Implementing reverse genetics in Rosaceae: analysis of T-DNA flanking sequences of insertional mutant lines in the diploid strawberry, Fragaria vesca.
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.
Oosumi
Teruko
T
Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
Ruiz-Rojas
Juan Jairo
JJ
Veilleux
Richard E
RE
Dickerman
Allan
A
Shulaev
Vladimir
V
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D031985:Fragaria / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D030821:Plants, Genetically Modified / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA