SNP calls from 275 cannabis accession using GBS.

Methods: Barcoded and multiplexed genomic DNA libraries were prepared from purified DNA samples based on the GBS methodology published by Gilchrist et al. (2022) and Poland et al. (2012). Paired-end (2 9 150bp) Illumina NextSeq sequencing and variant calling were performed according to Gilchrist et al. (2022), using Trimmomatic (Bolger et al., 2014) for read trimming and the GBS-SNP-CROP 2.0 pipeline (Melo et al., 2016) for parsing, demultiplexing, alignment, and variant calling on the ASM23057v2 Purple Kush genome assembly (Laverty et al., 2019).List of accession names and flowering phenotype are provided in Supplemental Table S1Bolger, A.M., Lohse, M. &amp; Usadel, B. (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30, 2114–2120. Available from: https://doi.org/10.1093/bioinformatics/btu170Gilchrist, E.J., Hegebarth, D., Wang, S., Quilichini, T.D., Sawler, J., Toh, S.Y. et al. (2022) A rapid method for sex identification in Cannabis sativa using high resolution melt analysis. Botany, 99,1–7. Available from: https://doi.org/10.1139/cjb-2021-0168Laverty, K.U., Stout, J.M., Sullivan, M.J., Shah, H., Gill, N., Holbrook, L. et al. (2019) A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci. Genome Research, 29, 146–156. Available from: https://doi.org/10.1101/gr.242594.118Melo, A.T., Bartaula, R. &amp; Hale, I. (2016) GBS-SNP-CROP: a referenceoptional pipeline for SNP discovery and plant germplasm characterization using variable length, paired-end genotyping-by-sequencing data. BMC Bioinformatics, 17,1–15. Available from: https://doi.org/10. 1186/s12859-016-0879-yPoland, J.A., Brown, P.J., Sorrells, M.E. &amp; Jannink, J.L. (2012) Development of high-density genetic maps for barley and wheat using a novel twoenzyme genotyping-by-sequencing approach. PLoS One, 7, e32253. Available from: https://doi.org/10.1371/journal.pone.0032253<br>