Abstract Transformation of plant genomes by biolistic methods has become routine over the past decade. How- ever, relatively little is known about how transgenes are physically integrated into the host genome. Using a high- resolution physical mapping technique, fluorescence in situ hybridization on extended DNA fibers (fiber-FISH), 13 independent transgenic wheat lines were analyzed to determine the structural arrangement of stably inherited transgenes in host-plant chromosomes. Twelve transgen- ic lines were transformed with a single plasmid and one line was co-transformed with two separate plasmids, which co-segregated genetically. Three basic integration patterns were observed from the fiber-FISH experiments: Type I, large tandemly repeated integration; Type II, large tandem integrations interspersed with unknown DNA; and Type III, small insertions, possibly inter- spersed with unknown DNA. Metaphase FISH showed that the integration of transgenes was in both hetero- and euchromatic, as well as proximal, interstitial and distal, regions of the chromosomes. In the transgenic plants, the type of promotor used, rather than the chromosomal site of transgene integration, was most critical for transgene expression. The integration of the transgenes was not as- sociated with detectable chromosomal rearrangements. Keywords Fiber-FISH · Physical mapping · Transgene structure · Wheat Introduction Transformation of plant genomes by biolistic methods or by Agrobacterium has become routine in plant research. Almost every species of economic importance is amena- ble to transformation, and the number of transformed crops being registered for testing and commercial pro- duction is increasing rapidly. Transformation of crop genomes is desirable for many purposes: increased nutri- tive value, plant disease resistance, insect resistance, her- bicide resistance, and production traits. Even though transformation has become fairly rou- tine, little is known about how transgenes physically in- tegrate into host genomes. Both the Agrobacterium and biolistic methods of transformation have resulted in the integration of multiple, rearranged copies of the trans- gene at a single physical locus. Multiple copies of the transgene can be head to head, head to tail, truncated, or interspersed with unknown DNA (Takano et al. 1997; Kohli et al. 1998, 1999; Pawlowski and Somers 1998). The mechanism by which multiple copies of a transgene are integrated into one physical locus by either method of transformation is not well understood. In biolistic transformation, it has been hypothesized to be due to ei- ther integration at replication forks, resulting in inter- spersed copies (Pawlowski and Somers 1998), or the multimerization of transgenes in the plant cell prior to integration into the host genome (Kohli et al. 1998). Traditionally, the copy number and structural infor- mation of stably inherited transgenes are analyzed by Southern blot analysis using restriction enzymes that cut the plasmid once. Alternatively, parts of the transgenes Communicated by H.C. Becker S.A. Jackson, P. Zhang and W.P. Chen contributed equally to this research S.A. Jackson · R.L. Phillips Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108, USA P. Zhang · B. Friebe ( ✉ ) · B.S. Gill The Wheat Genetics Resource Center and Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan, KS 66506, USA e-mail: friebe@ksu.edu W.P. Chen Cytogenetics Institute and Department of Agronomy, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, The People’s Republic of China S. Muthukrishnan Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA Theor Appl Genet (2001) 103:56–62 © Springer-Verlag 2001 ORIGINAL PAPER S.A. Jackson · P. Zhang · W.P. Chen · R.L. Phillips B. Friebe · S. Muthukrishnan · B.S. Gill High-resolution structural analysis of biolistic transgene integration into the genome of wheat Received: 25 August 2000 / Accepted: 31 October 2000