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.        flowcell lane sample_name barcode  6279UAAXX 1 M6 AAGTGA  6279UAAXX 1 SynOpDH0008 AGTTAAT  6279UAAXX 1 SynOpDH0018 CCACCA  6279UAAXX 1 SynOpDH0026 CTCGTCGT  6279UAAXX 1 SynOpDH0035 GCTAACA  6279UAAXX 1 SynOpDH0046 TCCGCA  6279UAAXX 1 Opata AATCG  6279UAAXX 1 SynOpDH0009 ATCATACCT  6279UAAXX 1 SynOpDH0019 CCACTGG  6279UAAXX 1 SynOpDH0027 CTTCCTCT  6279UAAXX 1 SynOpDH0036 GGAG  6279UAAXX 1 SynOpDH0047 TCTCA  6279UAAXX 1 SynOpDH0001 ACAGA  6279UAAXX 1 SynOpDH0010 ATGG  6279UAAXX 1 SynOpDH0020 CCATCCACT  6279UAAXX 1 SynOpDH0028 GACAG  6279UAAXX 1 SynOpDH0037 GGCCGA  6279UAAXX 1 SynOpDH0048 TGCGAGA  6279UAAXX 1 SynOpDH0002 ACCA  6279UAAXX 1 SynOpDH0011 ATGTTCAAT  6279UAAXX 1 SynOpDH0021 CCTG  6279UAAXX 1 SynOpDH0029 GACATCCA  6279UAAXX 1 SynOpDH0038 GTATA  6279UAAXX 1 SynOpDH0049 TGCTGAA  6279UAAXX 1 SynOpDH0003 AGAATGA  6279UAAXX 1 SynOpDH0013 ATTACA  6279UAAXX 1 SynOpDH0022 CGACG  6279UAAXX 1 SynOpDH0030 GACTCGG  6279UAAXX 1 SynOpDH0040 GTGCACCA  6279UAAXX 1 SynOpDH0050 TGGC  6279UAAXX 1 SynOpDH0005 AGGAG  6279UAAXX 1 SynOpDH0014 CACGACCA  6279UAAXX 1 SynOpDH0023 CGATGCGT  6279UAAXX 1 SynOpDH0031 GAGCGAA  6279UAAXX 1 SynOpDH0042 TATGGA  6279UAAXX 1 SynOpDH0051 TTGACCAG  6279UAAXX 1 SynOpDH0006 AGTCAAGA  6279UAAXX 1 SynOpDH0015 CAGGCCACT  6279UAAXX 1 SynOpDH0024 CGCTCA  6279UAAXX 1 SynOpDH0033 GATGA  6279UAAXX 1 SynOpDH0043 TATTCCACT  6279UAAXX 1 SynOpDH0052 TTGCGTCT  6279UAAXX 1 SynOpDH0007 AGTGTTAA  6279UAAXX 1 SynOpDH0017 CAGGCG  6279UAAXX 1 NA CTCACT  6279UAAXX 1 SynOpDH0034 GCGCCACT  6279UAAXX 1 SynOpDH0045 TCAT  6279UAAXX 1 SynOpDH0053 TTGTAG  6279UAAXX 2 SynOpDH0054 AAGTGA  6279UAAXX 2 SynOpDH0062 AGTTAAT  6279UAAXX 2 NA CCACCA  6279UAAXX 2 SynOpDH0078 CTCGTCGT  6279UAAXX 2 SynOpDH0086 GCTAACA  6279UAAXX 2 SynOpDH0094 TCCGCA  6279UAAXX 2 SynOpDH0055 AATCG  6279UAAXX 2 SynOpDH0063 ATCATACCT  6279UAAXX 2 SynOpDH0071 CCACTGG  6279UAAXX 2 SynOpDH0079 CTTCCTCT  6279UAAXX 2 SynOpDH0087 GGAG  6279UAAXX 2 SynOpDH0095 TCTCA  6279UAAXX 2 SynOpDH0056 ACAGA  6279UAAXX 2 SynOpDH0064 ATGG  6279UAAXX 2 SynOpDH0072 CCATCCACT  6279UAAXX 2 SynOpDH0080 GACAG  6279UAAXX 2 SynOpDH0088 GGCCGA  6279UAAXX 2 SynOpDH0096 TGCGAGA  6279UAAXX 2 SynOpDH0057 ACCA  6279UAAXX 2 SynOpDH0065 ATGTTCAAT  6279UAAXX 2 SynOpDH0073 CCTG  6279UAAXX 2 NA GACATCCA  6279UAAXX 2 SynOpDH0089 GTATA  6279UAAXX 2 SynOpDH0097 TGCTGAA  6279UAAXX 2 SynOpDH0058 AGAATGA  6279UAAXX 2 SynOpDH0066 ATTACA  6279UAAXX 2 SynOpDH0074 CGACG  6279UAAXX 2 SynOpDH0082 GACTCGG  6279UAAXX 2 SynOpDH0090 GTGCACCA  6279UAAXX 2 NA TGGC  6279UAAXX 2 SynOpDH0059 AGGAG  6279UAAXX 2 SynOpDH0067 CACGACCA  6279UAAXX 2 SynOpDH0075 CGATGCGT  6279UAAXX 2 SynOpDH0083 GAGCGAA  6279UAAXX 2 SynOpDH0091 TATGGA  6279UAAXX 2 SynOpDH0099 TTGACCAG  6279UAAXX 2 SynOpDH0060 AGTCAAGA  6279UAAXX 2 SynOpDH0068 CAGGCCACT  6279UAAXX 2 SynOpDH0076 CGCTCA  6279UAAXX 2 SynOpDH0084 GATGA  6279UAAXX 2 SynOpDH0092 TATTCCACT  6279UAAXX 2 SynOpDH0100 TTGCGTCT  6279UAAXX 2 SynOpDH0061 AGTGTTAA  6279UAAXX 2 SynOpDH0069 CAGGCG  6279UAAXX 2 SynOpDH0077 CTCACT  6279UAAXX 2 SynOpDH0085 GCGCCACT  6279UAAXX 2 SynOpDH0093 TCAT  6279UAAXX 2 SynOpDH0101 TTGTAG