Genotyping by Sequencing in Plants using Ion PGM™ Sequencer

The development of molecular markers in crops such as barley and wheat has always been a challenge due the massive and complex genomes and the lack of reference sequences. The development of new sequencing technologies has greatly increased the discovery of SNPs in many species including important model and non-model crop plants. The discovery of high-density molecular markers in crop species will lead to a better understanding of the genetic architecture of complex traits and its application in breeding programs for crop improvement. We demonstrate a Genotyping by Sequencing (GBS) workflow for barley using Ion PGM™ Sequencer and transfer the results towards the usage with Ion Proton™ System. Size and complexity of the barley  genome  were significantly reduced by the creation of species-specific fragments that are enriched prior to library construction. We created two libraries of each 24 bar-coded and pooled barley samples for a total of 48 samples. The libraries were sequenced on multiple Ion 318™ Chips. This design was chosen in order to simulate the capacity of an Ion PI™ Chip on Ion Proton™ Sequencer. Roughly 14,000 novel SNPs could be discovered. This workflow based on Ion semiconductor sequencing is considered to be well suitable for the GBS application due to its broad throughput range and speed at low cost to allow genotyping in large plant populations.