Chutimanitsakun et al. BMC Genomics 2011, 12:4 http://www.biomedcentral.com/1471-2164/12/4 be of significant benefit as a scaffold framework for placement of shotgun sequence reads and de novo gen- ome assembly refinement. Methods Plant material The mapping population consists of 93 doubled haploid (DH) lines. The DH lines were produced from the F1 of the cross of the Wolfe recessive and dominant marker stocks using the Hordeum bulbosum method [19]. In the course of this research we determined that nine sets of DH lines had identical genotypes. Specifically, the following sets of lines are identical: set1 = DH 1,4,27,62; set2 = DH 16,71; set3 = DH 5,18; set4 = DH 31,58; set5 = 35,50; set6 = DH 15, 47 set7 = DH 61, 88; set8 = DH 22,70; set9 = DH 80,77. Retention of one genotype per set (DH 4, 16, 18, 31, 35, 47, 61, 70 and 77) reduces the population size to 82. This report describes map- ping and QTL analysis using the OWB population of 82 lines. In order to ascertain the bias introduced by duplicate lines (an unintended consequence of the DH production process), all analyses were also conducted with a population size of n = 93 [20]. Genomic DNA was extracted from young leaf tissue of a single plant representing each DH line, and each of the parents, using DNeasy plant maxi kits (QIAGEN Inc. Califor- nia, USA). RAD protocols OWB genomic DNA from the selected mapping popula- tion was digested with the restriction endonuclease SbfI and processed into RAD libraries similarly to the method of Baird et al. [14]. Briefly, P0 (parental geno- types) and DH (progeny) genomic DNA (~300 ng; from each sample) was digested for 60 min at 37°C in a 50 μL reaction with 20 units (U) of SbfI (New England Biolabs [NEB]). Samples were heat-inactivated for 20 min at 65°C. 2.0 μL of 100 nM P1 Adapter(s), a modified Solexa© adapter (2006 Illumina, Inc., all rights reserved). SbfI P1 adapters each contained a unique multiplex sequence index (barcode) which is read during the first four nucleotides of the Illumina sequence read. 100 P1 nM adaptor were added to each sample along with 1 μL of 10 mM rATP (Promega), 1 μL 10× NEB Buffer 4, 1.0 μL (1000 U) T4 DNA Ligase (high concen- tration, Enzymatics, Inc), 5 μL H2O and incubated at room temperature (RT) for 20 min. Samples were again heat-inactivated for 20 min at 65°C, pooled and ran- domly sheared with a Bioruptor (Diagenode) to an aver- age size of 500 bp. Samples were then run out on a 1.5% agarose (Sigma), 0.5× TBE gel and DNA 300 bp to 700 bp was isolated using a MinElute Gel Extraction Kit (Qiagen). End blunting enzymes (Enzymatics, Inc) were Page 9 of 13 then used to polish the ends of the DNA. Samples were then purified using a Minelute column (Qiagen) and 15 U of Klenow exo- (Enzymatics) was used to add adenine (Fermentas) overhangs on the 3’ end of the DNA at 37°C. After subsequent purification, 1 μL of 10 μM P2 adapter, a divergent modified Solexa© adapter (2006 Illumina, Inc., all rights reserved), was ligated to the obtained DNA fragments at 18°C. Samples were again purified and eluted in 50 μL. The eluate was quantified using a Qubit fluorimeter and 20 ng of this product was used in a PCR amplification with 20 μL Phusion Master Mix (NEB), 5 μL of 10 μM modified Solexa© Amplifica- tion primer mix (2006 Illumina, Inc., all rights reserved) and up to 100 μL H2O. Phusion PCR settings followed product guidelines (NEB) for a total of 18 cycles. Sam- ples were gel purified, excising DNA 300-650 bp, and diluted to 1 nM. To promote SNP identification in low-copy, gene-rich regions of the barley genome, a species with ~90% ret- roelement content, selection of a restriction enzyme that does not fragment repetitive-class DNA is desirable. Pre- vious studies have documented epigenetic modification of CpG, CpNpG and CpNpN nucleotides with 5-methyl- cytosine (5 mC) in retroelement-dense regions of many plant genomes, including triticale [41-43]. Methylation- sensitive type II restriction endonucleases, which do not cleave 5 mC-modified DNA, can be used to specifically sample the hypomethylated genomic fraction and are commonly used in other restriction-enzyme based genetic marker systems [44]. We selected the restriction enzyme SbfI, (5’CCTGCA/GG’3) with a recognition site containing two CpNpG trinucelotide repeats for RAD sequencing of the barley genome. Illumina Sequencing The constructed OWB libraries were run on an Illumina Genome Analyzer II at the University of Oregon High Throughput Sequencing Facility. Illumina/Solexa proto- cols were followed for single read (1 × 36 bp) sequen- cing chemistry. A total of 20.4 M Illumina reads were obtained from sequencing of the population. Sequences are available at the Sequence Read Archive http://www. ncbi.nlm.nih.gov/Traces/sra/, at accession SRA020593. Sequence Analysis and SNP Discovery and Genotyping Internal Floragenex sequence tools and custom Perl- Scripts were used for processing of raw Illumina/Solexa data. Data from multiple Illumina/Solexa sequence channels was segregated by the appropriate four nucleo- tide multiplex identifier (MID) assigned to each sample. All reads were trimmed to 28 nucleotides from the 3’ end of genomic sequence to avoid using bases with a high Illumina sequence error rate.