DNA was extracted from the wheat leaves of 2–3 young two-week-old seedlings using BioSprint 96 DNA Plant Kits (Qiagen Valencia, California, USA) following the manufacturer’s instructions. The genotyping-by-sequencing (GBS) was performed as described by Poland et al. [48]. The SNPs were called using Tassel v5.2.40 GBS analysis pipeline with default parameters [12]. The GBS-tags were aligned to the reference genome using Burrows-Wheeler Aligner [36]. The reference genome v1.0 of the ‘Chinese Spring’ genome assembly from the International Wheat Genome Sequencing Consortium (IWGSC) was used in SNP calling. The raw sequence data of the 270 genotypes of the current study along with 6791 other genotypes previously genotyped in our program were combined for SNP calling in order to increase the coverage of the genome and read depth at SNP sites [9, 71]. A set of 200,064 SNPs were resulted from SNP calling. SNPs were removed from the dataset if they were either monomorphic, showed more than 20% missing values, had conflicting calls from SNP or exhibited minor allele frequencies (MAF) of less than 5% [30, 71]. Interestingly, none of our lines (270 F3,6) have missing information’s of more than 20%. The GBS data is available in (Supplementary Table S2).

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