Double-stranded RNAs purified from the V2356 (‘Successa’) sour cherry source of the Shirofugen stunt disease (SSD) were sequenced using a 454 pyrosequencing multiplex approach. The 15,646 reads obtained were assembled into 279 contigs, 5 of which, totaling almost 16.9 kbp and 5,332 reads (34% of sample reads), showed high Blast scores and homology to Little cherry virus 1 (LChV1). The five contigs were further assembled manually into three supercontigs spanning the full LChV1 genome with only two small gaps (17 and 55 bases). Completion of the sequencing of the viral genome was performed using targeted polymerase chain reaction and primers designed from the contigs. No evidence for the presence of other viral agents in the V2356 source could be obtained in the remaining contigs or singletons. The V2356 LChV1 isolate is only ≈76% identical with the reference complete LChV1 sequences and, in particular, with the ITMAR isolate associated with the Kwanzan stunting syndrome. However, it is highly homologous (97 to 100% identity) in two short genome regions with divergent LChV1 from North America, providing the first complete sequence for such divergent isolates. Although not providing a definite proof, the failure to detect any other viral agent in the V2356 SSD source and the identification of LChV1 in a second, independent, source of the disease suggests that LChV1 isolates could be responsible for the SSD syndrome.
Development and characterization of thirty two microsatellite markers for the anchovy, Engraulis ringens Jenyns, 1842 (Clupeiformes, Engraulidae) via 454 pyrosequencing
