For each of the three transgenic lines T7.1, T11.1 and T12.1, genotype inverse PCR (iPCR) was used to isolate genomic regions of ‘Gala’ flanking the RB, as well as the LB of the T-DNA insert in separate reactions, combining for each reaction the suitable restriction endonuclease and a corresponding pair of primers. The method of Triglia et al. (1988) was used with following modifications: 1 μg of DNA was digested in a total volume of 20 μL. For the self-ligation reaction, 100 ng (2 μL) of the digestion mixture was mixed with 10 μL 10X ligation buffer, 10 μL ATP (10 mm), and 4 μL T4 DNA ligase (3U) (New England Biolabs Inc., Beverly, MA) in a total volume of 100 μL and incubated overnight at 16 °C. The ligation reaction was deactivated by heating at 65 °C for 10 min. Five μL was taken from the ligation mixture and added directly to the PCR reaction. PCR reactions consisted of 1X Qiagen Multiplex PCR Master Mix, 0.5X Q-solution and 0.2 μm of each primer in a total volume of 50 μL. PCR reactions were performed applying the manufacturer's protocol conditions (95 °C for 15 min, followed by 35 cycles at 94 °C for 30 sec, 55 °C for 90 sec, 72 °C for 3 min, and a final extension at 72 °C for 10 min). The amplicons were analysed by agarose gel electrophoresis (1%), and a nested PCR was performed in a second PCR reaction. The reaction mixture and conditions were the same as for the first PCR reaction, with 5 μL of the PCR product used as the template. The amplicons were analysed by agarose gel electrophoresis (1%); the bands were cut out of the gel and sequenced (primer sequences are listed in Table 1) using the BigDye terminator kit 3.1 (Applied Biosystems, Foster City, CA). The obtained sequences, after trimming of the T-DNA sequences, were subjected to BLAST analysis against the whole genome sequence of ‘Golden Delicious’ (Velasco et al., 2010) in order to estimate the site of insertion in the Malus genome. Primers (T7.1_a, T7.1_c, T11.1_b, T11.1_d, T12.1_a, T12.1_d) were designed on the genomic DNA regions and used to amplify the corresponding region in untransformed ‘Gala’ to identify single nucleotide polymorphisms (SNPs) between the two alleles of ‘Gala’. In a second step, the primers were used for amplification on DNA of the transgenic ‘Gala’ lines T7.1, T11.1 and T12.1. As the inserted T-DNA (11 kbp) was very large for a normal PCR amplification, only amplicons from the allele in which the integration did not occur should be obtained from these lines. The developed primers were also used separately in combination with primers on the T-DNA (HcrVf2termrev2, pmf_IPCR_9; Table 1) to amplify the transition region between T-DNA and genomic DNA at the right and left borders, respectively. The amplicons were sequenced to verify the extent of the T-DNA integration. Finally, the developed primers were used to find out whether spontaneous recombination events had occurred, amplifying the DNA of the transgenic mother lines T7.1, T11.1 and T12.1. Spontaneous recombination is assumed to occur if the obtained amplicon is smaller in size than the expected T-DNA insert (11 kbp) and is shorter by the length of the excisable cassette (about 7.3 kbp shorter, Vanblaere et al., 2011). In the corresponding cisgenic line, the 11 kbp band should be absent.