ARS | Publication request: TRAP (TARGET REGION AMPLIFICAITON POLYMORPHISM), A NOVEL MARKER TECHNIQUE FOR PLANT GENOTYPING

Submitted to: International Congress of Plant Molecular Biology
Publication Type: Abstract Only
Publication Acceptance Date: March 1, 2003
Publication Date: June 23, 2003
Citation: HU, J., VICK, B.A. TRAP (TARGET REGION AMPLIFICAITON POLYMORPHISM), A NOVEL MARKER TECHNIQUE FOR PLANT GENOTYPING. INTERNATIONAL CONGRESS OF PLANT MOLECULAR BIOLOGY. 2003.

Technical 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 utilizes bioinformatics tools and EST database information to generate polymorphic markers around the targeted candidate gene sequences. This TRAP (Target Region Amplification Polymorphism) technique uses two primers of 18 nucleotides to generate markers. One of the primers, the fixed primer, is designed from the targeted EST sequence in the database, and the second primer, the random primer, is a random sequence but with either an AT- or GC-rich core to anneal with an intron or exon, respectively. The PCR amplification is run for the first five 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 about 50 scorable fragments with sizes ranging from 50 to 800 base pairs 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.