ISHS Acta Horticulturae 472: XVII International Symposium Virus and Virus-Like Diseases of Temperate Fruit Crops
COMPLETE NUCLEOTIDE SEQUENCE OF LITTLE CHERRY CLOSTEROVIRUS AND VIRUS DETECTION BY POLYMERASE CHAIN REACTION |
| Authors: | W. Jelkmann, R. Keim-Konrad, M. Vitushkina, B. Fechtner |
| Abstract:
The complete nucleotide sequence of a German isolate (UW1) of little cherry closterovirus (LChV) was determined. It consists of 16,934 nucleotides. This sequence was resolved from 88 cDNA clones that were obtained from the high molecular weight dsRNA associated with the disease. The terminal sequences were obtained by applying a 5'/3' RACE system to dsRNA. The sequence encompasses (from 5' to 3') eight open reading frames with molecular masses of 318 kDa (la/lb fusion product), 4kDa, 70 kDa (HSP70), 61 kDa, 46 kDa (CP), 76 kDa (CPd), 21 kDa and 27 kDa, respectively. In order to develop a sensitive PCR detection test, leaf tissue of symptomatic 'Canindex' and 'Sam' indicator plants grown in the field was collected in the autumn. Other samples were taken from fresh shoots forced during the dormant season. The samples were ground in liquid nitrogen and total RNA extracted using the Qiagen RNeasy method. Samples of isolates collected from England, The Netherlands, Switzerland and from several locations in Germany were subjected to RT-PCR. An amplification product of the expected size (276 bp) was observed in all samples that were positive for the disease by indexing on woody indicators. Cloned and sequenced RT-PCR fragments from nine isolates revealed sequence heterogeneity and variable length, suggesting nucleotide insertions or deletions in comparison to the UW1 isolate. | |
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