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.

Data are unprocessed, PEx150bp FASTQ reads generated from the sequencing run. FASTQ files were demultiplexed from the original base call files using Illumina's FASTQ generator analysis pipeline within Local Run Manager.

Line1415_LB includes all data for Camelina transgenic line #1415 using a left-border-specific primer. Sample identifiers represent the genome-walking primer used in the PCR, as indicated in the README file.

Line1415_RB includes all data for Camelina transgenic line #1415 using a right-border-specific primer. Sample identifiers represent the genome-walking primer used in the PCR, as indicated in the README file.

Line1416_LB includes all data for Camelina transgenic line #1416 using a left-border-specific primer. Sample identifiers represent the genome-walking primer used in the PCR, as indicated in the README file.

Line1416_RB includes all data for Camelina transgenic line #1416 using a right-border-specific primer. Sample identifiers represent the genome-walking primer used in the PCR, as indicated in the README file.

At_LB includes all data for the Arabidopsis T-DNA insertion mutant (GK-269g12) using a left-border-specific primer. Sample identifiers represent the genome-walking primer used in the PCR, as indicated in the README file.

At_RB includes all data for the Arabidopsis T-DNA insertion mutant (GK-269g12) using a right-border-specific primer. Sample identifiers represent the genome-walking primer used in the PCR, as indicated in the README file.

Cs_LB includes all data for wildtype Camelina using a left-border-specific primer. Sample identifiers represent the genome-walking primer used in the PCR, as indicated in the README file.

Cs_RB includes all data for wildtype Camelina using a right-border-specific primer. Sample identifiers represent the genome-walking primer used in the PCR, as indicated in the README file.