*4.2. Whole Genome Resequencing and SNP Calling*

Genotyping by sequencing (GBS) methodology was adopted to genotype the 407 accessions from the Canadian flax core collection. The 407 individuals were grown in pots in a greenhouse with a 16 h light and 8 h dark cycle. Young leaf tissues from single plants were collected for DNA extraction using the DNeasy 96 Plant kit (Qiagen, Mississauga, ON, Canada) according to manufacturer's instructions. Genomic DNA was quantified, sheared, size-selected, and libraries were constructed for each genotype by the Michael Smith Genome Sciences Centre of the BC Cancer Agency, Genome British Columbia (Vancouver, BC, Canada) which also sequenced the libraries generating 100 bp paired-end reads on the Illumina HiSeq 2000 platform (Illumina Inc., San Diego, CA, USA). A total of 26.875 billion 100 bp pair-end reads were generated, corresponding to 6587 MB sequences and 15.5× genome equivalents of the reference genome (~370 MB) [32,71], on average, per individual.

All reads from each individual of the population was aligned to the flax reference sequence (the flax pseudomolecules v2.0) [72] using BWA (v0.6.1) [73] with default parameters. The alignment file for each individual was used as input for SNP discovery using the software package SAMtools [74]. SAMtools converts the sequence alignment files from sequence alignment/map (SAM) to sorted binary alignment/map (BAM) format and call all potential variants (SNP and indels) into the pileup

files. Then, all variants were further filtered to get a set of high-quality SNPs. SNPs were filtered by the following criteria: (1) candidate SNP loci must be more than 10 bp away from each other; (2) each candidate SNP must have three mapped reads on the region; and (3) all the singleton SNPs were excluded. All steps of this procedure were implemented in an annotation-based genome-wide SNP discovery (AGSNP) pipeline [75,76]. The coordinates of all SNPs were extracted from the chromosome-based flax pseudomolecules v2.0 [72] totaling 1.7 million SNPs. Two hundred accessions, as previously mentioned, were selected for this study.
