Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/2003
Publication Date: 9/1/2003
Citation: HU, J., VICK, B.A. TARGET REGION AMPLIFICATION POLYMORPHISM: A NOVEL MARKER TECHNIQUE FOR PLANT GENOTYPING. PLANT MOLECULAR BIOLOGY REPORTER. 2003. 21:289-294 Interpretive Summary: This paper describes a new technique for molecular marker development in plants. Molecular markers are small pieces of DNA from the genome of an organism under investigation. The most popular way to generate molecular markers is to run a polymerase chain reaction (PCR) to amplify specific DNA sequences and visualize them with specialized equipment. Two of the widely used marker techniques, RAPD (random amplification polymorphic DNA) and AFLP (amplified fragment length polymorphism), have been playing an important role in the structural and functional genomics of animal, plant, and microbial species. They became popular because they do not require prior sequence information to run PCR. This was really an advantage at the time of the invention when sequence information was scarce for most of the economically important species. During the past decade, the advent of high throughput sequencing technology has generated abundant information on the DNA sequences for the genomes of many species. This includes the completion of the draft of the whole genome sequences for human, the model plant Arabidopsis, for one of the most important food crops, rice, and for many microbial species. In addition, the partial DNA sequences (expressed sequence tags, or ESTs) of genes of many other important crop species have now been generated, and powerful bioinformatics tools have annotated thousands of sequences as putative functional genes. The task of bridging this DNA sequence information with particular phenotypes relies on molecular markers. Improved from the recently published SRAP (sequence-related amplified polymorphism), the PCR-based TRAP (target region amplification polymorphism) technique described in this paper is both rapid and efficient. In addition, the TRAP technique utilizes bioinformatics tools and the EST database information to generate markers for specific gene sequences. Therefore, the TRAP method should be useful for genotyping germplasm and in generating markers associated with desirable agronomic traits in crop plants for marker-assisted breeding.
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 targeted candidate gene sequences. This TRAP (Target Region Amplification Polymorphism) technique was modified from a recently published sequence-related amplified polymorphism (SRAP). The SRAP technique uses two random primers; thus, it does not use existing sequence information. In contrast, the TRAP method uses one fixed and one random primer of 18 to 20 nucleotides to generate markers. The fixed primer is designed from a targeted EST sequence in the publicly accessible database, and 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 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 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.