Location: Crop Improvement and Genetics ResearchTitle: Measurement of Transgene Copy Number in Potatoes Using Droplet Digital PCR
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 7/23/2017
Publication Date: 7/1/2018
Citation: Mc Cue, K.F., Collier, R.A., Gardner, E.B., Thilmony, R.L. 2018. Measurement of Transgene Copy Number in Potatoes Using Droplet Digital PCR. Meeting Abstract. 95(3):208-229. https://doi.org/10.1007/s12230-018-9650-4.
Technical Abstract: Precise molecular breeding is a valuable tool for the improvement of crops. Knowing the exact nature of the insertion site in genetic transformation events is desirable and often necessary for commercialization of transgenic crops. While the precise nature of a genetic modification can be determined using high throughput sequencing, this is not an appropriate method for screening larger numbers of transformation events for those with one to a few copies of the newly inserted genes. Frequently the desired transformation contains a single or relatively few copies of the newly inserted gene. Historically, transgene copy numbers have been assessed using genomic blot hybridizations, the Southern blot procedure. More recently quantitative polymerase chain reaction (qPCR) has been applied for this purpose. A very recent development has been the digital PCR method that entails creation of many thousands of droplets for quantitation (Droplet digital PCR or ddPCR). We have applied this technique for the analysis of transgenic potato lines previously analyzed by the traditional Southern blotting methods. In most cases, ddPCR yielded integer values for transgene copy numbers that were in agreement with those determined by Southern analyses. The method was sensitive enough to detect single copies and distinguish 1 from 2 copies. This procedure provides an efficient tool to screen many transgenic events to rapidly identify a smaller number of candidates for further genetic and phenotypic analyses.