RAPD and other PCR-based analyses of plant genomes using DNA extracted from small leaf disks

Abstract

A nondestructive, early DNA diagnostic system to implement marker-assisted selection in plant breeding programs has been developed. The main components of the system are a rapid and simple DNA microextraction method and fast DNA polymorphism analyses based on site-specific or arbitrary DNA amplification. A small disk (5 mm diameter) is collected from one cotyledon or the first leaf of a young seedling using a common paper punch. Disruption of plant tissues is done by enzymatic digestion of cell walls. This ensures protection from sample-to-sample contamination and uniform DNA yield. DNA isolated from the resulting protoplasts is sufficient to perform a minimum of five and a maximum of 20 PCR reactions/sample. Total DNA, nuclear DNA, and RNA can be analyzed selectively. The system has been tested successfully with eight major crops. Amplification products generated with DNA prepared with this quick procedure are equivalent to those obtained from CsCl-purified DNA. Up to 120 plants can be treated in 2 days and the procedure lends itself to automation. Potential applications in plant breeding will be discussed.