Short Communication Physical localisation of transgenes on Vicia faba chromosomes R. J. Snowdon 1 , P. BÎttinger 2 , T. Pickardt 2 , W. KÎhler 1 & W. Friedt 1 1 Institut fÏr P£anzenbau und P£anzenzÏchtung I, Justus-Liebig-UniversitÌt Giessen, Heinrich-Buff-Ring 26^32, 35392 Giessen, Germany; Tel: +49 (0) 641 9937 438; Fax: +49 (0) 641 9937 429; E-mail: Rod.Snowdon@agrar.uni-giessen.de; 2 Institut fÏr Biologie ^ Angewandte Genetik, Freie UniversitÌt Berlin, Albrecht-Thaer-Weg 6, 14195 Berlin, Germany Received 5 April 2001; received in revised form and accepted for publication by Pat Heslop-Harrison 4 July 2001 Key words: DOP-PCR, £uorescence in-situ hybridisation, transgene detection, Vicia faba Geneticengineeringisbecominganimportanttool in the development of new crop varieties, and characterisation of the number and location of introgression sites is very important before transgeniclinescanbereleasedtothe¢eld.Along with genetic characterisation of insert sites, infor- mation on the physical location of introgressed genesintransgenicgenomesisnecessarytoenable a better understanding of the mechanisms under- lying transgene insertion and the potential for outcrossing to wild relatives of crop species. The use of £uorescence in-situ hybridisation (FISH)forthelocalisationoftransgeneconstructs in plant chromosomes has been described pre- viously (e.g. Wang et al. 1995, ten Hoopen et al. 1996, Moscone et al. 1996, Pedersen et al. 1997, ten Hoopen et al. 1999, Jakowitsch et al. 1999), but the resolution and reliability of signal detec- tion is not always reproducible in different laboratories. To overcome this, Salvo-Garrido et al. (2001) recently described a new approach where length and labelling of transgene probes is controlled by reducing the size of the plasmid prior to labelling by nick translation, in order tooptimisethehybridisationdynamicsandsignal detection. Here we describe how direct labelling of transgene constructs by PCR with degenerate oligonucleotide primers (DOP-PCR; Telenius et al. 1992) can also yield FISH probes with optimalprobelengthandlabellingthatarehighly suitable for physical detection of transgenes. Direct incorporation of 11-FITC-dUTP in the DOP-PCR reaction generated FISH probes approximately 300^500 bp in length which gave strong, reproducible signals in transgenic Vicia faba and allowed accurate physical location of the transgene with little to no background hybridisation. Cleanup of PCR products was not necessary when sheared V. faba DNA was added as competitor in probe solutions. The plants used were two independent transgenic lines of Vicia faba (designated as Vf12-1 and Vf12-2) which had been transformed using Agrobacterium tumefaciens (BÎttinger et al. submitted). The plants investigated were the offspring from homozygous (Vf12-2) and het- erozygous (Vf12-1) transgenic T 1 plants, respectively. Both lines contain the 8-kb T-DNA from the binary vector pAN109 (Karchi et al. 1993). The T-DNA was known to be present in one copy in the homozygous Vf12-2 parent and in four tandem copies in the heterozygous Vf12-1 parent. It was unknown, however, whether the individually investigated progeny offspring from theheterozygousVf12-1parentwerehomozygous positive, homozygous negative or heterozygous for the presence of the transgene. Also, the chromosomal location of the T-DNA in the Chromosome Research 9: 607^610, 2001. 607 # 2001 Kluwer Academic Publishers. Printed in the Netherlands