SNP markers used for QTL mapping in the inbred lines

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.