title: Genotyping-by-sequencing of Synthetic W9784 x Opata M85 DH population
design: Plant Material. Two community resource double-haploid (DH) populations were used for genetic mapping. In barley, the Oregon Wolfe Barley (OWB) DH population was developed from a cross between morphologically diverse genotypes that are differentiated by a number of dominant morphological markers [23,28]. We used a set of 82 DH lines from this population along with replicated samples of each parent (OWBdom and OWBrec, the dominant and recessive parents, respectively) to develop two 48-plex libraries. The OWB lines have been genotyped with 2,832 SNP, DArT (Diversity Arrays Technology Pty Ltd) and SSR markers and a combined reference map is available (www.barleyworld.org). In wheat, we used 147 lines from the Synthetic W9784 x Opata M85 DH population [24]. These lines have been genotyped with 1,351 DArT markers. DNA from each populations was extracted from seedling tissue using a standard CTAB protocol [29]. DNA was quantified in plates using PicoGreen and DNA concentrations were normalized to 20ng/ul.    Adapters. A set of 48 barcoded adapters with a PstI overhang was designed using a custom script in Java (sourceforge.net/projects/tassel/). The barcodes were designed with the following criteria 1) each barcode must be 2 or more bp different from all other barcodes, 2) barcodes can not contain a run of more than 2 of the same nucleotide, 3) barcodes can not contain or recreate (when ligated) the PstI or MspI restriction site. The full-set of barcodes was designed to optimize the uniformity of each nucleotide at each position. Nucleotide uniformity was accomplished by designing barcodes of different lengths (4bp to 9bp) and selecting nucleotides in the barcode that balanced bases in the restriction site [19]. An example of a barcoded adapter pair (barcode sequence shown with capitol bases):  A01_AAGTGA_top   5’ – gatctacactctttccctacacgacgctcttccgatctAAGTGAtgca – 3’  A01_AAGTGA_bot  5’ – TCACTTagatcggaagagcgtcgtgtagggaaagagtgtagatc – 3’  The full list of barcoded adapters for PstI is included in Table S1.      The common adapter was designed as a Y-adapter to prevent amplification of the more common MspI-MspI fragments and adapter-dimers formed by self-ligation of adapter fragments. During the PCR amplification the reverse primer is identical to the Y-tail of the common adapter and can only anneal if the complimentary strand has first been synthesized from the other end of the fragment containing Adapter 1 (Figure 1).   Standard, unmodified oligos were ordered in complimentary pairs and annealed in a high-salt solution to form the double-stranded adapter prior to use. The adapters were annealed by heating to 95C and then slowly cooling to 30C at a rate of -1C/minute in a BioRad DNA engine by programming a single step PCR cycle at 95C for 1 minute and then decreasing the temperature by 1C each cycle for 65 cycles. After ligation, the adapters were quantified using Quant-iTTM PicoGreen® (Molecular Probes / Invitrogen Eugene, OR 97402). The adapters were adjusted to a uniform concentration of 0.1 uM.     Restriction Digest. Genomic DNA (200ng) was digested in 20ul reaction volume of NEB Buffer 4 with 8U of HF-PstI (High-Fidelity) and 8U of MspI (New England BioLabs Inc., Ipswich, MA 01938).  The digest was conducted at 37C for 2h and then 65C for 20 min to inactivate the enzymes.     Ligation. The ligation reaction was completed in the same tube/plate as the digestion, again using NEB Buffer 4 with the addition of ATP. For wheat and barley, 0.1 pmol of the respective Adapter 1 (0.1 pmol for 200ng of genomic DNA) and 15pmol of the common Y-adapter were added to the samples. A master mix of NEB Buffer 4 (1x final), ATP (1mM final), and 200U T4 ligase (NEB T4 DNA Ligase #M0202) were added to each sample.  The ligation was completed at 22C for 2h and the ligase was inactivated prior to pooling the samples by holding at 65C for 20 min.     Multiplexing and Amplification.  Ligated samples were pooled and PCR-amplified in a single tube, producing a single library from 48 samples, which was sequenced on a single lane of Illumina GAII or HiSeq2000. The libraries were amplified for 18 cycles consisting of 95 (30 sec), 62C (30 sec), 68C (30 sec).     Sequencing. Two 48-plex libraries from the OWB population and four 48-plex libraries from the SynOpDH population were constructed for this study. To construct full libraries, the parents and a few of the DH lines were replicated. The barley libraries were each sequenced on one lane of Illumina GAII and one lane of Illumina HiSeq2000. The first two wheat libraries were sequenced on Illumina GAII and the second two libraries were sequenced on Illumina HiSeq2000.