*2.5. DNA Extraction, SLAF Library Construction, and High-Throughput Sequencing*

An improved CTAB method was utilized to extract genomic DNA from the young leaves of two parental lines and 150 F2 individuals that were at the five- to six-leaf stage [45]. We employed an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and performed 1.0% agarose gel electrophoresis to respectively measure DNA concentration and quality. The SLAF-seq library was constructed as detailed previously by Sun et al. [33], with only a few small changes. The restriction enzyme *Hae*III (New England Biolabs, NEB, USA) was utilized for digestion of the genomic DNA of the parental lines and individuals of the F2 population. We added polyA tails to the 3 ends of the digested fragments, which were then connected to duplex-labelled sequencing adapters and PCR amplified. PCR was performed with the diluted restriction-ligation DNA sample, Q5® High-Fidelity DNA polymerase (NEB), dNTPs, and PCR primers (forward, 5 -AATGATACGGCGACCACCGA-3 and reverse, 5 -CAAGCAGAAGACGGCATACG-3 ). The PCR products were purified with Agencourt AMPure XP beads (Beckman Coulter, High Wycombe, UK) and then resolved on a 2% agarose gel. Fragments that were 314 to 364 bp in size were separated and purified with a QIAquick gel extraction kit (Qiagen, Hilden, Germany). SLAF-seq was then conducted on an Illumina High-Seq 2500 sequencing platform (Illumina, San Diego, CA, USA) at Beijing Biomarker Technologies Corp. (Beijing, China, http://www.biomarker. com.cn, accessed on: 8 January 2019). We employed the *Oryza sativa* L. genome as reference for quality control and conducted library construction and sequencing using similar settings as that for the pepper mapping population.
