Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with "established' or "new breeding' techniques of genetic modification

Cisgenic plants must be free of exogenous genetic elements such as antibiotic or herbicide resistance genes commonly used in the selection phase of a gene transfer protocol. However, the use of a selection marker is essential for the transformation of many fruit crops, including apple (Malusxdomestica), for which the efficiency of DNA integration is very low. Currently, the approach with the highest chances of success relies on the removal of undesired exogenous genes by means of an inducible site-specific recombinase enzyme and its recognition sites. We developed a quantitative, rapid and cost-effective method based on real-time PCR to quantify the copy number of nptII marker gene in apple lines and to evaluate its elimination after the activation of the recombinase system. MdTOPO6 gene was chosen as endogenous reference gene for apple due to the single-copy presence in the haploid genome and to the species-specificity. A recombinant plasmid harboring specific sequences of both the reference gene and the target gene nptII was used as calibrator to build the standard curves. The limit of quantification of the method was evaluated, and precision and trueness of the quantification performances proved to be valid according to international reference criteria. Finally, this method was applied to characterize transgenic and cisgenic apple lines, and to investigate the possibility of removing an entire T-DNA cassette from the genome of edited apple plants.