GBS-seq of oryza sativa cultivar: Koniahu
Genotyping-by-sequencing (GBS) libraries were prepared following the protocol of Elshire et al. (2011) with minor modifications. Briefly, 100 ng of genomic DNA per sample was digested with ApeKI (New England Biolabs, Ipswich, MA, USA) at 75 C for 4 h in a 20 L reaction containing 1 NEB Buffer 3 and 3.6 U ApeKI. Barcoded adapters were ligated to the resulting sticky ends by adding 30 L of ligation mix containing 1.66 ligase buffer with ATP and T4 DNA ligase (New England Biolabs), followed by incubation at 22 C for 1 h and enzyme inactivation at 65 C for 30 min. A  uniquely barcoded samples were pooled (5 L per sample) and purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA), with final elution in 25 L. Restriction fragments were PCR-amplified in 50 L reactions containing 10 L pooled DNA, 25 L KAPA HiFi HotStart ReadyMix (KAPA Biosystems), and 1 L each of P5 and P7 dual-index primers (12.5 pmol). PCR products were purified using 0.9 AMPure XP beads (Beckman Coulter) to remove residual primers. The final library was quantified using an Agilent Bioanalyzer and sequenced on the Illumina HiSeq X10 platform using V4 sequencing chemistry.