Chutimanitsakun et al. BMC Genomics 2011, 12:4 http://www.biomedcentral.com/1471-2164/12/4 QTL analysis QTL analyses were performed for each of the nine traits using the RAD-only and RAD + 2383 locus maps as follows: For the RAD-only map, all data included in the linkage map were used. For the RAD + 2383 locus map, a skeleton map was developed using a single marker (selected at random) for an average marker density of 2 cM and a total of 624 markers. The QTL analyses were conducted with QTL Cartographer Version 2.5 [46] using Composite Interval Mapping (CIM) [47]. Up to seven cofactors for CIM were chosen, using a forward- selection backward-elimination stepwise regression pro- cedure with a significance threshold of 0.1. The walk speed was set to 1 cM, and the scan window to 50 cM beyond the markers flanking the interval tested. Experi- ment-wise significance (a = 0.05) likelihood ratio test (LR) thresholds for QTL identification were determined with 1,000 permutations, and expressed as LOD (LOD = 0.217 LR). Epistatic interactions between QTL were evaluated with the Multiple Interval Mapping (MIM) [48] method implemented in Windows QTL Cartographer using Baye- sian Information Criteria (BIC-M0). Broad-sense heritabil- ity values were estimated using the following formula: H 2 =  G2  G2 +  e2 r where  G2 represent the genetic variance,  e2 the resi- dual variance and r the number of replicates per genotype. Additional material Additional file 1: Table S1: Oregon Wolf Barley DH Sequencing Summary. The aggregate sequence reads obtained for both parents and each member of the OWB mapping population are provided. Sequencing coverage for each sample is also calculated based on the formula (Number of SbfI genome sequences from Barley genome/raw sequences obtained). Clustering of RAD data from multiple individuals indicates there are approximately 10,000 SbfI sequences in the typical Hordeum genome. Additional file 2: Table S2: Oregon Wolf Barley DH RAD Marker Sequences. The sequence data for each RAD marker positioned on the genetic map is provided in this spreadsheet. Additional file 3: Figure S1: Linkage map of Oregon Wolfe Barley population based on RAD markers. Additional file 4: Table S4: Oregon Wolf Barley RAD EST/Genome Alignments. Bowtie alignments of OWB RAD markers to three sequence databases are provided: The Hordeum gene index (HvGI v10.2) from the Dana-Farber Cancer Institute, the MSU Rice Genome Annotation Project Release 6.0 (January 30, 2009) and the 8× Brachypodium Genome Assembly from brachypodium.org. The table columns detail, from left to right: the OWB marker name, sequence alignment orientation, the name (either EST/contig/chromosome identifier) and position (in bp) of the sequence alignment within the reference assembly, the sequence of the RAD marker and any variations observed between query (RAD marker) and reference. Variations are reported as: position in read, reference allele and query allele. Page 11 of 13 Additional file 5: Table S5: Syntenic Oregon Wolf Barley/ Brachypodium RAD Marker Sequences. Bowtie alignments of OWB RAD markers to the 8× Brachypodium Genome Assembly from brachypodium.org are shown. OWB RAD markers have been ordered by linkage group and map position. The corresponding alignment positions for each marker on the Bd21 assembly are shown in columns at right, with chromosome, alignment position, sequence and observed sequence variation between Brachypodium and OWB RAD markers. List of abbreviations DArT: Diversity Array Technology; DH: Doubled haploid; EST: Expressed Sequence Tag; GBS: Genotyping by-sequencing; LOD: Logarithm of odds; OWB; Oregon Wolfe Barley; QTL: Quantitative Trait Locus; RAD: Restriction site Associated DNA; SNP: Single Nucleotide Polymorphism; Acknowledgements The authors wish to thank Jason Boone, Tressa Atwood and Jenna Gribbin for their insights and critical review of this manuscript. Author details 1Crop and Soil Science Department, Oregon State University, Corvallis, Oregon, USA. 2Floragenex, Inc., Eugene, Oregon, USA. 3Departamento de Genética y Producción Vegetal. Estación Experimental de Aula Dei. Consejo Superior de Investigaciones Científicas. Zaragoza, Spain. Authors’ contributions EAJ handled raw sequence analysis and developed PerlScripts for barley sequence genotyping. RWN drafted portions of the manuscript and performed alignments and comparative genomic analysis of the short read data. PMH drafted portions of the manuscript. AC-M drafted portions of the manuscript and performed linkage and QTL mapping analyses. CY performed linkage and QTL analyses, and assembled the final draft. LC, AC, TF assisted in generating data and provided insightful suggestions for analysis and interpretation. All authors have read and approved the final version of this manuscript. Competing interests RWN is an employee of, and EJ a shareholder in, Floragenex - an organization which offers commercial RAD sequencing services. This organization is not financing the manuscript. Received: 10 July 2010 Accepted: 4 January 2011 Published: 4 January 2011 References 1. Tanksley SD, Ganal MW, Prince JP, de-Vicente MC, Bonierbale MW, Broun P, Fulton TM, Giovannoni JJ, Grandillo S, Martin GB, Messeguer R, Miller JC, Miller L, Paterson AH, Pineda O, Roder MS, Wing RA, Wu W, Young ND: High density molecular linkage maps of the tomato and potato genomes. Genetics 1992, 132(4):1141-1160. 2. 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