侵染地黄的瓜类褪绿黄化病毒基因组序列克隆及系统进化分析

Complete nucleotide sequence and phylogenetic analysis of the cucurbit chlorotic yellows virus infecting Chinese foxglove Rehmannia glutinosa

To develop virus-free Chinese foxglove Rehmannia glutinosa and control the cucurbit chlorotic yellows virus, 60 leaf samples with typical symptoms of mosaic, yellowing and chlorotic were collected from the main production region including Wenxian, Wuzhi and Yuzhou City of Henan Province. Mixed samples were further analyzed with high-throughput sequencing (HTS). Sixty samples were detected by using RT-PCR with primer pairs CCYV-CPF/CPR and CCYV-P22F/P22R. The genomic nucleotide sequences of CCYV infecting R. glutinosa were amplified and cloned by using nine primer pairs. Molecular variability was analyzed by using DNAMAN 6.0 software and phylogenetic trees were constructed using MEGA X 10.0. The result of HTS showed that BBWV2, ReMV, YoMV and CCYV were detected from the 60 leaf samples of R. glutinosa from Henan Province. CCYV was detected from 22 of the 60 samples with a detection rate of 36.7%. The nucleotide and amino acid identities of CP gene were respectively 99.3%-100.0% and 98.4%-100.0% in 21 CCYV-Rg isolates, 94.6%-100.0% and 96.8%-100.0% between CCYV-Rg isolates and other isolates. The nucleotide and amino acid identities of P22 gene were respectively 98.4%-100.0% and 96.3%-100.0% in 17 CCYV-Rg isolates, 96.1%-100.0% and 94.1%-100.0% between CCYV-Rg isolates and other isolates. Phylogenetic analysis based on nucleotide sequences of CP and P22 genes demonstrated that these isolates were grouped into two clades. The genome of CCYV R. glutinosa isolate (CCYV-Rg) consist of two segments and the length of RNA1 and RNA2 segments are 8 607 and 8 041 nt, respectively. The genome encode 12 proteins and their nucleotide sequences are highly conserved. The CCYV-Rg isolate had 99.7%-100.0% (RNA1 and RNA2 segments) nucleotide identities with other isolates deposited in GenBank. Phylogenetic analysis based on genome showed that the nucleotide sequences of CCYV-Rg isolate was grouped into one cluster together with CCYV isolates from other hosts but not with other virus species in crinivirus.