Small RNA sequencing and RT-PCR detection of viral pathogens infecting Cynanchum chinense

Cynanchum chinense is used in traditional Chinese medicine. To identify the causes of mosaic symptoms in the leaves of C. chinense, sRNA deep sequencing technology with RT-PCR was used to identify pathogens causing mosaic symptoms in Taigu, Shanxi, and the resulting viral sequence was analyzed using bioinformatics. The results showed that a total of 15 039 334 original sequences were obtained from the C. chinense samples by sRNA deep sequencing technology. The contigs were compared and annotated with the virus database in NCBI, and the results showed that they were alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). The complete sequences of the coat protein (CP) and movement protein (MP) genes of AMV and CMV were cloned using specific primers and named AMV-BR and CMV-BR, respectively. Through sequence alignment analysis, it was found that the amino acid sequence and nucleotide sequence of AMV-BR CP were 100% higher than those of AMV isolates AMV-Gyn (MH332899), Dich-rep (MW835989), and VIC-320 (MF075254), respectively. The amino acid sequence and nucleotide sequence of MP have the highest similarity with AMV isolate AMV-Gyn (MH332899) at 99.3%. The amino acid and nucleotide sequences of CMV-BR CP had the highest similarity with the CMV isolate YA17 (MH119159) in CMV subgroup IA (100% and 99.1%, respectively), and the MP amino acid and nucleotide sequences had the highest similarity with the CMV subgroup I isolate PV-0185 (ON013887) (98.2% and 96.4%, respectively). Phylogenetic analysis of the MP and CP amino acid sequences showed that CMV-BR belongs to the CMV subgroup I. This is the first time that AMV and CMV have been detected in C. chinense in Shanxi Province. This study will help to better understand the molecular evolution of AMV and CMV and provide a theoretical basis for preventing and treating C. chinense viral diseases.