A rapid PCR-based method for genetically mapping ESTs |
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| Authors: | S A Cato R C Gardner J Kent T E Richardson |
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| Institution: | (1) Forest Research, Private Bag 3020, Rotorua, New Zealand Fax: +64-7-3435444 e-mail: sheree.cato@forestresearch.co.nz, NZ;(2) School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand, NZ |
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| Abstract: | A simple, semi-automatable procedure was developed for converting expressed sequence tags (ESTs) into mappable genetic markers.
The polymerase chain reaction is used to amplify regions immediately 5′ or 3′ to the coding regions of genes in order to maximise
sequence variability between alleles. Fragment length and nucleotide substitution polymorphisms among amplified alleles can
be detected using either ethidium bromide staining or automated laser-based fluorescence. A 6% non-denaturing acrylamide gel,
analysed with an ABI 377 DNA sequencer, proved capable of resolving homoduplexes and heteroduplexes formed between amplified
alleles containing nucleotide substitutions as well as resolving allelic length differences. With this approach 75% of 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 four alleles were detected in each pedigree for 42% of the EST markers.
Received: 4 January 2000 / Accepted: 26 May 2000 |
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| Keywords: | Expressed sequence tag Polymerase chain reaction Fragment length polymorphism Single nucleotide polymorphism Heteroduplex Pinus radiata Pinus taeda |
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