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

Abstract

BackgroundGenotype 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.ResultsWe 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).ConclusionOur 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.

Identification, characterization, and gene expression analysis of nucleotide binding site (NB)-type resistance gene homologues in switchgrass

“…Allele-specific PCR is a high throughput and cost-effective way of distinguishing SNPs at a particular locus without the need for DNA sequencing [49]. A total of 8 primer pairs, 4 for ‘Alamo’ and 4 for ‘Dacotah’ (Additional file 9: Table S7), were designed to validate 16 SNPs identified by RNA-seq analysis.…”

“…Allele-specific SNP PCR primers for both ‘Alamo’ and ‘Dacotah’ were designed as previously described [49]. Both the forward and reverse primers for each locus accounted for a SNP at the 3′ end.…”

“…Both the forward and reverse primers for each locus accounted for a SNP at the 3′ end. The 3 rd base pair from the 3′ end of each primer was changed according to the recommendations from Hirotsu et al [49] in order to maximize allele-specificity. Four sets of forward and reverse primers that detected 16 SNPs were designed and used for PCR amplification in a total reaction volume of 20 μL (Additional file 2: Table S2).…”

Clade analysis of enterohemorrhagic Escherichia coli serotype O157:H7/H- strains and hierarchy of their phylogenetic relationships

“…Briefly, the first cycle was at 95°C for 5 min; followed by 5 cycles of 95°C for 30 s, annealing at Tm+5°C for 30 s (with the annealing temperature decreased 1°C for each cycle), and 72°C for 30 s; 35 cycles of 95°C for 30 s, annealing at Tm for 30 s, and 72°C for 30 s; and 1 cycle at 72°C for 7 min. After amplification, the presence of amplified DNA was confirmed using Sybre Green I (TaKaRa, Kyoto, Japan) as described by Hirotsu et al (2010). Analysis by agarose gel electrophoresis was carried out if it was difficult to confirm PCR amplification with Sybre Green I.…”

Markers for distinguishing Orostachys species by SYBR Green-based real-time PCR and verification of their application in commercial O. japonica food products

“…It is very difficult to design primer pairs for the development of PCR-based molecular markers if the SNP has only one of the forward or reverse primers. Some researchers have suggested that these difficulties could be resolved by increasing the primer specificity, by adding mismatches to the base pairs around the SNPs [25,26]. Therefore, we attempted such addition of mismatches to the base pairs of the designed primers for increasing the specificity.…”