Droplet Digital PCR for Estimation of Allele Frequencies in Barley: Evaluation Using F1 Hybrid DNA

The grain industry requires reliable methods to assess varietal purity and variety composition of malting barley to provide assurances and facilitate transactions. At present, such analyses are commonly performed on an individual kernel basis, with multiple kernels analyzed per sample to assess variety composition. Alternatively, methods that operate on bulk ground samples of grain would be advantageous because a large number of kernels may be represented in a single analysis, potentially allowing more precise estimation of composition. Previously we demonstrated that droplet digital PCR (ddPCR) using a RainDance RainDrop system showed promise as an effective means to estimate marker allele frequencies in barley mixtures. However, a lack of well characterized reference materials challenged our ability to effectively assess the accuracy of ddPCR-derived estimates. Here we report on the performance of ddPCR assays under improved reaction conditions and using DNA of F1 barley hybrids as PCR template. Hybrid DNA is an ideal reference material for evaluation of accuracy and consistency because it has exact 50:50 allele ratios at heterozygous loci. Eight selected TaqMan genotyping assays were evaluated using template DNA from correspondingly heterozygous hybrid lines under a single set of PCR conditions. Resulting allele frequency estimates were very close to expected values and variability among replicates and among markers was low (CV ≤ 1%).  Examination of five of these assays in a DNA mixture prepared by combining DNA of two barley varieties at a proportion more relevant to the malt barley industry (95:5) found deviations to be of a similar magnitude.