Estimation of Allele Frequencies in Barley Mixtures Using Droplet Digital PCR

Rapid and reliable methods to assess varietal purity of malting barley are required by the grain industry to provide assurances when transactions include varietal purity (typically 95% or greater) as a contract specification. As a potential means to this end, we are exploring the application of droplet digital PCR (ddPCR) to the estimation of marker allele frequencies in barley mixtures. TaqMan genotyping assays were designed targeting SNPs or small indels in barley and their response to various DNA mixtures was investigated using a RainDance RainDrop ddPCR system. Seven assays were examined in DNA mixtures prepared by combining the DNA of two barley varieties at various proportions (99:1, 95:5, 90:10, 10:90, 5:95 and 1:99). Two combinations of varieties were sufficient to provide alternate alleles appropriate for the assessment of all seven assays. Allele frequency estimates obtained using ddPCR were linear over the range of frequencies considered and variability among three replicates was low (CV <2%, <4% and <9% for expected minor allele frequencies of 10%, 5% and 1%, respectively). However, considerable variation of allele frequency estimates was evident among different markers examined within the same DNA mixture. We are investigating approaches to reducing this variability.