Detection of Chloroplast Genome Integrations

The inverted repeat region (IR) of the chloroplast genome of potato (S. tuberosum; GenBank: NC_008096.2; from 104457 to 104978 bp) was used to design a DNA probe for detection of Gen1 vector integration. The KanR gene sequence from the vector backbone pMK GeneArt (Thermo Fisher Scientific) was used to design the probe to detect extra‐plastomic DNAs (_eGen1 or _eGen2). The aadA gene (GenBank: ARK38551.1) was used to design to probe to detect the selection cassette. The IR, KanR, and aadA DNA‐probes labelled with digoxigenin‐(DIG)‐sUTP were synthesized using the PCR DIG Probe Synthesis Kit (Roche, Indianapolis, IN) and the pair of primers 21 Fw/Rv, 22 Fw/Rv and 23 Fw/Rv, respectively. 1 µg of total genomic DNA from leaf tissue was digested using KasI/HindIII or FspI/FseI restriction enzymes, for detection of IR and aadA or KanR fragments, respectively. DNA samples were separated on 0.9% agarose gel, depurinated, denatured, and then transferred on Hybond‐N+ nylon membrane (GE Healthcare, Life Sciences, Marlborough, MA) (Lin et al., 2014 (link)). The anti‐digoxigenin‐AP Fab fragments detection kit (Roche) was used for detection of the DIG‐labelled probe signal.