Droplet digital PCR for verification of interspersed refuge in midge tolerant wheat varietal blends1

Analysis of bulk ground samples by droplet digital PCR (ddPCR) was investigated as an alternative to individual kernel testing for assessment of interspersed refuge in midge [Sitodiplosis mosellana (Géhin)] tolerant wheat (Triticum aestivum L.) varietal blends. Four genotyping assays were selected such that at least one assay was informative for each of 15 varietal blends registered for production in Canada. The assays were examined in DNA of each of the constituent varieties to assess intravarietal polymorphism and in DNA mixtures simulating each varietal blend. Seed mixtures corresponding to a range of refuge proportions in two different varietal blends were prepared and assessed by ddPCR on two different platforms (RainDrop Digital PCR System, RainDance Technologies, Billerica, MA, and QX200 AutoDG Droplet Digital PCR System, Bio-Rad Laboratories, Hercules, CA). Both systems yielded refuge estimates that were very close to the targeted proportions across the range simulated. Standard deviations among estimates made with the QX200 were, on average, about 60% greater than those among corresponding estimates made with the RainDrop system, but in either case, the coefficients of variation generally remained under 5%. For a ddPCR-based assessment of interspersed refuge in midge tolerant wheat varietal blends, any advantage held by the RainDrop system with respect to precision may be offset by higher throughput achievable with the QX200.