Chutimanitsakun et al. BMC Genomics 2011, 12:4 http://www.biomedcentral.com/1471-2164/12/4 RAD clusters from the dominant and recessive lines were then compared using a custom k-mer matching algo- rithm permitting exact sequence matches (monomorphic loci), single mismatch (one SNP per read) and two nucleotide mismatches (two SNPs per read) per 28 bp sequence. An initial panel of 530 SNPs with fixed geno- types in both parents were identified using these criteria and alleles for each marker were assigned to their respec- tive parental donor (Additional file 2: Table S2). The putative 530 SNP marker panel was then used to score RAD sequences obtained from each of the DH individuals. As alleles are fixed within each member of the doubled haploid OWB population, we posited sequence genotypes could be accurately determined at low sequence coverage (<5×) [16]. To further minimize genotyping miscalls due to possible sequencing errors, a minimum of two independent sequence reads were required over any locus to assign any SNP genotype. Putative genotypes developed for individual samples were converted into JoinMap 4 [17] compatible format using custom PerlScript. Loci lacking sufficient sequence coverage or with conflicting genotype data were coded as missing data. Linkage map We used the following criteria to assess the quality of the RAD markers for linkage map construction. First, with the RAD-only map we considered the (i) total number of loci detected, (ii) the percentage of poly- morphic loci, (iii) the number of missing allele calls for polymorphic loci, (iv) the percentage of codominant loci, (v) segregation distortion, (vi) the number of signif- icant singletons (vii) linkage map length, and (viii) the number, location, interaction and effect of significant QTL. For purposes of comparison, we used the map reported by Szűcs et al. [3]. Subsequently, we added the RAD data to the 2383 locus map and assessed criteria v - vii, above. For criteria viii, however, we used a skele- ton map, as described in the Methods. First, we will pre- sent results in terms of criteria i - vii; the QTL results will be presented separately. Of approximately 10,000 RAD sequence clusters inter- rogated, 530 loci (5.3%) were classified as codominant markers where two distinct alleles were explicitly observed between the OWB parents. A number of dominant-style markers, which are sequences present in one parent but not the other, were also observed within the data but were not used for map construction as dominant markers have reduced genotyping quality. Of the codominant RAD marker class, 67 (13%) were excluded from further analysis due to missing data (≥ 15% missing data points). This left 463 (4.6% of the total) RAD loci, plus the nine morphological markers, for map construction. Twenty-seven RAD markers Page 3 of 13 remained unlinked at LOD 5.0 and the remaining 436 formed seven linkage groups, together with the nine morphological markers. Based on visual assessment of locus orders, there were 22 loci showing apparent dou- ble crossover events. Of these, 23 singletons data points were re-coded as missing data for 20 loci where these occurred, except for two loci where distances between flanking markers were large enough to expect recombi- nation. The final map is composed of 436 RAD and nine morphological markers. The total length of the RAD map is 1260 cM. Excluding co-segregating mar- kers, the average marker density is 5 cM (Additional file 3: Figure S1). Significant segregation distortion was observed on chromosomes 2H, 3H, 6H, and 7H (Figure 1). On chromosomes 2H and 3H the segregation distortion was in favor of the OWB recessive parent allele and on chromosomes 6H and 7H it was in favor of the OWB dominant parent allele. The lengths (in Haldane cM) for each linkage group are shown in Table 1. For construction of the RAD + 2383 locus map, the same 463 RADs selected initially for the RAD-only map were added to the 2383-locus data set reported by Szűcs et al. [3]. The 23 singletons were replaced by missing values. The combined map therefore consists of 2846 loci and has a total length of 1286 cM (Table 1). Marker orders for the non-RAD markers are consistent between the 2383 and 2846-locus maps. Seventy-eight percent (359) of the RAD markers co-segregate with one or more of the previous markers. There were examples of gap-filling: for example, FGX_OWB00091, mapped to a 17 cM gap on chromosome 7H in the Szűcs et al. [3] and incorporation of this marker reduced the distance between the two flanking markers to 10 cM. Segregation distortion was observed at the same positions as in the RAD-only map (Figure 1). The lengths (in Haldane cM) for each linkage group are shown in Table 1. The same lines identified as identical with the RAD-only data (see Methods, Plant material) were confirmed as identical using the 2383 data points reported by Szűcs et al. [3]. Although a significant number of RAD loci were eliminated based on lack of polymorphism and missing sequence data, the genome scan uncovered over 400 high quality loci that were available for map construc- tion. By way of comparison, there are 722 DArT loci on the Szűcs et al. OWB map [3], out of the 1,500 loci that were genotyped. The RAD loci are codominant whereas DArTs are dominant markers [18]. In the case of domi- nant markers, missing data due to error vs. allele absence cannot be distinguished, and this leads to a higher frequency of apparent singletons in map con- struction. The high quality of the RAD data is further confirmed by the comparable linkage map lengths for the RAD only, RAD + prior marker, and Szűcs et al. OWB map [3](Table 1). Segregation distortion was