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

Random amplified polymorphic DNA (RAPD) analysis has proven to be an e ffective procedure for molecular marker applications in plant breeding , although non-specific amplification may limit its utility in some sp ecies. 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 amplif ication buffer, suggesting that the restriction endonuclease can be ad ded 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. Genet ic segregation of two polymorphic markers tested in F-5 inbred progeny fit expected 1:1 ratios. These results suggest that heritable DNA mar kers may be generated with reduction in non-specific amplification whe n restriction-endonuclease digestion of template DNA is conducted as p art of the RAPD procedure.