Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase

We have investigated the organization of transgenes delivered by particle bombardment into the wheat genome, combining conventional molecular analysis with fluorescence in situ hybridization (FISH) and three-dimensional confocal microscopy. We selected a representative population of transformed wheat lines and carried out molecular and expression analysis. FISH on metaphase chromosomes showed that transgene integration sites were often separated by considerable lengths of genomic DNA (>1 Mbp), or could even be on. opposite chromosome arms. Plants showing multiple integration sites on a single chromosome were selected for three-dimensional confocal analysis of interphase nuclei in root and embryo tissue sections. Confocal microscopy revealed that these sites lay in close physical proximity in the interphase nuclei. Our results clearly show that multiple transgenes physically separated by large intervening regions of endogenous DNA at metaphase can be brought together at interphase. This may reflect the original physical organization of the endogenous DNA at the moment of transformation, with DNA strand breaks introduced into several co-localized DNA loops by the intruding gold particles. Alternatively, the transgenes may be brought together after transformation, either by an ectopic homologous pairing mechanism, or by recruitment to a common transcription site.