|DASGUPTA, KASTURI - University Of California|
|XING, YAN-PING - Inner Mongolian Agriculture University|
|HERNANDEZ, BRYAN - University Of California|
|SHAO, MIN - University Of California|
|ROHOZINSKI, DOMINICA - University Of California|
|KOVAK, EMMA - University Of California|
|DE OLIVEIRA, MARIA LUIZA - Agrosource, Inc|
|Thomson, James - Jim|
Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2017
Publication Date: 2/23/2017
Citation: Collier, R.A., Dasgupta, K., Xing, Y., Hernandez, B., Shao, M., Rohozinski, D., Kovak, E., Lin, J.W., De Oliveira, M., Stover, E.W., Mc Cue, K.F., Harmon, F.G., Blechl, A.E., Thomson, J.G., Thilmony, R.L. 2017. Accurate measurement of transgene copy number in crop plants using droplet digital PCR. Plant Journal. 90:1014-1025. https://doi.org/10.1111/tpj.13517.
Interpretive Summary: Genetic transformation provides scientists with many plants that are potentially useful for basic and applied research, but a significant amount of time and effort is needed to sort through them to find those with only one copy of the added DNA. Such “single-copy transformants” are desirable because they are usually more stable in both the inheritance and expression of the added genes than transformed plants with multiple copies. The research in this paper describes best practices for using the digital drop Polymerase Chain Reaction (ddPCR) method to determine gene copy numbers in genomic DNA of six crops: citrus, potato, maize, rice, tomato and wheat. The resulting copy number measurements are shown to be as good as or better than those obtained by previous methods, with enough precision to distinguish one from two copies and enough sensitivity to detect single gene copies even in genomes as large as 17 billion base pairs. Among the critical parameters identified are primer design and the quality and amounts of input DNA. This method provides an experimentally validated resource that the plant research community can use for simple and accurate determination of gene copy numbers in plant genomes.
Technical Abstract: Technical abstract: Genetic transformation is a powerful means for the improvement of crop plants, but requires labor and resource intensive methods. An efficient method for identifying single copy transgene insertion events from a population of independent transgenic lines is desirable. Currently transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one and two copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR (ddPCR)-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato, and wheat. The method utilizes specific primers to amplify target transgenes, and species-specific reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.