Using Droplet Digital PCR (ddPCR) to Detect Copy Number Variation in Sugarcane, a High-Level Polyploid

Copy number variation (CNV) generally refers to duplications or deletions of sections of DNA, including genes.  It has been implicated in a number of important diseases in humans.  In polyploid plant species, CNV can also take the form of multiple copies of an allele at a given locus.  This variation in dosage has been shown to alter gene expression and phenotype.  As copy number rises, it becomes increasingly challenging to quantify dosage changes.  A new technique, known as droplet digital PCR (ddPCR), has been used for measuring CNV.   Digital PCR partitions a PCR reaction into many thousands to millions of discrete reactions (emulsified droplets in the case of ddPCR).   This partitioning has the effect of diluting the target DNA, such that some droplets contain zero copies of the target, while others contain one or more copies.   Post-PCR, this results in a binary (positive or negative) outcome, hence the term digital.  Partitioning the reaction into a large number of discrete tests also increases sensitivity and dynamic range.  In this article, ddPCR was employed to survey sugarcane (Saccharum spp.) germplasm for dosage variation for the Bru1 locus, which imparts resistance to the fungal disease known as brown rust.   The breeding nursery at the USDA-ARS Sugarcane Field Station in Canal Point, FL was surveyed for the presence of Bru1 using standard methods.  Those clones which were Bru1-positive were then analyzed via ddPCR.  Out of 80 Bru1-positive clones, the majority (60) were simplex or single copy.  Eighteen clones were duplex for Bru1, and there were two clones that were triplex.  The progeny of several test-crosses supported the results of ddPCR; a simplex parent produced approximately 50% positive progeny, while parents that were duplex produced more than 80%.  Further verification was done via quantitative PCR.  Results generally agreed with ddPCR, in that three groups could be distinguished, but the groups were relative, and not quantitative.  This analysis of dosage variation has important implications for both breeding and molecular biology research.  Clones with more than one copy of Bru1 (or any other gene of interest) will pass on the gene at much higher frequencies than close that are simplex.  In addition, the identification of clones with varying gene dosages allows for formal testing of dosage effects.  This is the first report of ddPCR being used to measure copy number/dosage variation in a high-level polyploid plant species.