ENHANCEMENT OF RAPD ANALYSIS BY RESTRICTION-ENDONUCLEASE DIGESTION OF TEMPLATE DNA IN WHEAT

Random amplified polymorphic DNA (RAPD) analysis has proven to be an effective procedure for molecular marker applications in plant breeding, although non-specific amplification may limit its utility in some species. The objective of this study was to determine the effectiveness of restriction-endonuclease digestion of template DNA for elimination of non-specific amplification and generation of heritable RAPD markers. Restriction endonucleases digested wheat DNA to completion in amplification buffer, suggesting that the restriction endonuclease can be added directly to the reaction mixture prior to amplification. A 1-h 37 degrees C step was programmed into the thermal cycler for restriction-endonuclease digestion which was followed immediately by amplification. Non-specific amplification was reduced and DNA marker patterns were altered in digested samples when compared to undigested samples. Genetic segregation of two polymorphic markers tested in F-5 inbred progeny fit expected 1:1 ratios. These results suggest that heritable DNA markers may be generated with reduction in non-specific amplification when restriction-endonuclease digestion of template DNA is conducted as part of the RAPD procedure.