DOI: 10.5061/dryad.8sf7m0crw
title: SNP markers used for QTL mapping in the inbred lines

description: 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.
description: # SNP markers used for QTL mapping in the inbred lines Dataset DOI:
 [10.5061/dryad.8sf7m0crw](10.5061/dryad.8sf7m0crw) ## Description of the
 data and file structure The dataset consists of
 a Brassica_napus_SNP-SNP5743_6.csv SNP discovery using genotype by
 sequencing Young leaves of the 184 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 a 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 the SIGMA DNA extraction kit (Sigma-Aldrich, St. Louis, MO, USA)
 following manufacture’s instruction. DNA concentration and purity of the
 samples was 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 191
 (184 lines + 7 parents) treated DNA was pooled for construction of tGBS
 library and sequencing. The raw sequence data was 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. Of the 184 inbred lines genotyped by SNP markers,
 nine lines had missing data greater than 37%; therefore, they were not
 included in association analysis. Thus, a total of 175 inbred lines were
 used in GWAS and further analyses; these lines derived from 34 F2 and 22
 BC1 plants of the above-mentioned six interspecific crosses. Normally,
 GWAS is carried out using a larger size population carrying wide diversity
 of alleles. However, the use of a small size inbred population derived
 from a single cross, for example, 122 doubled haploid lines of barley
 (Hordeum vulgare L.) (Hu et al., 2018), has shown the potential for
 mapping of different agronomic traits following association mapping
 approach. As mentioned above, the population that we used in this study,
 was theoretically, expected to be segregating for a part of the C genome
 and carrying allelic diversity of six B. oleracea accessions; therefore,
 the use of 175 lines in association mapping could be justified
 Code/software Excel