Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.
Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.
Plant Breeding, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. henk.schouten@wur.nl.
Henk J
http://orcid.org/0000-0003-4495-1951
HJ
Schouten
Business Unit Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
Henri
H
Vande Geest
Business Unit Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
Sofia
S
Papadimitriou
Business Unit Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
Marian
M
Bemer
Plant Breeding, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
Jan G
JG
Schaart
Plant Breeding, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
Marinus J M
MJ
Smulders
Business Unit Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
Gabino Sanchez
GS
Perez
Business Unit Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
Elio
E
Schijlen
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D004269:DNA, Bacterial / Q000235:genetics*; D015321:Gene Rearrangement / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D030821:Plants, Genetically Modified; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA
Agrobacterium tumefaciens-mediated transformation; Arabidopsis thaliana; Molecular characterization; Mutation frequency; Next-generation sequencing; Splinter