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=4x=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 forN. 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.
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Ambros PF, Matzke AJM, Matzke MA (1986a) Localization ofAgrobacterium rhizogenes T-DNA in plant chromosomes byin situ hybridization. EMBO J 5: 2073–2077
Ambros PF, Matzke MA, Matzke AJM (1986b) Detection of a 17 kb unique sequence (T-DNA) in plant chromosomes by in situ hybridization. Chromosoma 94: 11–18
Anamthawat-Jónsson K, Reader SM (1995) Pre-annealing of total genomic DNA probes for simultaneous genomic in situ hybridization. Genome 38: 814–816
Bennett MD (1995) The development and use of genomic in situ hybridization (GISH) as a new tool in plant biosystematics. In: Brandham PE, Bennett MD (eds) Kew Chromosome Conference IV. Royal Botanic Gardens, Kew, England, pp 167–183
Finnegan J, McElroy D (1994) Transgene inactivation: plants fight back! Biotechnology 12: 883–888
Fransz PF, Stam M, Montijn B, Ten Hoopen R, Wiegant J, Kooter JM, Oud O, Nanninga N (1996) Detection of single-copy genes and chromosome rearrangements inPetunia hybrida by fluorescence in situ hybridization. Plant J 9: 767–774
Gill BS (1995) The molecular cytogenetic analysis of economically important traits in plants. In: Brandham PE, Bennett MD (eds) Kew Chromosome Conference IV. Royal Botanic Gardens, Kew, England, pp 47–53
Jiang J, Gill BS (1994) Nonisotopic in situ hybridization and plant genome mapping: the first ten years. Genome 37: 717–725
Kenton A, Parokonny AS, Gleba YY, Bennett MD (1993) Characterization of theNicotiana tabacum L. genome by molecular cytogenetics. Mol Gen Genet 240:159–169
Kenton A, Khashoggi A, Parokonny A, Bennett MD, Lichtenstein C (1995) Chromosomal location of endogenous geminivirus-related DNA sequences inNicotiana tabacum L. Chromosome Res 3: 346–350
Kilby NJ, Leyser HM, Furner IJ (1992) Promoter methylation and progressive transgene inactivation inArabidopsis. Plant Mol Biol 20: 103–112
Matzke AJM, Neuhuber F, Park Y-D, Ambros PF, Matzke MA (1994) Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes. Mol Gen Genet 244: 219–229
Matzke MA, Matzke AJM (1995) How and why do plants inactivate homologous (trans)genes? Plant Physiol 107: 679–685
Neuhuber F, Park Y-D, Matzke AJM, Matzke MA (1994) Susceptibility of transgene loci to homology-dependent gene silencing. Mol Gen Genet 244: 230–241
Papp I, Iglesias VA, Moscone EA, Michalowski S, Spiker S, Park Y-D, Matzke MA, Matzke AJM (1996) Structural instability of a transgene locus in tobacco is associated with aneuploidy. Plant J 10: 469–478
Park Y-D, Papp I, Moscone EA, Iglesias VA, Vaucheret H, Matzke AJM, Matzke MA (1996) Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity. Plant J 9: 183–194
Parokonny AS, Kenton AY (1995) Comparative physical mapping and evolution of theNicotiana tabacum L. karyotype. In: Brandham PE, Bennett MD (eds) Kew Chromosome Conference IV. Royal Botanic Gardens, Kew, England, pp 301–320
Register JC, Peterson DJ, Bell PJ, Bullock WP, Evans IJ, Frame B, Greenland AJ, Higgs NS, Jepson I, Jiao S, Lewnau CJ, Sillick JM, Wilson HM (1994) Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment. Plant Mol Biol 25: 951–961
Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison JS (1989) In situ localization of parental genomes in a wide hybrid. Ann Bot 64: 315–324
Strehl S, Ambros PF (1993) Fluorescence in situ hybridization with immunohistochemistry for highly sensitive detection of chromosome 1 aberrations in neuroblastoma. Cytogenet Cell Genet 63: 24–28
Vaucheret H (1993) Identification of a general silencer for 19S and 35S promoters in a transgenic tobacco plant: 90 bp of homology in the promoter sequence are sufficient for trans-inactivation. C R Acad Sci Paris 316: 310–323
Vaucheret H, Kronenberger J, Lepingle A, Vilaine F, Boutin J-P, Caboche M (1992) Inhibition of tobacco nitrite reductase activity by expression of antisense RNA. Plant J 2: 559–569
Wang J, Lewis ME, Whallon JH, Sink KC (1995) Chromosome mapping of T-DNA inserts in transgenePetunia by in situ hybridization. Transgenic Res 4: 241–246
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Moscone, E.A., Matzke, M.A. & Matzke, A.J.M. The use of combined FISH/GISH in conjuction with DAPI counterstaining to identify chromosomes containing transgene inserts in amphidiploid tobacco. Chromosoma 105, 321–326 (1996). https://doi.org/10.1007/BF02524650
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DOI: https://doi.org/10.1007/BF02524650