Chutimanitsakun et al. BMC Genomics 2011, 12:4 http://www.biomedcentral.com/1471-2164/12/4 Sequence Alignment and Comparative Genomics The short-read alignment program Bowtie [21] was used for mapping of polymorphic barley RAD sequence loci (Additional file 4: Table S4) to the comprehensive Hordeum gene index (HvGI v10.2) database from the Dana-Farber Cancer Institute [22]. Both tentative con- sensus (TC) and singleton expressed sequence tags (ESTs) were used in analysis. Briefly, sequences corre- sponding to all 530 polymorphic RAD loci were aligned against the HvGI assembly. Two criteria were imposed for sequence mapping. First, a maximum of three nucleotide mismatches and no gaps between the RAD sequence and reference were permitted for any align- ment. Second, each sequence had to anchor to a single unique position to be scored. For macro-scale syntenic mapping of barley RAD sequences to other grass gen- omes, we extended the CIP/CALP (Conserved Identity Percentage/Conserved Alignment Percentage) method previously used in Triticale comparative analysis [26]. 30 bp RAD sequences ordered by the linkage map were aligned against the Oryza sativa and Brachypodium dis- tachion chromosome assemblies using relaxed Bowtie alignment parameters. Bowtie is able to tolerate up to three nucleotide mismatches between query and refer- ence, translating to minimum values of 90% and 90% respectively for CIP and CALP. Linkage mapping Two linkage maps were constructed. The first map was built with only the RAD data and data for nine morpho- logical markers (Table 4). The morphological marker data were reported by Szűcs et al. [3] and were included because they provide anchors for equating linkage groups with six of the seven barley chromosomes. A second map was built using RAD data and all 2383 data points reported by Szűcs et al. [3]. Each linkage map was constructed using JoinMap 4 [17]. Linkage groups were identified using minimum LOD values of 5. The Monte Carlo Maximum Likelihood (ML) mapping algo- rithm was used to determine the orders of markers Table 4 Anchor markers for RAD-only map construction Chromosome Phenotype Page 10 of 13 within each linkage group. Map distances were calcu- lated using the Haldane’s mapping function. Maps were drawn using MapChart v2.2 [45]. Data used for linkage map construction are available at Oregon Wolfe Barley Data and GrainGenes Tools http://wheat.pw.usda.gov/ ggpages/maps/OWB/. Phenotyping In order to assess the utility of the RAD and RAD + SNP map for quantitative trait locus (QTL) detection, data on phenological and reproductive fitness pheno- types were obtained for the 93 DH lines and the two parents. Individual plants were grown in 13.5 cm pots at the Oregon State University greenhouses (Corvallis, Oregon USA). Supplemental light was used to maintain a 16 h light/24 h photoperiod. Temperatures were main- tained at a constant 18 ± 2°C day and night tempera- ture. Each DH and parental line was replicated twice. Eight traits were measured on each plant. The trait abbreviations and definitions are as follows: (1) Final leaf number (FLN) was recorded as the total number of leaves on the main stem of each plant; (2) Plant height (PH) was measured as the distance (in cm) from the soil surface to the tip of the tallest inflorescence (spike), exclusive of awns, if present; (3) Spike number (SN) was the actual count of the total number of fertile spike on each plant. Three stems with fertile spikes were selected at random from each plant for determining the follow- ing traits, and the individual values were averaged: (4) Spike length (SL) was measured as the length (in cm) from the first rachis internode to the top of the final fully formed floret, exclusive of awn; (5) Floret number (FS) was the count of the number of florets (fertile and sterile) per spike; (6) Grain number (GN) was the count of the number of seed-containing florets per spike; (8) Hundred grain weight (HGW) was the weight (in g) of 100 grains. Grain yield per plant (GY) was estimated by the function GY = SN*GN*HGW. Phenotype data are available at Oregon Wolfe Barley Data and GrainGenes Tools http://wheat.pw.usda.gov/ggpages/maps/OWB/. Locus VRS-1 ZEO-1 ALM HSH SRH ROB WX NUD LKS2 Gene HvHox1 GBSS-I 2H 2H 3H 4H 5H 6H 7H 7H 7H Two-row inflorescence (Vrs1Vrs1)/six-row inflorescence (vrs1vrs1) Dwarf plant with compact head (Zeo1)/normal height and head length (zeo1) Green lemma and nodes (Alm)/albino lemma and nodes (alm) Hairs on lower leaf sheaths (Hsh)/lack of hair on lower leaf sheaths (hsh) Long hairs on rachilla (Srh/short hairs on rachilla (srh) Green lemma and nodes (Rob)/orange lemma and nodes (rob) Wild type endosperm starch (Wx)/waxy endosperm starch (wx) Hulled seed (Nud)/hulless seed (nud) Long awn (Lks2)/short awn (lks2) Gene [49] GenBank:[AB489122.1] [50] GenBank:[AF486518.1] [51] GenBank:[AP009567] Nine morphological marker loci used for linkage map construction, together with RAD loci, in the Oregon Wolfe Barley mapping population, showing chromosome assignments, phenotypes, and genes (if known).