The use of combined FISH/GISH in conjunction with DAPI counterstaining to identify chromosomes containing transgene inserts in amphidiploid tobacco

Abstract

We have used combined fluorescent and genomic in situ hybridization (FISH/GISH) together with 4',6-diamidino-2-phenylindole (DAPI) counterstaining to determine simultaneously the chromosomal integration site and subgenomic allocation of a transgene insert in amphidiploid tobacco (Nicotiana tabacum, 2n = 4 chi = 48). The procedure provides sufficient information on physical markers to identify at least 20 out of 24 chromosome pairs of two tobacco cultivars commonly used in studies on transgene expression and silencing (cv. Petit Havana SR1 and cv. Gatersleben). The chromosomes can be distinguished on the basis of diploid parental ancestry, size, morphology, the presence of rDNA loci and/or intergenomic exchanges, and the DAPI banding pattern, which is shown here for the first time for N. tabacum. From a single ISH experiment, it should now be possible in most cases to identify a tobacco chromosome carrying a transgene insert, thus permitting systematic studies of how the chromosomal location of transgenes influences expression levels.