*2.1. Re-Sequencing and Genome-Wide SNPs*

In the present study, a set of 260 genotypes (97 from the recombinant inbreeding line (RIL) population from a cross between CDC Bethune and Macbeth (BM), 91 from the RIL population from a cross between E1747 and Viking (EV) and 72 from the doubled haploid population from a cross between SP2047 and UGG5-5 (SU) along with the 5 of 6 parents except for the reference CDC Bethune) were re-sequenced using GBS to identify genome-wide single nucleotide polymorphism (SNP) markers on the chromosome-based flax pseudomolecules [45]. An average of ~57.7 million paired end reads were generated for each individual, corresponding to 5754 Mb sequences or 19.2× genome equivalents of the reference scaffolds (~302 Mb) [46] (Table S1). Paired-end reads of each genotype were aligned to the flax scaffolds [46], resulting in a total of 536,186 SNPs. After filtering off SNPs with minor allele frequency (MAF) <0.05 and genotyping rate <60% [47,48], 17,288 SNPs were retained on the flax pseudomolecules [45] (Table S2). Out of these, 15,284 segregated in BM, 15,397 in EV and 7568 in SU. The SNPs were mostly uniformly distributed across all 15 chromosomes (chr), ranging from 601 on chr11 to 1572 on chr13 (Figure 1, Table S2). Approximately 71.1% of all SNPs were located in intergenic regions, 16.2% were in introns and 12.7% were in exons (Table S2). These SNPs were used for further population structure analysis, GWAS and GW3S.
