Polymerase Chain Reaction-Based Markers

Abstract

Restriction fragment length polymorphism (RFLP) was the first DNA-based marker, and it was once widely used in biology and, to some extent, plant breeding. But due to the need for high technical skill, considerable marker development work, and some other limitations, the search continued for more user-friendly DNA marker systems. With the discovery of polymerase chain reaction (PCR) technique, the new generation of PCR-based DNA markers was developed. Initially arbitrary primers of different sizes were used to amplify genomic DNA to generate fingerprints of different individuals. Randomly amplified polymorphic DNAs (RAPDs), DNA amplification fingerprinting (DAF), and arbitrary-primed PCR (AP-PCR) are examples of marker systems based on arbitrary primers. Amplified fragment length polymorphism (AFLP) marker system detects polymorphism due to the sequence variation in and around the recognition sites of restriction endonucleases and uses PCR for marker assay. Refinements in the DNA sequencing technology supported the discovery and development of marker systems, which exploit the sequence variation in specific fragments of DNA using the PCR technology. Sequence-tagged site (STS) markers, including microsatellite or simple sequence repeats (SSR) markers, are an example of this group. The SSR markers revolutionized the marker application in crop improvement in view of their abundance, codominant nature, user-friendliness, and other desirable features. But the development of SSR markers is expensive and complicated so that several other simpler PCR-based marker systems like sequence-related amplification polymorphism (SRAP; it uses open reading frame sequences), target region amplification polymorphism (TRAP; it uses expressed sequence tags), etc. were developed. In addition, some markers exploit variation in RNA sequences of a species: cDNA AFLP, cDNA-SSCP, etc. are examples of RNA-based markers. This chapter describes these and other PCR-based marker systems in some details in addition to introducing the technique of PCR.