Digital PCR as New Approach to SNP Genotyping in Sugar Beet

Real-time PCR (qPCR) allelic discrimination and high-resolution melting (HRM) methods are widely adopted for the detection of single nucleotide polymor- phisms (SNP). Digital PCR (dPCR) is a new method recently proposed for the detection of low-frequency and/ or rare SNP. The molecular marker SNP_BvBTC1 is used in sugar beet to distinguish between annual and biennial flowering plants. The CC genotype at this SNP locus is associated with biennial beets, while the CA and AA genotypes are typically found in annual beets. In this study, we compared the sensitivity of qPCR, HRM and dPCR in detecting the allele A from two pools of bulk beet DNA composed of 90 biennial ? 10 annual plants (B1) and 99 biennial ? 1 annual plant (B2), respectively. All annual plants were found to have the AA genotype. qPCR could not detect allele A in either the B1 or B2 pool and HRM detected allele A only at moderate frequencies (10 %), in the B1 pool. dPCR, on the contrary, was able to detect allele A in both pools. We therefore concluded that dPCR is a suitable method for the quantitation of SNP_BvBTC1 within bulked DNA samples of sugar beet.