Genetic variation and population structure of Cucumber green mottle mosaic virus

Cucumber green mottle mosaic virus (CGMMV) is a single-stranded, positive sense RNA virus infecting cucurbitaceous plants. In recent years, CGMMV has become an important pathogen of cucurbitaceous crops including watermelon, pumpkin, cucumber and bottle gourd in China, causing serious losses to their production. In this study, we surveyed CGMMV infection in various cucurbitaceous crops grown in Zhejiang Province and in several seed lots purchased from local stores with the dot enzyme-linked immunosorbent assay (dot-ELISA), using a CGMMV specific monoclonal antibody. Seven CGMMV isolates obtained from watermelon, grafted watermelon or oriental melon samples were cloned and sequenced. Identity analysis showed that the nucleotide identities of the seven complete genome sequences ranged from 99.2 to 100%. Phylogenetic analysis of seven CGMMV isolates as well as 24 other CGMMV isolates from the GenBank database showed that all CGMMV isolates could be grouped into two distinct monophyletic clades according to geographic distribution, i.e. Asian isolates for subtype I and European isolates for subtype II, indicating that population diversification of CGMMV isolates may be affected by geographical distribution. Site variation rate analysis of CGMMV found that the overall variation rate was below 8% and mainly ranged from 2 to 5%, indicating that the CGMMV genomic sequence was conservative. Base substitution type analysis of CGMMV showed a mutational bias, with more transitions (A↔G and C↔T) than transversions (A↔C, A↔T, G↔C and G↔T). Most of the variation occurring in the CGMMV genome resulted in non-synonymous substitutions, and the variation rate of some sites was higher than 30% because of this mutational bias. Selection constraint analysis of CGMMV ORFs showed strong negative selection acting on the replication-associated protein, similar to what occurs for other plant RNA viruses. Finally, potential recombination analysis identified isolate Ec as a recombinant with a low degree of confidence.