Flowering, glasshouse-cultivated plants were maintained and manually pollinated as described previously (17). Seeds were germinated in soil, and genomic DNA was extracted from emerging leaf material [modified from (59)]. PCR amplification of regions of the T-DNA was performed (described in the “PCR amplification of T-DNA in putative transgenic lines” section). PCR amplification of GBSS alleles consisted of 1× Q5 reaction buffer, 200 μM dNTPs, 0.5 μM forward primer, 0.5 μM reverse primer, 200 ng of genomic DNA, 1 U of Q5 High-Fidelity DNA Polymerase (New England Biolabs), and sterile nuclease-free water. Conditions were as follows: initial step at 98°C (1 min 30 s); then 25 cycles of 98°C (10 s), 60 to 67°C (depending on oligonucleotides Tm) (10 s), and 72°C (20 s); and a final step of 72°C for 2 min. Reaction products were cloned using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) and used to transform DH5α E. coli following the manufacturer’s guidelines. Plasmids from independent bacterial colonies for each of the cloned GBSS amplicons were Sanger-sequenced (Microsynth AG), and the data were mapped to similarly amplified and cloned WT control sequence.

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