Figure 4: Evidence of pAA1 cassette integration in several independent hygromycin resistant C. truncatum pathotype F8-3B fungal genome. (a) PCR analysis of genomic DNA with primers specific for the amplification of an internal 544-bp fragment of the hph marker gene (top panel) and the 207 bp sgfp gene (lower panel). Wild type, indicated as FW was used as negative control and pAA1 plasmid was used as positive control. M, 1 kb Plus DNA Ladder (bars from top to bottom: 650 bp, 500 bp, 300 bp and 200 bp). (b) Southern analysis of the hygromycin-resistant transformants. Genomic DNA (8 μg) was digested with HindIII and probed with DIG-labelled 540 bp cutinase sense fragment. Closed triangle indicates the single copy endogenous cutinase gene (hybridised at ~8.0 kb) in the fungal genome. Open triangle represents the exogenous cutinase silencing transcriptional unit (2386 bp) within the T-DNA fragment. Non-transformed C. truncatum (FW) was used as negative control. HindIII+EcoRI-digested pAA1 plasmid was used as positive control. Arrow corresponds to the undigested plasmid.