*2.2. Sequencing Library Construction and Genotyping*

Genomic DNA was extracted from the fresh young leaves of the Koshihikari × Baegilmi RILs using the CTAB (cetyl trimethylammonium bromide) method [16] with minor modifications. The quality and quantity of the extracted DNA were checked using the DeNovix DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA) and the Quant-iTTM dsDNA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The genotyping-by-sequencing (GBS) library was constructed according to [17]. Briefly, each DNA sample was digested with the *ApeKI* restriction enzyme (New England Biolabs, Ipswitch, MA, USA), ligated with barcode adapters, pooled, and purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). The pooled libraries were amplified by PCR with an adapter-specific primer set, analyzed for the target length of 170–350 bp using BioAnalyzer 2100 (Agilent, Santa Clara, CA, USA), and sequenced using the Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA). The sequencing reads were mapped to the Nipponbare IRGSP-1.0 reference using the BWA aligner [18] and single nucleotide polymorphism (SNPs) were extracted in the format of a VCF (variant calling format) file. The SNP filtering and genotype calling of each RIL were carried out using TASSEL-GBS v2 [19] with the filtering conditions (mapping quality ≥30, base quality ≥20, and coverage ≥7) as described in [20]. Genotype calling for each SNP was conducted by defining a homozygous genotype when the same sequence rate is over 0.90 and a heterozygous genotype when the alternative sequence rate is 0.25–0.27.
