(P0652) Genotyping-By-Sequencing and Reference Genome Enabled Variant Discovery in Octoploid Strawberry Genotyping-by-sequencing (GBS) approaches have enabled routine high-density genome-wide DNA variant discovery in numerous agriculturally important species. Applications of GBS in octoploid (2n = 8x = 56) strawberry (Fragaria × ananassa) have been hindered by the absence of a reference genome for physically mapping DNA sequences; for discovering variants with sub-genome resolution, or effectively distinguishing homologous from homeologous variation. High-quality reference genome assemblies have recently emerged, supplying the foundation for this study, which focused on demonstrating the utility of GBS for calling sub-genome specific DNA variants in octoploid strawberry. To reduce genomic DNA complexity, double-digest protocols were tested on diverse accessions with two restriction enzyme combinations (PstI-MseI and HindIII-MseI). GBS libraries were sequenced on an Illumina HiSeq 4000 using a 150 bp paired-end protocol. For the purpose of this study, we describe the deployment of a flexible bioinformatic pipeline for GBS-facilitated variant discovery in octoploid strawberry. The percentage of uniquely mapped reads ranged from 51.41% for PstI-MseI to 55.56% for HindIII- MseI resulting in 1,591,764 and 2,362,556 unique locations, respectively. The number of discovered variants was 2.5-fold greater for HindIII-MseI (491,811) than PstI-MseI (199,486). The GBS protocols uncovered a dense genome-wide landscape of DNA variants for high- precision genetic mapping, identification of DNA variants associated with agriculturally important phenotypes, genomic-enabled breeding, and other applications in octoploid strawberry. Poster: PDF of poster and abstract for PAG 2018 (P0652) Figures: PNGs of figures on the poster Scrips: Txt files of SLURM scripts used to generate the follow .vcf files. - 8x_GBS_0_index: Uses BWA to index the reference genome (Edger et al 2019) for later use. - 8x_GBS_1_Demultiplex: Used Sabre to demultiplex fastq.gz files. Demultiplex_key_PE links individuals to their unique barcode. - 8x_GBS_2_Main: adapter removal, sequence alignment, and individual variant calling as a SLURM array. results in a .gvcf file for individuals. - 8x_GBS_3_Variant: population-level variant calling to a final .vcf file VCF: Two VCF files from the two enzyme experiments. H = HindIII-MseI; P = PstI-MseI