Ion Torrent sequencing as a tool for mutation discovery in the flax (Linum usitatissimum L.) genome

Detection of induced mutations is valuable for inferring gene function and for developing novel germplasm for crop improvement. Many reverse genetics approaches have been developed to identify mutations in genes of interest within a mutagenized population, including some approaches that rely on next-generation sequencing (e.g. exome capture, whole genome resequencing). As an alternative to these genome or exome-scale methods, we sought to develop a scalable and efficient method for detection of induced mutations that could be applied to a small number of target genes, using Ion Torrent technology. We developed this method in flax (Linum usitatissimum), to demonstrate its utility in a crop species.

We used an amplicon-based approach in which DNA samples from an ethyl methanesulfonate (EMS)-mutagenized population were pooled and used as template in PCR reactions to amplify a region of each gene of interest. Barcodes were incorporated during PCR, and the pooled amplicons were sequenced using an Ion Torrent PGM. A pilot experiment with known SNPs showed that they could be detected at a frequency > 0.3% within the pools. We then selected eight genes for which we wanted to discover novel mutations, and applied our approach to screen 768 individuals from the EMS population, using either the Ion 314 or Ion 316 chips. Out of 29 potential mutations identified after processing the NGS reads, 16 mutations were confirmed using Sanger sequencing.

The methodology presented here demonstrates the utility of Ion Torrent technology in detecting mutation variants in specific genome regions for large populations of a species such as flax. The methodology could be scaled-up to test >100 genes using the higher capacity chips now available from Ion Torrent.