Abstract
The advent of large-scale DNA sequencing technology has generated a tremendous amount of sequence information for many important organisms. We have developed a rapid and efficient PCR-based technique, which uses bioinformatics tools and expressed sequence tag (EST) database information to generate polymorphic markers around targeted candidate gene sequences. This target region amplification polymorphism (TRAP) technique uses 2 primers of 18 nucleotides to generate markers. One of the primers, the fixed primer, is designed from the targeted EST sequence in the database; the second primer, the arbitrary primer, is an arbitrary sequence with either an AT-or GC-rich core to anneal with an intron or exon, respectively. PCR amplification is run for the first 5 cycles with an annealing temperature of 35°C, followed by 35 cycles with an annealing temperature of 50°C. For different plant species, each PCR reaction can generate as many as 50 scorable fragments with sizes ranging from 50–900 bp when separated on a 6.5% polyacrylamide sequencing gel. The TRAP technique should be useful in genotyping germplasm collections and in tagging genes governing desirable agronomic traits of crop plants.
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Abbreviations
- EST:
-
expressed sequence tag
- nt:
-
nucleotide
- PCR:
-
polymerase chain reaction
- TRAP:
-
target region amplification polymorphism
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Hu, J., Vick, B.A. Target region amplification polymorphism: A novel marker technique for plant genotyping. Plant Mol Biol Rep 21, 289–294 (2003). https://doi.org/10.1007/BF02772804
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DOI: https://doi.org/10.1007/BF02772804