Development of PCR-based assays for allelic discrimination in maize by using the 5′-nuclease procedure

We describe the development of non-electrophoresis-based PCR assays for the allelic discrimination at two linked loci flanking an important QTL controlling days to pollen shed in maize. The assays are based on the fluorogenic 5'-nuclease procedure (TaqMan), which allows for the direct detection of the PCR product by the release of a fluorescent reporter dye as a result of DNA amplification. The assays were developed after sequencing the alleles at both loci, by designing suitable primers and probes based on single nucleotide or insertion/deletion polymorphisms. The TaqMan procedure allowed for a fast and highly reproducible analysis directly in the PCR vials. The lack of any fragment separation step allows for an almost complete automation of the process thus making this technique particularly valuable for the large-scale screening required for map-based cloning projects and for marker-assisted selection, particularly when the results are needed in a short time.