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Research Project: Characterizing Circadian Regulatory Networks in Grain Crops to Establish their Role in Development and Abiotic Responses

Location: Plant Gene Expression Center

Title: Accurate measure of transgene copy number in crop plants using droplet digital PCR

Author
item Collier, Ray
item Xing, Yan-ping - University Of California
item Lin, Jeanie
item Mccue, Kent
item Blechl, Ann
item Thomson, James - Jim
item Thilmony, Roger
item Dasgupta, Kasturi - University Of California
item Hernandez, Bryan - University Of California
item Shao, Min - University Of California
item Oliveira, Maria
item Stover, Ed
item Novak, Emma - University Of California
item Harmon, Frank
item Rohozinski, Dominica - University Of California

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2017
Publication Date: 6/15/2017
Citation: Collier, R.A., Xing, Y., Lin, J.W., Mc Cue, K.F., Blechl, A.E., Thomson, J.G., Thilmony, R.L., Dasgupta, K., Hernandez, B.T., Shao, M., Oliveira, M.L., Stover, E.W., Novak, E., Harmon, F.G., Rohozinski, D. 2017. Accurate measure of transgene copy number in crop plants using droplet digital PCR. Plant Journal. 9(5):1014-1025 doi: 10.1111/TPJ.13517.

Interpretive Summary: A goal of biotechnology is to develop crop plants with superior performance, such as improved drought tolerance, increased grain production, or enhanced levels of beneficial nutrients. An important tool for biotechnology is genetic modification of plants by genetic transformation. When a new type of plant is made by genetic transformation, the properties of the genetic changes caused by transformation must be known. Traditional methods for this type of assessment are not efficient, mainly because these are time consuming and expensive. This study found an improved method to assess plants that have been genetically transformed. The new method is efficient, simple, and accurate. The method is useful for several types of important crop plants, including rice, rice, citrus, potato, corn, tomato and wheat. This new method is an advance for the entire plant research community, especially for researchers employing biotechnology to improve crop plants.

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-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 endogenous 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.