RAD-seq of Populus alba and Populus tremula hybrids
DNA was extracted from 15-20 mg of silica-dried leaf material with the Qiagen DNeasy Plant Mini Kit (Valencia, CA). The concentration of DNA was measured with a Qubit 2.0 Fluorometer using the dsDNA HS assay kit (Invitrogen), and its integrity verified with electrophoresis on 1.5% agarose gels (1X TBE). Concentrations were standardized to 20 ng/l and individual samples were submitted for library preparation and Restriction site Associated DNA sequencing (RAD-seq) to Floragenex (Eugene, OR). There, all extractions of the individuals of the first set, as well as the replicate extractions of F039_05 and I373_A, were processed (together with additional samples prepared in the same way), in five libraries of 95 individuals each. These libraries were prepared according to Floragenex standard commercial protocol: genomic DNA was digested with the restriction endonuclease PstI (chosen according to previous studies on these species - Stlting et al. 2013; Christe et al. 2016) and RAD libraries were prepared with a method similar to the one described in Baird et al. (2008). This protocol included 18 PCR cycles, after which DNA fragments ranging from 300 to 500 bp were retained. All five libraries were sequenced in a single run on an Illumina HiSeq2500 instrument, but on individual lanes (named C436, C437, C438, C439 and C440).