Penta-Primer Amplification Refractory Mutation System (PARMS) with Direct PCR-Based SNP Marker-Assisted Selection (D-MAS)

The penta-primer amplification refractory mutation system (PARMS) is a high-throughput, low-cost, and automated genotyping assay system that utilizes competitive allele-specific polymerase chain reaction (AS-PCR) combined with a homogeneous fluorescence-based reporting system to detect genetic variation occurring at single-nucleotide polymorphism (SNP). It is flexible in terms of the number of SNPs and samples to be analyzed, and the whole process only needs standard liquid handling, thermal cycling instruments, and plate reading instruments, which are present in many labs. Its compatibility with DNA samples prepared from a variety of sources and extraction technologies, such as alkaline lysis, makes it suitable for a direct PCR-based SNP marker-assisted selection system (D-MAS), a simple, cost- and labor-saving, and robust SNP genotyping system. It combines rapid and high-throughput DNA extraction through modified alkaline lysis with PARMS to dramatically reduce the time of manual operation and result analysis in the molecular breeding of major crops.