Analysis of flanking regions adjacent to the left or right border of T-DNA.

<p>(A) The diagram illustrates the PCR strategy used to obtain sequences flanking the T-DNA insertion sites. Restriction enzymes (shown as lightning) with recognition sites near the T-DNA border sequences (LB and RB) were used to digest DNA from the T-DNA insertion lines. Enzymes cut both within the T-DNA, and ligations were performed to circularize purified digestion products with <i>Bfa</i> I adapter. PCR1 using primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172936#pone.0172936.s001" target="_blank">S1 Table</a>) specific to adapter and T-DNA (AP1+R1 for RB analysis or AP1 + Fa1 for LB analysis). PCR2 using nested primer pairs (AP2+R2 for RB analysis or AP2+Fa2 for LB analysis). (B) PCR2 products were separated by gel electrophoresis. The first lane of each row contains a molecular marker (size of bottom five bands: 200, 400, 600, 800 and 1,000 nt).