Analysis of transgene copy number by Southern blotting. (A) Schematic diagram of marker-free vector-based hairpin RNAi structure. (B) Southern blot using a probe carrying the CaMV 35S double enhancers. WT control or transgenic plant DNAs were digested with Eco RV. Probe prepared from the 35S double enhancers detected the transgene copy number irrespective of the marker status. Cre, the bacteriophage P1 Cre recombinase with an intron; Frag, fragment; Hpt, a hygromycin-resistance marker gene driven by nopaline synthase (nos) promoter (Pnos); ICMV, Indian cassava mosaic virus; loxP, specific recognition sites of Cre; OlexA-46, eight copies of the LexA DNA-binding site fused to the −46 CaMV 35S promoter; XVE, a chimeric transactivator containing the regulator domain of an estrogen receptor. Arrows inside transcription units indicate the direction of transcription.