High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer

Amplified fragment length polymorphism (AFLP) analysis is currently the mos t powerful and efficient technique for the generation of large numbers of a nonymous DNA markers in plant and animal genomes. We have developed a proto col for high-throughput AFLP analysis that allows up to 70000 polymorphic m arker genotype determinations per week on a single automated DNA sequencer. This throughput is based on multiplexed PCR amplification of AFLP fragment s using two different infrared dye-labeled primer combinations. The multipl exed AFLPs are resolved on a two-dye, model 4200 LI-COR(R) automated DNA se quencer, and the digital images are scored using semi-automated scoring sof tware specifically designed for complex AFLP banding patterns (AFLP-Quantar (TM)). Throughput is enhanced by using high-quality genomic DNA templated o btained by a 96-well DNA isolation procedure.