Digital PCR for Genotyping of SNP_BvBTC1 in DNA Bulks of Sugar Beet

Discovery and validation of SNP markers linked to the annual habit in Beta vulgaris is essential for breeding of sugar beet. Pin et al. 2012 (Curr. Biol. 22:1095-1101) found a SNP variant in exon 8 (nucleotide position 79) of the gene BvBTC1 (BOLTING TIME CONTROL 1) that helps to discriminate between annual and biennial plants. A TaqManTM assay (SNP_BvBCT1) was designed to detect this SNP variant. SNP_BvBTC1 could be genotyped to differentiate annual from biennial flowering plants: CC genotypes are mainly present in biennial beets, while AA and CA genotypes are typically found in annual beets. However, screening many populations for SNP polymorphisms is time-consuming and expensive when performed as individual genotyping. An option to reduce the cost of SNP_BvBTC1 screening for removal of off-type plants is to use DNA bulks, where plant DNA is pooled by group and genotyping is performed on the bulk rather than on individual plants. In this study, we compared the accuracy of the SNP_BvBTC1 genotyping by qPCR, HRM and digital PCR (dPCR) in two DNA bulks constituted by pooling 90 biennial + 10 annual plants (B1) and 99 biennial + 1 annual plants (B2) to simulate high or low contamination with wild beets, respectively. qPCR could not detect the minor allele in either the B1 or B2 pool; HRM detected the allele A only at moderate frequencies (10%), in the B1 pool. dPCR, on the contrary, was able to detect the allele A in both pools. This study provides evidence that dPCR is suitable for the quantitation of SNP_BvBTC1 from pooled DNA samples in sugar beet.