Abstract
Development of agricultural biotechnology requires rapid and convenient methods for crop plant genotyping. Real-time PCR is sensitive and reliable, and has been a routine technique in plant research. However, its application is limited by the cumbersome DNA template preparation procedures. We tested three PCR master mixes for direct amplification of crude seed DNA extracts without extensive purification. One mix had higher resistance to plant-derived PCR inhibitors and was shown to be applicable to various important crop plants. Furthermore, this method is capable of detecting single-copy genes from 2 mg pieces of seeds repetitively. Meanwhile, melting curve analysis could detect amplicons directly without electrophoresis manipulations. Taken together, this direct real-time PCR method provides a rapid and convenient tool for seed genotypic screening in crop plants.
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