Genotyping-by-Sequencing of RIL Population

The genomic DNA isolated using cetyltrimethyl ammonium bromide (CTAB) method (Lodhi et al., 1994 (link)) from the young leaves of parental lines and 166 RIL population were used for the preparation of GBS libraries (Elshire et al., 2011 (link)). In brief, 100 ng DNA was digested for 4.0 h at 75 °C with ApeKI (New England Biolabs, Ipswitch, MA) in 20 µL volume containing 1 × NEB Buffer3 and 3.6U ApeKI. The purified 168-plex final DNA library was quantified using Bioanalyzer (Agilent Technologies) and was sequenced on Novaseq 6000 (Illumina^® Inc., San Diego, CA, USA). Library construction and sequencing were done by NGB Diagnostics Pvt Ltd (India) and SNPs were identified. Variant calling was done using UGBS-GATK pipeline (version v3.6, https://gatk.broadinstitute.org/hc/en-us). Further SNPs were filtered based on minor allele frequency (MAF) of 5%, being present in at least 50% of the population, the proportion of heterozygosity, and polymorphism information,