title: Invasive sunflower GBS
design: Genomic DNA was extracted from the young leaves of a single seedling for each genotype. Pre-warmed CTAB buffer (100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 2% w/v CTAB, 0.5% w/v NaHSO3) was added and the samples were incubated at 65oC for 60 minutes. After a short cool-down, cold Phenol/Chloroform/IAA (25:24:1) mixture was added and the samples were vigorously vortexed and centrifuged for 5 minutes at 14000rpm in 4oC. The upper phase was transferred into a new tube, and Chloroform/IAA (24:1) mixture was added. Samples were again vigorously vortexed and centrifuged for 5 minutes at 14000rpm in 4oC. Next, the upper phase was transferred again into a new tube and treated with 5l RNase A (10mg/ml, abm-G117) for 30 minutes at 37oC. Once again Chloroform/IAA (24:1) was added, vortexed, centrifuged and upper phase was transferred to a new tube followed by NaCl/Isopropanol precipitation for 30 minutes at -20oC and two ice-cold 70% ethanol washes. Finally, samples were eluted in 50l Low TE buffer (10mM Tris HCl pH 8.0 and 0.1mM EDTA pH 8.0). For GBS library preparation, 200 ng of genomic DNA was digested with two restriction enzymes (PstI, NEB-R3140 and MspI, NEB-R0106) for 5 hours at 37oC (Poland et al., 2012). Each sample was ligated with a unique barcoded adapter and a common adapter for 2 hours at 22oC, and was cleaned and concentrated using 1.6X volumes of SPRI beads (Fisher Scientific #09-981-123), prepared following Rohland and Reich (2012). Next, each sample was amplified using 2X KAPA HiFi HotStart ReadyMix (KAPABIOSYSTEMS - KK2600) and Illumina_PE forward and reverse primers, and quantified using the Qubit dsDNA BR Assay Kit (Thermo-Q32850). From each of the 178 libraries, 100 ng were pooled and concentrated, and quantified using the Qubit dsDNA BR Assay Kit. Pooled libraries were loaded on 1.5% agarose gel and DNA fragments in the 400-600 bp range were cut and extracted using Wizard SV Gel and PCR Clean-Up System (Promega-A9281) and quantified with the Qubit dsDNA BR Assay Kit. We used duplex-specific nuclease (DSN) normalization (Shagina et al., 2010) to reduce the portion of repetitive sequences in the samples, and re-amplified the pooled-depleted library with the Illumina_PE primers. Fragment size was analyzed using the Agilent High Sensitivity D1000 ScreenTape (Agilent-5067-5584). The pooled-library was first sequenced on a MiSeq system (Illumina) using the V2 Nano kit, to validate and confirm normalization between samples, and then on 4 lanes on the Illumina NextSeq550 to generate 2X150bp reads in the high output mode.