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.