Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species

Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment. We have developed a simple method for multi-sample or multi-locus SNP genotyping using allele-specific primers (ASP). More specifically, we (1) improved the design of allele-specific primers, (2) established a method to detect PCR products optically without electrophoresis, and (3) standardized PCR conditions for parallel genomic assay using various allele-specific primers. As an illustration of multi-sample SNP genotyping using ASP, we mapped the locus for lodging resistance in a typhoon (lrt5). Additionally, we successfully tested multi-locus ASP-PCR analysis using 96 SNPs located throughout the genomes of rice (Oryza sativa) cultivars 'Koshihikari' and 'Kasalath', and demonstrated its applicability to other diverse cultivars/subspecies, including wild rice (O. rufipogon). Our ASP methodology allows characterization of SNPs genotypes without electrophoresis, expensive probes or specialized equipment, and is highly versatile due to the flexibility in the design of primers. The method could be established easily in any molecular biology laboratory, and is applicable to diverse organisms.