RAD-Seq analysis of Plantago ovata var. fastigiata for landscape genomics
Total genomic DNA was extracted at California Botanic Garden (Claremont CA) from silica dried plant leaf tissue using a modified CTAB method, amended to address polysaccharides present in tissue with a pectinase incubation step. DNAs were normalized to 20 ng/ml to obtain 1 mg of DNA for genomic library preparation.  Restriction-site Associated DNA Sequencing (RADseq) libraries were prepared and sequenced by Floragenex (Portland, OR), with PstI as the single-digest restriction enzyme. Samples were barcoded and multiplexed in two libraries (380 samples per library), and single-end sequencing was run on a 1  100 bp Illumina HiSeq4000 platform at the University of Oregon Genomics facility. Lanes were 100% allocated, and samples were run in replicate to improve depth of coverage. Genotypes were derived from raw data using Stacks 2.0 (Catchen et al. 2013) to assemble RAD-tags and to call genotypes. Raw sequences were trimmed of restriction site sequence, demultiplexed, and binned into individual sample files using process radtags. The read data from the two replicate runs were then merged to a single data file for each individual.