Confirmation of gene stacking by Southern hybridization. Genomic DNA of B53 T1 plants, homozygous RMCE B53-1 and B53-2, hemizygous (RMCE/excision) B53-3 and B53-4, and excision B53-5 and B53-6, the T0 plant B531-1 (RMCE/excision), and homozygous B5 and B ancestor plants were digested with MfeI and hybridized sequentially with probes SCP1, HPT, FATB-5, and DGAT1. The expected sizes of Southern bands are specific to transgenes integrated at the B target site, where the transgenic QC288A lost 17-bp 5′ end and 49-bp 3′ end sequences. There is an MfeI site 2,131 bp upstream and 1,230 bp downstream, respectively, of the transgenes that contain MfeI sites (). A, The SCP1 probe hybridized to the expected 12,205-bp band of QC288A436A in B53-1 and B53-2, the 3,987-bp band of QC288ME in B53-5 and B53-6, and both bands in hemizygous plants B53-3 and B53-4. The same 3,987-bp QC288ME band and a 3,681-bp QC288A436A438A band were hybridized in B531-1. As expected, the same 3,681-bp band also specific to QC288A329A was detected in B5 and a 7,839-bp QC288A band was detected in B. B, The HPT probe hybridized to the same 12,205-bp QC288A436A band in B53-1, B53-2, B53-3, and B53-4 and to the same 7,839-bp QC288A band in B. C, The FATB-5 probe hybridized to two endogenous bands in all samples in addition to the same 12,205-bp QC288A436A band in B53-1, B53-2, B53-3, and B53-4. Instead of the expected 12,931-bp band of QC288A436A438A, a larger band, likely the 16,402-bp band expected from modified QC288A436A438A with its 2,946-bp DHPS-MYB2-UBIQ10 deleted, overlapped with the top wild-type band (wt) in B531-1. D, The DGAT1 probe detected in B531-1 the same 16,402-bp band of the modified QC288A436A438A instead of an expected 6,352-bp QC288A436A438A band. The DIGVII DNA markers are 8,576, 7,427, 6,106, 4,899, and 3,639 bp (Roche).