Accurate measurement of transgene copy number in crop plants using droplet digital PCR

Abstract

SUMMARYGenetic 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.

CRISPR‐S: an active interference element for a rapid and inexpensive selection of genome‐edited, transgene‐free rice plants

“…Furthermore, we proved that all bentazon-susceptible plants were transgenic, while all bentazon-resistant plants lacked T-DNA. The segregation of T 1 plants derived from a homozygous or biallelic genome-edited T 0 plant is presented in Figure 1e, in which the genome-edited, transgene-free plants are resistant to bentazon because the #2 #3 #4 #5 #7 #9 #11 #13 #18 #19 #20 #22 #23 #26 #27 #29 RNAi (Collier et al, 2017). The segregation ratio observed for bentazon susceptibility was consistent with this fact in all T 1 lines except for Line #19 (Figure 1g).…”

“…Transgenic plants produced by A. tumefaciens ‐mediated transformation often carry one or two copies of T‐DNA (Collier et al ., 2017). The segregation ratio observed for bentazon susceptibility was consistent with this fact in all T 1 lines except for Line #19 (Figure 1g).…”

TaPIMP2, a pathogen-induced MYB protein in wheat, contributes to host resistance to common root rot caused by Bipolaris sorokiniana

“…The primers used in ddPCR assay were the same with those used in PCR detection for transgenic plants. The ddPCR has proven to be a reliable and reproducible method in estimation of transgene copy number in wheat 27 and tobacco 28 . TaSTOP1 - A was used as the reference with two copies in hexaploid wheat 29 .…”

The AVR4 effector is involved in cercosporin biosynthesis and likely affects the virulence of Cercospora cf. flagellaris on soybean

“…The primer and probe sequences used for droplet digital PCR are presented in Table 1. The accuracy of using droplet digital PCR to determine target gene copy number in comparison to Southern blot analysis has been well documented in earlier studies (Collier et al, 2017;Głowacka et al, 2016).…”