Young leaves of the 175 inbred lines and their seven parents were collected from seedlings grown in a greenhouse. About 200 mg bulk leaf sample from three plants of a line was placed in 2 ml safe-lock Eppendorf tube and stored at ‒80 ˚C for one night prior to crushing using a Mixer Mill (TissueLyser II, Qiagen, Germany). Genomic DNA was extracted using SIGMA DNA extraction kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instruction. DNA concentration and purity of the samples were assessed using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The samples were processed and sequenced using tunable genotyping-by-sequencing (tGBS®) method by Data2Bio (Ames, IW, USA). Genomic DNA was digested using two restriction enzymes NSpI (5′-RCATG^Y-3′) and BfuCI/Sau3AI (5′-^GATC-3′) which created 3´and 5´overhangs, respectively. Two single-stranded oligos, one containing a sample-specific internal barcode and the other a universal oligo, were ligated to the complementary 3´ and 5´ overhangs, respectively. All 175 inbred lines' and seven parents' treated DNA was pooled for construction of the tGBS library and sequencing. The raw sequence data were demultiplexed by barcode, which was subsequently removed bioinformatically from each sequence. The barcode-trimmed sequence reads of genotype were further trimmed using the trimming software, Lucy (Chou & Holmes, 2001; Li & Chou 2004) to remove low-quality reads based on Phred quality scores of Q15.

The quality trimmed reads were aligned to the reference B. napus GCA_000751015.1 (Chalhoub et al. 2014, Brassica_napus_v4.1) with bowtie2 version 2.2.0 using the –local, --sensitive, -k 50 and --score-min L, 0, 0.8 parameters (Langmead & Salzberg, 2012). SNP calling was done based on the reads that align to a single location in the reference genome using the Genome Analysis Toolkit (GATK) version 3.2.0 UnifiedGenotyper tool with parameters -glm BOTH and -ploidy 2 (DePristo et al., 2011). SNPs with minor allele frequency (MAF) less than 5% and heterozygous calls (heterozygous loci) were considered as missing data, and the inbred lines with more than 24% missing data were eliminated from the analysis. Based on this, a total of 5,743 SNP markers were retained and used for association mapping.