High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes

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 T-1 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.

Automated summary

This study developed a simple method using T-DNA-specific primers and high-throughput sequencing to identify transgene insertion sites in the T-1 generation of any crop transformed via Agrobacterium. The example using allohexaploid oil-seed plant Camelina sativa demonstrated the feasibility of determining insertion site location of two transgenes. This new methodology allows for early selection of desirable transgene location and copy number to generate homozygous lines within two generations.