Sequence-tagged high-density genetic maps of Zoysia japonica
The RAD-Seq libraries were constructed according to the protocol described by Zhang et al.(Zhang et al., 2014). Approximately 1 µg of genomic DNA was digested with restriction endonuclease Nsi I and Mse I (New England Biolabs, Ipswich, MA) overnight at 37?. The reactions were stopped by incubation at 80? for 20 minutes. P1 and P2 adapters containing unique identifying sequences (Molecular Identifier, MID, supplemental Table S1) were ligated to the digested DNA samples and the ligation reaction was performed with 30µl of digested genomic DNA, 2µl of NEB buffer 3, 8µl of 0.1µM Adapter P1, 1µl of 10µM Adapter P2, 5µl of 10mM ATP, 2.75µl of H2O, and 500 U T4 Ligase (NEB) at room temperature for 2 hours or at 4?overnight.  The ligation reaction was deactivated at 65?for 20 minutes, and then purified using Promega Wizard SV Gel and PCR Clean- up Kit (Promega Corporation, Madison, WI). The purified products were amplified using Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA), and then size-selected on a 1% agarose gel to isolate DNA fragments spanning 200-500 bp. Selected DNA fragments were then purified with the Promega Wizard SV Gel and PCR Clean- up Kit again (Promega Corporation, Madison, WI). The purified RAD-Seq libraries were quantified by a Qubit Fluorometer using Qubit dsDNA HS Assay kit (Life Technologies, Grand Island, NY), and diluted to 10nM for pooling and sequencing.  The pooled DNA concentration was further confirmed by qPCR and sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA).