A GenomeWalker Universal kit (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to find the unknown genomic DNA sequences adjacent to a transposable element using the manufacturer’s instructions. DNA (2.5 μg) was independently digested with four restriction enzymes (DraI, EcoRV, PvuII, and StuI) to generate blunt ended fragments (S2 Fig). After phenol:chloroform::1:1 purification, digested genomic DNA was ligated to the GenomeWalker adaptor to generate genomic DNA libraries. The four genomic libraries, generated through digestion of DNA with four restriction endonucleases for each germinal revertant were used for the first PCR (PCR 1) using the outer adaptor primer (AP1) and an outer Tgm9-specific primer (Trans R1) (S2 Fig; S1 Table). The PCR 1 mixture was then diluted to 100 times and used as a template for a second or “nested” PCR (PCR 2). PCR 2 reactions were conducted using the nested adaptor primer (AP2) and a nested Tgm9-specific primer (Trans R2) (S2 Fig). For visualization, the resulting PCR products were separated on a 1.5% agarose gel at 100 V for 1 hour. As there are some residual/truncated copies of Tgm9 in T322, only unique bands of individual mutants were sequenced (S2 Fig). Residual/truncated copies were displayed as common bands among the selected mutants (S3 Fig). Unique PCR products were sequenced and evaluated for presence of over 100 bp Tgm9-end specific sequences.