Genomic DNA was extracted from 95 individual F2 plants and five plants of each parent using a CTAB method (Murray and Thompson 1980 (link)). DNA concentrations were quantified using the QubitTM dsDNA BR Assay Kit (Life Technologies, Carlsbad, CA, USA) with a Qubit fluorometer 2.0 (Invitrogen, Grand Island, NY, USA), according to the manufacturers’ directions and were adjusted to a final concentration of 20 ng μL–1.
The double-digest restriction-site associated DNA (RAD) library (Peterson et al. 2012 (link)) was generated as described (Sakaguchi et al. 2015 ) with slight modifications. Genomic DNA (10 ng) was digested with EcoRI-HF and BglII (New England Biolabs, Ipswich, MA, USA), ligated to barcoded adapters and purified with AMPure®XP (Beckman Coulter, Pasadena, CA, USA). Libraries were amplified using KAPA HiFi HS ReadyMix (Kapa Biosystems, Wilmington, MA, USA) and the PCR products were purified using AMPure®XP. Fragments 200–1,000 bp in size were selected with E-Gel Size Select (Life Technologies, Carlsbad, CA, USA); the average size of the selected fragments was 337 bp (CV 21.0%). The library was constructed by Clockmics Inc. (Osaka, Japan) and sequenced with 50 bp single-end reads in one lane of an Illumina HiSeq2000 (Illumina, San Diego, CA, USA) by Macrogen (Seoul, South Korea).
The double-digest restriction-site associated DNA (RAD) library (Peterson et al. 2012 (link)) was generated as described (Sakaguchi et al. 2015 ) with slight modifications. Genomic DNA (10 ng) was digested with EcoRI-HF and BglII (New England Biolabs, Ipswich, MA, USA), ligated to barcoded adapters and purified with AMPure®XP (Beckman Coulter, Pasadena, CA, USA). Libraries were amplified using KAPA HiFi HS ReadyMix (Kapa Biosystems, Wilmington, MA, USA) and the PCR products were purified using AMPure®XP. Fragments 200–1,000 bp in size were selected with E-Gel Size Select (Life Technologies, Carlsbad, CA, USA); the average size of the selected fragments was 337 bp (CV 21.0%). The library was constructed by Clockmics Inc. (Osaka, Japan) and sequenced with 50 bp single-end reads in one lane of an Illumina HiSeq2000 (Illumina, San Diego, CA, USA) by Macrogen (Seoul, South Korea).
