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Transgene integration and organization in Cotton (Gossypium hirsutum L.) genome

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Abstract

While genetically modified upland cotton (Gossypium hirsutum L.) varieties are ranked among the most successful genetically modified organisms (GMO), there is little knowledge on transgene integration in the cotton genome, partly because of the difficulty in obtaining large numbers of transgenic plants. In this study, we analyzed 139 independently derived T0 transgenic cotton plants transformed by Agrobacterium tumefaciens strain AGL1 carrying a binary plasmid pPZP-GFP. It was found by PCR that as many as 31% of the plants had integration of vector backbone sequences. Of the 110 plants with good genomic Southern blot results, 37% had integration of a single T-DNA, 24% had two T-DNA copies and 39% had three or more copies. Multiple copies of the T-DNA existed either as repeats in complex loci or unlinked loci. Our further analysis of two T1 populations showed that segregants with a single T-DNA and no vector sequence could be obtained from T0 plants having multiple T-DNA copies and vector sequence. Out of the 57 T-DNA/T-DNA junctions cloned from complex loci, 27 had canonical T-DNA tandem repeats, the rest (30) had deletions to T-DNAs or had inclusion of vector sequences. Overlapping micro-homology was present for most of the T-DNA/T-DNA junctions (38/57). Right border (RB) ends of the T-DNA were precise while most left border (LB) ends (64%) had truncations to internal border sequences. Sequencing of collinear vector integration outside LB in 33 plants gave evidence that collinear vector sequence was determined in agrobacterium culture. Among the 130 plants with characterized flanking sequences, 12% had the transgene integrated into coding sequences, 12% into repetitive sequences, 7% into rDNAs. Interestingly, 7% had the transgene integrated into chloroplast derived sequences. Nucleotide sequence comparison of target sites in cotton genome before and after T-DNA integration revealed overlapping microhomology between target sites and the T-DNA (8/8), deletions to cotton genome in most cases studied (7/8) and some also had filler sequences (3/8). This information on T-DNA integration in cotton will facilitate functional genomic studies and further crop improvement.

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References

  • Brunaud V, Balzergue S, Dubreucq B, Aubourg S, Samson F, Chauvin S, Bechtold N, Cruaud C, DeRose R, Pelletier G, Lepiniec L, Caboche M, Lecharny A (2002) T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites. EMBO Rep 3:1152–1157

    Article  PubMed  CAS  Google Scholar 

  • Cluster PD, O’Dell M, Metzlaff M, Flavell RB (1996) Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression. Plant Mol Biol 32:1197–1203

    Article  PubMed  CAS  Google Scholar 

  • Crane CF, Price HJ, Stelly DM, Czeschin DG, McKnight TD (1993) Identification of a Homeologous Chromosome Pair by in-Situ DNA Hybridization to Ribosomal-Rna Loci in Meiotic Chromosomes of Cotton (Gossypium-Hirsutum). Genome 36:1015–1022

    CAS  PubMed  Google Scholar 

  • De Buck S, De Wilde C, Van Montagu M, Depicker A (2000) Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants. Mol Plant Microbe Interact 13:658–665

    Article  PubMed  Google Scholar 

  • De Buck S, Jacobs A, Van Montagu M, Depicker A (1999) The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. Plant J 20:295–304

    Article  PubMed  Google Scholar 

  • De Neve M, De Buck S, Jacobs A, Van Montagu M, Depicker A (1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11:15–29

    Article  PubMed  Google Scholar 

  • Forsbach A, Schubert D, Lechtenberg B, Gils M, Schmidt R (2003) A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome. Plant Mol Biol 52:161–176

    Article  PubMed  CAS  Google Scholar 

  • Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994

    Article  PubMed  CAS  Google Scholar 

  • Haseloff J, Amos B (1995) GFP in plants. Trends Genet 11:328–329

    Article  PubMed  CAS  Google Scholar 

  • Iglesias VA, Moscone EA, Papp I, Neuhuber F, Michalowski S, Phelan T, Spiker S, Matzke M, Matzke AJ (1997) Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco. Plant Cell 9:1251–1264

    Article  PubMed  CAS  Google Scholar 

  • Jakowitsch J, Papp I, Moscone EA, van der Winden J, Matzke M, Matzke AJ (1999) Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters in trans. Plant J 17:131–140

    Article  PubMed  CAS  Google Scholar 

  • Jeong DH, An S, Park S, Kang HG, Park GG, Kim SR, Sim J, Kim YO, Kim MK, Kim J, Shin M, Jung M, An G (2006) Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice. Plant J 45:123–132

    Article  PubMed  CAS  Google Scholar 

  • Kirik A, Salomon S, Puchta H (2000) Species-specific double-strand break repair and genome evolution in plants. Embo J 19:5562–5566

    Article  PubMed  CAS  Google Scholar 

  • Kohli A, Griffiths S, Palacios N, Twyman RM, Vain P, Laurie DA, Christou P (1999) Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant J 17:591–601

    Article  PubMed  CAS  Google Scholar 

  • Kohli A, Leech M, Vain P, Laurie DA, Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci U S A 95:7203–7208

    Article  PubMed  CAS  Google Scholar 

  • Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J 11:945–957

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Fladung M (2000) Transgene repeats in aspen: molecular characterisation suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome. Mol Gen Genet 264:20–28

    Article  PubMed  CAS  Google Scholar 

  • Lange M, Vincze E, Moller MG, Holm PB (2006) Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation. Plant Cell Rep 25:815–820

    Article  PubMed  CAS  Google Scholar 

  • Li XB, Fan XP, Wang XL, Cai L, Yang WC (2005) The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation. Plant Cell 17:859–875

    Article  PubMed  CAS  Google Scholar 

  • Matzke AJ, Matzke MA (1998) Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol 1:142–148

    Article  PubMed  CAS  Google Scholar 

  • Newell CA (2000) Plant transformation technology. Developments and applications. Mol Biotechnol 16:53–65

    Article  PubMed  CAS  Google Scholar 

  • Olhoft PM, Flagel LE, Somers DA (2004) T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method. Plant Biotechnol J 2:289–300

    Article  PubMed  CAS  Google Scholar 

  • Parinov S, Sevugan M, Ye D, Yang WC, Kumaran M, Sundaresan V (1999) Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis. Plant Cell 11:2263–2270

    Article  PubMed  CAS  Google Scholar 

  • Parinov S, Sundaresan V (2000) Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project. Curr Opin Biotechnol 11:157–161

    Article  PubMed  CAS  Google Scholar 

  • Paterson AH (1993) A rapid method for extraction of cotton Genomic DNA suitable for RFLP or PCR analysis. Plant Mol Biol Reptr 1:122–127

    Article  Google Scholar 

  • Raina S, Mahalingam R, Chen F, Fedoroff N (2002) A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana. Plant Mol Biol 50:93–110

    Article  PubMed  CAS  Google Scholar 

  • Stahl R, Horvath H, Van Fleet J, Voetz M, von Wettstein D, Wolf N (2002) T-DNA integration into the barley genome from single and double cassette vectors. Proc Natl Acad Sci U S A 99:2146–2151

    Article  PubMed  CAS  Google Scholar 

  • Thomas CM, Jones DA, English JJ, Carroll BJ, Bennetzen JL, Harrison K, Burbidge A, Bishop GJ, Jones JD (1994) Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction. Mol Gen Genet 242:573–585

    Article  PubMed  CAS  Google Scholar 

  • Topping JF, Wei W, Clarke MC, Muskett P, Lindsey K (1995) Agrobacterium-mediated transformation of Arabidopsis thaliana. Application in T-DNA tagging. Methods Mol Biol 49:63–76

    PubMed  CAS  Google Scholar 

  • Tzfira T, Li J, Lacroix B, Citovsky V (2004) Agrobacterium T-DNA integration: molecules and models. Trends Genet 20:375–383

    Article  PubMed  CAS  Google Scholar 

  • Wenck A, Czako M, Kanevski I, Marton L (1997) Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol Biol 34:913–922

    Article  PubMed  CAS  Google Scholar 

  • Yin Z, Wang GL (2000) Evidence of multiple complex patterns of T-DNA integration into the rice genome. Theor Appl Genet 100:461–470

    Article  CAS  Google Scholar 

  • Zambryski P (1988) Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu Rev Genet 22:1–30

    Article  PubMed  CAS  Google Scholar 

  • Zambryski PC (1992) Chronicles from the Agrobacterium-Plant Cell-DNA Transfer Story. Annu Rev Plant Physiol Plant Mol Biol 43:465–490

    Article  CAS  Google Scholar 

  • Zhao X, Wing RA, Paterson AH (1995) Cloning and characterization of the majority of repetitive DNA in cotton (Gossypium L.). Genome 38:1177–1188

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This project was supported by an internal research grant of Temasek Life Sciences Laboratory, Singapore. We would like to thank Tan Jason and Ng Khar Meng for technical help.

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Correspondence to Yan Hong.

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Zhang, J., Cai, L., Cheng, J. et al. Transgene integration and organization in Cotton (Gossypium hirsutum L.) genome. Transgenic Res 17, 293–306 (2008). https://doi.org/10.1007/s11248-007-9101-3

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  • DOI: https://doi.org/10.1007/s11248-007-9101-3

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