Background

Plant genetic transformation has been widely used for investigating gene function in wheat as well as the production of environmentally friendly and disease-resistant wheat varieties (Vasil, 2007). During genetic transformation in wheat embryos, the delivery number of transgenes into the plant genome cannot be controlled. Moreover, the expression level of transgenes and, consequently, the phenotype can be influenced by copy numbers. Thus, the validation of the copy number of transgenes is essential for research projects involving transgenic plants (Hobbs et al., 1993; Sivamani et al., 2015). The transgenic wheat plants with a single, full-length copy of a gene at a single locus is the most desirable event because the transgenes typically segregate in a predictable Mendelian fashion (Srivastava et al., 1996). Additionally, copy number determination can also be used to track inheritance and zygosity (Wu et al., 2017).

Gene copy number analysis could be determined by southern blot analysis (Southern, 1975), quantitative polymerase chain reaction (qPCR) (Higuchi et al., 2013), thermal asymmetric interlaced PCR (TAIL-PCR) (Y. G. Liu et al., 1995) and, most recently, digital droplet PCR (ddPCR). However, these methods vastly differ in precision, reproducibility, and potential to scale up. Among these, southern blot analysis was regarded as a powerful method for estimating copy number and loci complexity in transgenic plants. In this assay, genomic DNA was digested, separated by electrophoresis, blotted onto a membrane, and then detected with a radioactive, fluorescent, or chemiluminescent-labelled probe. However, it is hard to detect the copy number of transgenes using this method, which are inserted at a single locus. Moreover, the southern blot assay is impractical for examining large populations of transgenic plants because it is unable to automate and requires substantial skills, especially in species with large genomes like wheat plants. In qPCR, the concentration of the transgene in each sample is compared with either a standard curve or an endogenous gene of a known copy number, to estimate the copy number and zygosity of the transgenic allele(s) (Higuchi et al., 2013). Estimation of copy number by qPCR is very sensitive to PCR efficiency because of direct coupling between the PCR amplification and quantification. TAIL-PCR has also been used to establish the copy numbers of transgenes by amplifying the flanking sequence of the transgenic allele(s) (Y. G. Liu et al., 1995; Hanhineva and Krenlampi, 2007).

The ddPCR method has been used to detect the copy number of transgenes with high accuracy (Gowacka et al., 2015). Similar to qPCR, the detection principle of ddPCR is based on a fluorescent dye or probe and requires an endogenous reference gene with a known copy number (Collier et al., 2017). In ddPCR, the PCR reaction partitions individual into separate compartments, such as oil-bounded droplets, and then detects their endpoint amplification products. Poisson probability distribution is also used to derive the template concentration. Accordingly, the ddPCR method enables a more effective calculation of gene target enumeration and accurate quantification of nucleic acid targets. In qPCR, logarithmic PCR template quantification may limit the ability to identify small copy number differences (Bubner and Baldwin, 2004). However, in ddPCR, the decoupling of amplification and quantification with the linearity of the quantification scale allows the detection of small copy differences (Hindson et al., 2013; Bharuthram et al., 2014). In recent years, ddPCR has been successfully applied for the accurate determination of transgene copy number in different plant species. Here, we summarize the protocol of ddPCR used to determine the transgene copy number in wheat plants (P. Liu et al., 2021).