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Transgene excision from wheat chromosomes by phage phiC31 integrase

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Abstract

The Streptomyces phage phiC31 integrase was tested for its ability to excise transgenic DNA from the wheat genome by site-specific recombination. Plants that stably express phiC31 integrase were crossed to plants carrying a target construct bearing the phiC31 recognition sites, attP and attB. In the progeny, phiC31 recombinase mediates recombination between the att sites of the target locus, which results in excision of the intervening DNA. Recombination events could be identified in 34 independent wheat lines by PCR and Southern blot analysis and by sequencing of the excision footprints. Recombinant loci were inherited to the subsequent generation. The results presented here establish the integrase-att system as a tool for catalysing the precise elimination of DNA sequences from wheat chromosomes.

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Abbreviations

ALS:

Acetolactate synthase

attB :

Bacterial attachment site, phiC31 target recombination sequence

attP :

Phage attachment site, phiC31 target recombination sequence

attR, attL:

phiC31 integrase recombination products

DH:

Doubled haploid

pICH14313, pICH13130:

Vectors containing a Streptomyces phage phiC31 integrase coding sequence

ICH14313, ICH13130:

Genomic locus containing the Streptomyces phage phiC31 integrase coding sequence

pICH27371:

Vector containing phiC31 integrase target recombination sequences

ICH27371:

Genomic target locus carrying phiC31 integrase target recombination sequences

ICH27371-N, ICH27371-C:

Derivative genomic locus resulting from phiC31 integrase-mediated site-specific recombination

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Acknowledgments

The authors are grateful to Dr. Heike Schmuths (Saaten Union Biotec Gatersleben, Germany) for logistic cooperation and Kerstin Denzin, Linda Tillack, Christin Meinhardt and Erika Grützemann for laboratory support and plant care. Furthermore, we acknowledge Wolf v. Rhade, Dr. Ralf Schachschneider (Nordsaat GmbH Böhnshausen, Germany), Dr. Jens Weyen (Saaten Union Biotec, Leopoldshöhe, Germany) and Dr. Frank Wolter (PflanzenInnovationsAgentur; PIA) for constant support. We thank anonymous reviewers for helpful comments. In particular, the authors also wish to thank Dr. Renate Schmidt for her comments on the manuscript and for many stimulating discussions. The research was done at the Leibniz Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK) Gatersleben with funding from the Bundesministerium für Bildung und Forschung (BMBF, GABI-FUTURE grant 0315043A).

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Correspondence to Mario Gils.

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Katja Kempe and Myroslava Rubtsova contributed equally to the paper.

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Kempe, K., Rubtsova, M., Berger, C. et al. Transgene excision from wheat chromosomes by phage phiC31 integrase. Plant Mol Biol 72, 673–687 (2010). https://doi.org/10.1007/s11103-010-9606-7

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