Fluorescence in situ hybridization on extended DNA fibres as a tool to analyse complex T‐DNA loci in potato

In this study the T-DNA composition of four antisense potato transformants showing complete or very strong inhibition of granule-bound starch synthase (GBSS) activity was analysed in detail. By Southern blot hybridizations, it was determined that all four transformants contained T-DNAs on multiple linkage groups and that most linkage groups contained multiple T-DNA copies, often in combination with non-T-DNA vector sequences. Subsequently, fluorescence in situ hybridization was performed on extended DNA fibres (‘fibre-FISH’) of three progeny plants each containing a single linkage group with a complex T-DNA organization. By using two differently labelled probes, one consisting of T-DNA sequences and the other of vector DNA sequences, it was possible to visualize the composition of complex loci. DNA sequences of 5–6 kb were well distinguishable. With this technique it is possible to determine T-DNA copy number, and arrangement of T-DNA and vector DNA sequences in a locus, more accurately than by Southern blot analysis alone. Therefore, fibre-FISH is a valuable supplementary tool to study T-DNA integrations in detail.