Targeted sequencing of T-DNA borders in OCP1xOGC transgenic lines of Camelina

Background: Genetic engineering of crop plants has been successful in
transferring traits into elite lines beyond what can be achieved with
breeding techniques. Introduction of transgenes originating from other
species has conferred resistance to biotic and abiotic stresses, increased
efficiency, and modified developmental programs. The next challenge is now
to combine multiple transgenes into elite varieties via gene stacking to
combine traits. Generating stable homozygous lines with multiple
transgenes requires selection of segregating generations which is time
consuming and labor intensive, especially if the crop is polyploid.
Insertion site effects and transgene copy number are important metrics for
commercialization and trait efficiency. Results: We have developed a
simple method to identify the sites of transgene insertions using
T-DNA-specific primers and high-throughput sequencing that enables
identification of multiple insertion sites in the T1 generation
of any crop transformed via Agrobacterium. We present an example
using the allohexaploid oil-seed plant Camelina
sativa to determine insertion site location of two transgenes.
Conclusion: This new methodology enables the early selection of desirable
transgene location and copy number to generate homozygous lines within two
generations.