PCR-based Assays for Validation of Single Nucleotide Polymorphism Markers in Rice and Mungbean

Background: Single nucleotide polymorphism (SNP) markers are the method of choice for genetic analyses including diversity and quantitative trait loci (QTL) studies. Marker validation is essential for QTL studies, but the cost and workload are considerable when large numbers of markers need to be verified. Marker systems with low development costs would be most suitable for this task.

Results: We have tested allele specific polymerase chain reaction (PCR), tetra markers and a genotyping tool based on the single strand specific nuclease CEL-I to verify randomly selected SNP markers identified previously either with a SNP array or by genotyping by sequencing in rice and mungbean, respectively. The genotyping capacity of allele-specific PCR and tetra markers was affected by the sequence context surrounding the SNP; SNPs located in repeated sequences and in GC-rich stretches could not be correctly identified. In contrast, CEL-I digestion of mixed fragments produced from test and reference DNA reliably pinpointed the correct genotypes, yet scoring of the genotypes became complicated when multiple SNPs were present in the PCR fragments. A cost analysis showed that as long the sample number remains small, CEL-I genotyping is more cost-effective than tetra markers.

Conclusions: CEL-I genotyping performed better in terms of genotyping accuracy and costs than tetra markers. The method is highly useful for validating SNPs in small to medium size germplasm panels.