DNA of Primary recombinant
Selected 65 primary recombinants along with 66 randomly selected non-recombinants were used for genotype-based sequencing that was performed on Ion Torrent sequencing platform at Western Regional Small Grain Genotyping Laboratory (https://plantpath.wsu.edu/see-lab/). In brief, ~100ng DNA from each plant was used for restriction digestion using PstI and MspI, and the library was prepared as previously described (28,29). Barcode ligation was performed on the fragmented DNA followed by pooling and amplification of pooled samples using PCR followed by cleaning using QIAquick PCR purification kit (QIAGEN, Valencia, CA, USA). Fragment size selection (~200bp) was made on E-Gel iBaseTM, and E-Gel Safe ImagerTM combo kit using E-Gel SizeSelectTM Agarose Gel (2%) Fragments were eluted in ddH2O (Thermo Fisher Scientific, Waltham, MA, USA). The library was checked for size distribution and quantification in Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA). Ion sphere particle emulsion PCR was conducted on Ion OneTouchTM 2 system and template ion sphere particles were enriched on Ion OneTouchTM ES following the protocol from Ion PI template OT2 200 kit V2 user Guide. Ion sphere quality control assay on Qubit 2.0 fluorometer was used to determine the percentage of templated ion sphere particles. Libraries with the optimum percent of template ion spheres were used for sequencing on Ion Proton Sequencer following Ion PI Sequencing 200 Kit V3 user guide.  Samples were loaded on Ion PI Chip v2 for sequencing. Parents were replicated four times to increase the sequencing depth.
SUB7786959
P28