Chutimanitsakun et al. BMC Genomics 2011, 12:4 http://www.biomedcentral.com/1471-2164/12/4 order between the n = 93 and n = 82 maps and map lengths are comparable [20]. EST and genome mapping of RAD sequence markers The RAD technique develops sequence from regions adjacent to restriction endonuclease digestion sites in a target genome [14]. To establish if sequence-based RAD markers from the OWB genetic map would anchor to existing Hordeum genomic resources, we used the short-read aligner Bowtie to map RAD sequences onto a barley gene index [21,22]. Using this database, we suc- cessfully identified unique alignment positions for 51 of 436 sequenced RAD loci (11.0%). An additional 22 RAD loci (4.7%) mapped to multiple positions in the gene index. A list of summary alignments for all RAD mar- kers in this database can be found in additional file 4: Table S4. Although the gene index contains approxi- mately 54 Mb of putative coding sequence distributed across 80,723 tentative assemblies, this database spans only a small fraction (~0.1%) of the 5.0 Gb barley hap- loid genome. As Ty3 and Copia retrotransposon families are believed to inhabit a large portion of the barley gen- ome, we postulated some percentage of RAD sequences might originate from repetitive-class sequences [23]. However, several attempts to align the 463 RAD sequence loci to the 1.3 Mb TIGR Hordeum repeat database under a variety of thresholds did not reveal any successful alignments. A larger percentage of RAD sequences could be positioned on candidate genes than would expected by random sampling, suggesting that RAD markers are significantly enriched in the gene space. The absence of any alignments to known repeti- tive sequences also hints that RAD markers are clus- tered within recombinatorially active regions of the genome. Comparative Genome Analysis To examine if assembled grass genomes would serve to anchor other RAD markers, we aligned polymorphic Page 5 of 13 sequences to the 430 Mb Oryza sativa and 300 Mb Brachypodium distachyon genomes using a modified CIP/CALC method [24-26]. Bowtie alignment results using relaxed parameters indicate that only 16 and 24 of the 463 OWB RAD sequences mapped to either the rice or Brachypodium chromosome assemblies, respectively. Despite the small number of orthologous RAD sequences and the short Illumina read of 28 bp, align- ments of RAD markers ordered by the genetic map against the finished Brachypodium genome (Figure 2 and additional file 5: Figure S5) agree with macro-scale syntenic relationships established by previous efforts [25]. Although this study has relatively few sequence loci available for comparison, our findings suggest that a denser RAD marker scan, using a more frequently cut- ting restriction enzyme would interrogate more genome sequence and interrogate more sequence for compara- tive analyses. Overall, we were able to assign 74 of 463 RAD sequence loci (15.9%) to at least one of the three sequence refer- ences, leaving the genomic origin of the remaining bar- ley RAD tags (389 loci, 84.1%) unknown. We postulate the large numbers of RAD sequences placed on the OWB linkage without homology or orthology to known sequences are a result of two factors. First, the lack of a contiguous barley genome, which would allow us to explicitly determine the location of all RAD sequences, restricts our analysis to the small fraction of the haploid genome that has been sequenced. Second, despite estab- lished syntenic relationships between the Oryza, Hordeum and Brachypodium genomes, the inefficient mapping of barley sequenced RAD markers across spe- cies is likely a result of the majority of RAD loci ema- nating from areas of the barley genome which have significantly diverged at the nucleotide level since the speciation of the Poaceae [27,28]. A cohesive explanation for the results observed in the genetic map and comparative genome analysis is that the majority of RAD loci are linked with, but lie outside Figure 2 Macro-scale syntenic relationships between barley and Brachypodium revealed with sequenced RAD markers. RAD sequences anchored by linkage analysis are distributed across the seven Hordeum linkage groups. Alignments to orthologous sequence loci in Brachypodium are shown. Solid lines denote relationships supported by EST sequence comparison. Two dashed lines indicate sequence alignments that do not coincide with expected chromosomal relationships.