SLAF-seq for Genetic Mapping

SLAF-seq method^{13 (link)} was used. First, the genome of the parents and F2 population was digested by Hpy166II (New England Biolabs, MA, USA). Then polyA as dATP was ligated to the end of the digested fragment by employing the Klenow fragment (3′–5′ exo-) (New England Biolabs) at 37 ℃. Next, the PAGE-purified dual-label sequencing markers (Life Technologies, CA, USA) were ligated to the newly added terminal polyA utilizing the T4-DNA ligase. PCR was performed with the diluted DNA samples, and the forward: 5′-AATGATACCGACCACCGA-3′ and reverse: 5′-CAAGCAGAAGACGGCATA-3′ primers, Q5^® High-Fidelity DNA Polymerase (NEB), and dNTPs. The PCR products were purified and collected by Agencourt AMPure XP beads (Beckman Coulter, High Wycombe, UK) and separated by electrophoresis on a 2% agar gel. The DNA fragment (with indices and adaptors) between 264 and 464 bp was electrophoresed again, and the band was extracted from the gel employing a QIAquick^® gel extraction kit (Qiagen, Hilden, Germany). The paired, terminal 125 bp sequences obtained were analyzed on a Hi-Seq 2500 system (Illumina Inc., CA, USA).