Measurement of Transgenes Copy Number in Wheat Plants Using Droplet Digital PCR

Genetic transformation is a powerful method for the investigation of gene function and improvement of crop plants. The transgenes copy number in the transgenic line is involved in gene expression level and phenotypes. Additionally, identification of transgene zygosity is important for quantitative assessment of phenotype and for tracking the inheritance of transgenes in progeny generations. Several methods have been developed for estimating the transgene copy number, including southern blot assay and quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization, although convincing and reliable, is a time-consuming method through which the examination of the copy number is challenging in species with large genomes like wheat plants. Although qPCR is potentially simpler to perform, its results lack accuracy and precision, especially to distinguish between one and two copy events in transgenic plants with large genomes. The droplet digital PCR (ddPCR)-based method for investigation of transgenes copy number has been widely used in an array of crops. In this method, the specific primers to amplify target transgenes and reference genes are used as a single duplexed reaction, which is divided into tens of thousands of nanodroplets. The copy number in independent transgenic lines is determined by detection and quantification of droplets using sequence-specific fluorescently labeled probes. This method offers superior accuracy and reliability with a low cost and scalability as other PCR techniques in the investigation of transgenes copy number. This protocol was validated in: Mol Plant (2021), DOI: 10.1016/j.molp.2021.03.022 Graphical abstract Flow chart for the ddPCR protocol.