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Implementing reverse genetics in Rosaceae: analysis of T-DNA flanking sequences of insertional mutant lines in the diploid strawberry, Fragaria vesca

Authors

  • Teruko Oosumi,

    1. Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
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    • Present address: Simplot Company, Simplot Plant Sciences, Boise, ID 83706, USA.

  • Juan Jairo Ruiz-Rojas,

    1. Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
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  • Richard E. Veilleux,

    1. Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
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  • Allan Dickerman,

    1. Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
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  • Vladimir Shulaev

    Corresponding author
    1. Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
    2. Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
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e-mail: vshulaev@vbi.vt.edu

Abstract

Reverse genetics is used for functional genomics research in model plants. To establish a model system for the systematic reverse genetics research in the Rosaceae family, we analyzed genomic DNA flanking the T-DNA insertions in 191 transgenic plants of the diploid strawberry, Fragaria vesca. One hundred and seventy-six T-DNA flanking sequences were amplified from the right border (RB) and 37 from the left border (LB) by thermal asymmetric interlaced PCR. Analysis of the T-DNA nick positions revealed that T-DNA was most frequently nicked at the cleavage sites. Analysis of 11 T-DNA integration sites indicated that T-DNA was integrated into the F. vesca genome by illegitimate recombination, as reported in other model plants: Arabidopsis, rice and tobacco. First, deletion of DNA was found at T-DNA integration target sites in all transgenic plants tested. Second, microsimilarities of a few base pairs between the left and/or right ends of the T-DNA and genomic sites were found in all transgenic plants tested. Finally, filler DNA was identified in four break-points. Out of 191 transgenic plants, T-DNA flanking sequences of 79 plants (41%) showed significant similarity to genes, elements or proteins of other plant species and 67 (35%) of the sequences are still unknown strawberry gene fragments. T-DNA flanking sequences of 126 plants (66%) showed homology to plant ESTs. This is the first report of T-DNA integration in a sizeable population of a rosaceous species. We have shown in this paper that T-DNA integration in strawberry is not random but directed by sequence microsimilarities in the host genome.

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