevine rupestris stem pitting-associated virus (GRSPaV); RFLP; SSCP; genetic diversity analysis.

Occurrence and genetic diversity analysis of apple stem pitting virus isolated from apples in China.

Two primer pairs were used to detect apple stem pitting virus (ASPV) using a reverse transcription (RT)-PCR test. 82 out of the 141 randomly collected samples, from ten orchards in five provinces and regions of China, tested positive. In the positive samples forty-five (55%) were infected by ASPV and two other viruses. The full coat protein (CP) and the triple gene block (TGB) gene 1, 2 and 3 of partial ASPV isolates were subsequently cloned. The nucleotide and amino acid identities of 39 CP sequence variants from 31 Chinese apple samples were compared with that of previously reported ASPV isolates and were 67.4-96.0% and 68.4-97.7%, respectively. All ASPV sequence variants from Chinese apples separated into two clades with CP- and TGB-based phylogenetic trees, whilst the grouping of TGB2 and TGB3 trees was the same. Three recombinants (FS06-2, X5-2, and XLF-C-2) for CP and six (TH2-5, X8-2, FS05-2, X6-2 and XLF-A-1) recombinants for TGB were identified from the Chinese apple isolates. Two recombinants were found in the TGB sequence of isolate XLF-A-1. The results presented here may assist in the development of a more comprehensive screening tool for apple viruses.

Detection and genetic variation analysis of grapevine fanleaf virus (GFLV) isolates in China.

To investigate the prevalence and genetic variation of grapevine fanleaf virus (GFLV) in China, 142 grapevine samples from 13 provinces and regions were tested using DAS-ELISA, RT-PCR, and nested RT-PCR. Of the samples, 38% tested positive for GFLV by DAS-ELISA, and 26.8% tested positive by RT-PCR and nested RT-PCR. Movement protein (MP) and coat protein (CP) gene PCR products were cloned and sequenced. The MP or CP nucleotide and protein sequences shared identities that ranged from 94.9% to 100%. Phylogenetic analysis revealed that Chinese GFLV isolates obtained in this study were distinct from the isolates reported in GenBank.

Molecular characterizations of two grapevine rupestris stem pitting-associated virus isolates from China.

The complete nucleotide sequences of two isolates of grapevine rupestris stem pitting-associated virus (LSL and JF) collected from grapevine of Xingcheng in Liaoning Province, China, were determined. The genomes of both LSL and JF were found to contain five open reading frames (ORFs). Sequence alignments showed that the genomic sequences of JF were 76.1 %-83.5 % identical to the other ten GRSPaV isolates that have been reported previously and that the nucleotide sequence identity of isolate LSL to other isolates was no more than 78 %. Phylogenetic analysis based on the complete genome sequence indicated that JF belongs to group III and that LSL belongs to a new group (group IV). The average genetic distances of the new genetic lineage from groups I, II and III were 0.34, 0.32 and 0.33, respectively.

Genetic diversity and recombination analysis of grapevine leafroll-associated virus 1 from China.

Grapevine leafroll-associated virus 1 (GLRaV-1) is one of the causal agents of grapevine leafroll disease (GLD). To investigate the prevalence and genetic variation of GLRaV-1 in China, 132 grapevine samples from 14 Chinese provinces and regions were tested using reverse transcription PCR (RT-PCR) and reverse transcription nested PCR (RT-nPCR). The samples included symptomatic and asymptomatic cultivars, and 36.4% of them tested positive for GLRaV-1. 'Beida' samples, previously identified as virus-free rootstocks, were also found to be infected with GLRaV-1 with an incidence of 40 . GLRaV-1 coat protein (CP) genes and heat-shock protein 70 (HSP70) genes from 43 GLRaV-1 isolates were selected and sequenced. Phylogenetic analysis of global CP and HSP70 gene sequences showed that all variants belonged to eight and seven groups, respectively. For CP gene sequence variants, group 4 was a new group that included only Chinese isolates. The results also showed that natural selection, rather than random processes, led to the evolution of variants belonging to CP gene sequence variants in group 2 and group 8. Furthermore, three new recombination events were identified in the GLRaV-1 CP gene population. This is the first report on the genetic variation of GLRaV-1 isolates in China, and this study will benefit grape clean-plant programs in China.