Present address: Simplot Company, Simplot Plant Sciences, Boise, ID 83706, USA.
Implementing reverse genetics in Rosaceae: analysis of T-DNA flanking sequences of insertional mutant lines in the diploid strawberry, Fragaria vesca
Article first published online: 15 AUG 2010
Copyright © Physiologia Plantarum 2010
Volume 140, Issue 1, pages 1–9, September 2010
How to Cite
Oosumi, T., Ruiz-Rojas, J. J., Veilleux, R. E., Dickerman, A. and Shulaev, V. (2010), Implementing reverse genetics in Rosaceae: analysis of T-DNA flanking sequences of insertional mutant lines in the diploid strawberry, Fragaria vesca. Physiologia Plantarum, 140: 1–9. doi: 10.1111/j.1399-3054.2010.01378.x
- Issue published online: 15 AUG 2010
- Article first published online: 15 AUG 2010
- Received 1 November 2009; revised 21 April 2010
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.