CLONING AND MAPPING OF VARIETY-SPECIFIC RICE GENOMIC DNA-SEQUENCES - AMPLIFIED FRAGMENT LENGTH POLYMORPHISMS (AFLP) FROM SILVER-STAINED POLYACRYLAMIDE GELS

An efficient technique for cloning DNA from silver-stained denaturing polyacrylamide gels was developed to allow the isolation of specific b ands obtained from selective restriction fragment amplification (SRFA) . This method proved as reliable as cloning radioactively labelled SRF A bands from the same gels. Rice DNA was used as a template, both with and without [P-32]dCTP, using the same PCR profiles. Amplified produc ts were separated using denaturing polyacryamide gel electrophoresis a nd visualized either by silver staining of gels or by autoradiography of P-32-labelled products. We cloned specific polymorphic SRFA bands d irectly from the denaturing polyacrylamide gels with one round of PCR amplification and confirmed that the sequences of the bands from silve r-stained gels were identical to the corresponding P-32-labelled bands . The bands that were chosen represented amplified fragment length pol ymorphisms (AFLPs) between japonica and indica rice varieties. We stud ied the ability of two cloned AFLP bands to serve as heritable genetic markers by mapping them as RFLPs in an interspecific rice population and found that they represented single-copy DNA at unique loci in the rice genome.