A rapid PCR-based method for genetically mapping ESTs

A simple, semi-automatable procedure was developed for converting expressed sequence tags (ESTs) into mappable genetic markers, The polymerase chain r eaction is used to amplify regions immediately 5' or 3' to the coding regio ns of genes in order to maximise sequence variability between alleles. Frag ment length and nucleotide substitution polymorphisms among amplified allel es can be detected using either ethidium bromide staining or automated lase r-based fluorescence. A 6% non-denaturing acrylamide gel, analysed with an ABI 377 DNA sequencer, proved capable of resolving homoduplexes and heterod uplexes formed between amplified alleles containing nucleotide substitution s as well as resolving allelic length differences. With this approach 75% o f 60 ESTs from a range of Pinus species could be genetically mapped in each of three pedigrees from P. radiata and P. taeda. Furthermore, three or fou r alleles were detected in each pedigree for 42% of the EST markers.