A Targeted Resequencing Pipeline Defines Insertion Events for InnateTM Potatoes

At JR Simplot Company, we produced a first generation of Innate potato (Solanum tuberosum) lines that displayed reduced black spot bruise, remained white upon peeling, and contained up to 75% less acrylamide when processed into chips or fries. Retransformation of the three best performing lines yielded a second generation of lines with further reductions in acrylamide and reducing sugars and, importantly, enhanced tolerance against late blight. Defining the insertion events within these lines using traditional methods, Southern blotting and PCR, proved complex and labor-intensive. The DNA used for both transformations was generally derived from potato sequences, so molecular analyses identified both endogenous and added copies of the genetic elements employed. Taking advantage of advances in next-generation sequencing and high throughput bioinformatic analyses, we developed a faster and more accurate approach to analyzing multiple lines. This approach is based on paired-end Illumina sequencing of DNA captured in solution by SureSelect baits that target the inserted sequences. Junction-finding algorithms that discriminate between endogenous and inserted DNA were used successfully to assemble reads into 1 kb-contigs containing approximately 0.5 kb of DNA sequence flanking the insertion sites. The sequence of these contigs was confirmed with conventional PCR. Interestingly, we also frequently identified insertion-fragment rearrangements that would have easily escaped detection using conventional approaches. We propose replacing standard methods by those described here for molecular analyses of genetically engineered crops considered for deregulation.