Efficiency of randomly amplified polymorphic DNA to sequence characterized amplified region marker conversion and their comparative polymerase chain reaction sensitivity in cucumber.

摘要

The conversion of randomly amplified polymorphic DNA (RAPD) markers to sequence characterized amplified region (SCAR) markers, and the effects of differing polymerase chain reaction (PCR) conditions were studied in cucumber (Cucumis sativus). Attempts were made to clone and sequence 75 RAPD PCR products to produce SCAR primers (16-22 nucleotides) designed to amplify original RAPD PCR products. The influence of template DNA source, purity, and concentration, MgCl2 concentration, Taq polymerase source, and type of thermocycler upon RAPD and SCAR marker performance was evaluated. Conversion of RAPD to SCAR markers was not universally successful, and SCAR primers reacted differently to varying PCR conditions. Only 48 (64%) of 75 RAPD markers were successfully converted to SCAR markers and 11 (15%) of these reproduced the polymorphism observed with the original RAPD PCR product. Moreover, some SCAR primer pairs produced multiple polymorphic PCR products. The band intensity of SCAR markers were brighter than their corresponding RAPD markers with only one exception. The SCAR markers examined were less influenced by MgCl2 concentration than their corresponding RAPD markers. However, some SCAR markers were more sensitive to reaction impurities than theirRAPD counterparts and SCAR markers tended to be less readily visualized (decrease in frequency of visible PCR product) with low concentrations (1 and 2 mM) of template DNA than their corresponding RAPD markers. Neither the source of Taq nor the type ofthermocycler used affected the performance of SCAR and RAPD markers. These data suggest that although SCAR markers may demonstrate enhanced performance over the RAPD markers from which they are derived, careful consideration must be given to both the costs and potential benefits of SCAR marker development in cucumber.