ddRAD Library Preparation and Sequencing

Genomic DNA was extracted from young leaves of a single plant for each inbred line. ddRAD libraries for all inbred lines were constructed as previously described [21 (link)]. Briefly, 200 ng genomic DNA was digested with SacI and MseI. Restriction fragments for each individual were then ligated to the SacAD and MseAD adaptors with unique barcodes. The final ligates of 12 individuals were pooled to form a library and separated on a 2% agarose gel. Fragments in a size range between 220 and 500 bp, which corresponded to restriction fragments in a size range of 140–420 bp before adaptor ligation, were recovered from the gel and purified with a Qiagen gel purification kit (Qiagen Inc., Valenica, CA, USA). The pooled libraries were amplified and PCR products were separated on a 2% agarose gel. Fragments in a size range of 270–550 bp were purified with a Qiagen gel purification kit and submitted for sequencing on a HiSeq2000 platform with paired-end (PE) reads of 90 bp.