title: Genotyping-by-sequencing of Oregon Wolfe Barley DH
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. 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).         gbs.flowcell gbs.lane dna.sample_id barcodes.well dna.sample_name barcodes.barcode  628P8AAXX 3 DNA100511P01_A01 1A OWB002 AAGTGA  628P8AAXX 3 DNA100511P01_A02 2A OWB011 AGTTAAT  628P8AAXX 3 DNA100511P01_A03 3A OWB021 CCACCA  628P8AAXX 3 DNA100511P01_A04 4A OWB031 CTCGTCGT  628P8AAXX 3 DNA100511P01_A05 5A OWB039 GCTAACA  628P8AAXX 3 DNA100511P01_A06 6A OWB047 TCCGCA  628P8AAXX 3 DNA100511P01_B01 1B OWB003 AATCG  628P8AAXX 3 DNA100511P01_B02 2B OWB013 ATCATACCT  628P8AAXX 3 DNA100511P01_B03 3B OWB023 CCACTGG  628P8AAXX 3 DNA100511P01_B04 4B OWB032 CTTCCTCT  628P8AAXX 3 DNA100511P01_B05 5B OWB040 GGAG  628P8AAXX 3 DNA100511P01_B06 6B OWB048 TCTCA  628P8AAXX 3 DNA100511P01_C01 1C OWB004 ACAGA  628P8AAXX 3 DNA100511P01_C02 2C OWB014 ATGG  628P8AAXX 3 DNA100511P01_C03 3C OWB024 CCATCCACT  628P8AAXX 3 DNA100511P01_C04 4C OWB033 GACAG  628P8AAXX 3 DNA100511P01_C05 5C OWB041 GGCCGA  628P8AAXX 3 DNA100511P01_C06 6C OWB049 TGCGAGA  628P8AAXX 3 DNA100511P01_D01 1D OWB006 ACCA  628P8AAXX 3 DNA100511P01_D02 2D OWB016 ATGTTCAAT  628P8AAXX 3 DNA100511P01_D03 3D OWB025 CCTG  628P8AAXX 3 DNA100511P01_D04 4D OWB034 GACATCCA  628P8AAXX 3 DNA100511P01_D05 5D OWB042 GTATA  628P8AAXX 3 DNA100511P01_D06 6D OWB051 TGCTGAA  628P8AAXX 3 DNA100511P01_E01 1E OWB007 AGAATGA  628P8AAXX 3 DNA100511P01_E02 2E OWB017 ATTACA  628P8AAXX 3 DNA100511P01_E03 3E OWB026 CGACG  628P8AAXX 3 DNA100511P01_E04 4E OWB035 GACTCGG  628P8AAXX 3 DNA100511P01_E05 5E OWB043 GTGCACCA  628P8AAXX 3 DNA100511P01_E06 6E OWB052 TGGC  628P8AAXX 3 DNA100511P01_F01 1F OWB008 AGGAG  628P8AAXX 3 DNA100511P01_F02 2F OWB018 CACGACCA  628P8AAXX 3 DNA100511P01_F03 3F OWB028 CGATGCGT  628P8AAXX 3 DNA100511P01_F04 4F OWB036 GAGCGAA  628P8AAXX 3 DNA100511P01_F05 5F OWB044 TATGGA  628P8AAXX 3 DNA100511P01_F06 6F OWB053 TTGACCAG  628P8AAXX 3 DNA100511P01_G01 1G OWB009 AGTCAAGA  628P8AAXX 3 DNA100511P01_G02 2G OWB019 CAGGCCACT  628P8AAXX 3 DNA100511P01_G03 3G OWB029 CGCTCA  628P8AAXX 3 DNA100511P01_G04 4G OWB037 GATGA  628P8AAXX 3 DNA100511P01_G05 5G OWB045 TATTCCACT  628P8AAXX 3 DNA100511P01_G06 6G OWB054 TTGCGTCT  628P8AAXX 3 DNA100511P01_H01 1H OWB010 AGTGTTAA  628P8AAXX 3 DNA100511P01_H02 2H OWB020 CAGGCG  628P8AAXX 3 DNA100511P01_H03 3H OWB030 CTCACT  628P8AAXX 3 DNA100511P01_H04 4H OWB038 GCGCCACT  628P8AAXX 3 DNA100511P01_H05 5H OWB046 TCAT  628P8AAXX 3 DNA100511P01_H06 6H OWB055 TTGTAG  628P8AAXX 4 DNA100511P01_A07 7A OWB056 AAGTGA  628P8AAXX 4 DNA100511P01_A08 8A OWB066 AGTTAAT  628P8AAXX 4 DNA100511P01_A09 9A OWB075 CCACCA  628P8AAXX 4 DNA100511P01_A10 10A OWB084 CTCGTCGT  628P8AAXX 4 DNA100511P01_A11 11A OWB093 GCTAACA  628P8AAXX 4 DNA100511P01_A12 12A OWB002 TCCGCA  628P8AAXX 4 DNA100511P01_B07 7B OWB057 AATCG  628P8AAXX 4 DNA100511P01_B08 8B OWB067 ATCATACCT  628P8AAXX 4 DNA100511P01_B09 9B OWB076 CCACTGG  628P8AAXX 4 DNA100511P01_B10 10B OWB085 CTTCCTCT  628P8AAXX 4 DNA100511P01_B11 11B OWB094 GGAG  628P8AAXX 4 DNA100511P01_B12 12B OWB003 TCTCA  628P8AAXX 4 DNA100511P01_C07 7C OWB059 ACAGA  628P8AAXX 4 DNA100511P01_C08 8C OWB068 ATGG  628P8AAXX 4 DNA100511P01_C09 9C OWB077 CCATCCACT  628P8AAXX 4 DNA100511P01_C10 10C OWB086 GACAG  628P8AAXX 4 DNA100511P01_C11 11C OWBDom GGCCGA  628P8AAXX 4 DNA100511P01_C12 12C OWB004 TGCGAGA  628P8AAXX 4 DNA100511P01_D07 7D OWB060 ACCA  628P8AAXX 4 DNA100511P01_D08 8D OWB069 ATGTTCAAT  628P8AAXX 4 DNA100511P01_D09 9D OWB078 CCTG  628P8AAXX 4 DNA100511P01_D10 10D OWB087 GACATCCA  628P8AAXX 4 DNA100511P01_D11 11D OWBRec GTATA  628P8AAXX 4 DNA100511P01_D12 12D OWB006 TGCTGAA  628P8AAXX 4 DNA100511P01_E07 7E OWB061 AGAATGA  628P8AAXX 4 DNA100511P01_E08 8E OWB070 ATTACA  628P8AAXX 4 DNA100511P01_E09 9E OWB079 CGACG  628P8AAXX 4 DNA100511P01_E10 10E OWB089 GACTCGG  628P8AAXX 4 DNA100511P01_E11 11E OWBDom GTGCACCA  628P8AAXX 4 DNA100511P01_E12 12E OWB007 TGGC  628P8AAXX 4 DNA100511P01_F07 7F OWB063 AGGAG  628P8AAXX 4 DNA100511P01_F08 8F OWB072 CACGACCA  628P8AAXX 4 DNA100511P01_F09 9F OWB081 CGATGCGT  628P8AAXX 4 DNA100511P01_F10 10F OWB090 GAGCGAA  628P8AAXX 4 DNA100511P01_F11 11F OWBDom TATGGA  628P8AAXX 4 DNA100511P01_F12 12F OWB008 TTGACCAG  628P8AAXX 4 DNA100511P01_G07 7G OWB064 AGTCAAGA  628P8AAXX 4 DNA100511P01_G08 8G OWB073 CAGGCCACT  628P8AAXX 4 DNA100511P01_G09 9G OWB082 CGCTCA  628P8AAXX 4 DNA100511P01_G10 10G OWB091 GATGA  628P8AAXX 4 DNA100511P01_G11 11G OWBRec TATTCCACT  628P8AAXX 4 DNA100511P01_G12 12G OWB009 TTGCGTCT  628P8AAXX 4 DNA100511P01_H07 7H OWB065 AGTGTTAA  628P8AAXX 4 DNA100511P01_H08 8H OWB074 CAGGCG  628P8AAXX 4 DNA100511P01_H09 9H OWB083 CTCACT  628P8AAXX 4 DNA100511P01_H10 10H OWB092 GCGCCACT  628P8AAXX 4 DNA100511P01_H11 11H OWBRec TCAT  628P8AAXX 4 DNA100511P01_H12 12H OWB010 TTGTAG