Plant Molecular Biology 51: 83–98, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 83 Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome D. Gidoni 1,∗ , E. Fuss 2,3,a,∗ , A. Burbidge 4,∗ , G-J. Speckmann 5,∗ , S. James 6,∗ , D. Nijkamp 7,∗ , A. Mett 1,∗ , J. Feiler 1,∗ , M. Smoker 8,∗ , M.J. de Vroomen 7,∗ , D. Leader 6,c,∗ , T. Liharska 7,∗ , J. Groenendijk 5,∗ , E. Coppoolse 7 , J.J.M. Smit 7 , I. Levin 1 , M. de Both 5 , W. Schuch 6,d , J.D.G. Jones 8 , I.B. Taylor 4 , K. Theres 2,3,b and M.J.J van Haaren 5,∗∗ 1 Department of Plant Genetics, Institute of Field Crops, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel; 2 Institute for Genetics, University of Cologne, Cologne, Germany, 3 Max Planck Institute for Plant Breeding Research, Cologne, Germany; 4 Department of Physiology and Environmental Science, Sutton Bonington Cam- pus, The University of Nottingham, UK; 5 Keygene N.V., Agro Business Park 90, P.O. Box 216, NL-6700 AE Wageningen, The Netherlands; 6 Zeneca Plant Science, Jealott’s Hill Research Station, Bracknell Berkshire, UK; 7 Free University of Amsterdam, Department of Genetics, Institute for Molecular and Biological Sciences (IMBW), BioCentrum Amsterdam, The Netherlands; 8 John Innes Centre, The Sainsbury Laboratory, Norwich, UK; present address: a Institut fuer Entwicklungs- und Molekularbiologie der Pflanzen, Heinrich-Heine-Universitaet Duessel- dorf, Duesseldorf, Germany; b Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, D-50829 Cologne, Germany; c Wheat Improvement Centre, Syngenta, Norwich Research Park, Colney, Norwich NR4 7UH, UK; d CellFor Inc. 355 Burrard Street, Vancouver, British Columbia V6C 2G8, Canada ( ∗ Equal contributors to this work; ∗∗ author for correspondence; e-mail: mark.van-haaren@keygene.com) Received 24 April 2002; accepted 17 May 2002 Key words: chromosomal rearrangements, site-specific recombination, T-DNA, tomato, transposon Abstract In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunc- tional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well. Introduction Recently the complete genomes of Arabidopsis thaliana (The Arabidopsis Genome Initiative, 2000) and rice (Oryza sativa; Syngenta Co.) have been se- quenced and additionally, a large number of expressed sequence tags (ESTs) from these and different other organisms have become available in sequence data-