Chutimanitsakun et al. BMC Genomics 2011, 12:4 http://www.biomedcentral.com/1471-2164/12/4 Background Plant breeders and geneticists have benefited from the availability of tools for the rapid and cost-effective devel- opment of molecular marker-based linkage maps. As predicted by Tanksley et al. [1], linkage maps have proven to be useful for discovering, dissecting and manipulating the genes that determine simple and complex traits in crop plants. Barley (Hordeum vulgare) is a model for plant breeding and genetics because it is diploid (2n = 2x = 14) and has a long history of genetics research. Over the past decade, increasingly dense maps of the barley genome have been constructed using multiple populations and many types of molecular markers [2]. Most recently, Szűcs et al. [3] reported an integrated 2383-locus linkage map developed in the Oregon Wolfe Barley (OWB) mapping population based on representative early generation mar- kers (e.g. morphological loci, RFLPs, and SSRs) and single nucleotide polymorphisms (SNPs). SNP markers have become increasingly important tools for molecular genetic analysis, as single base-pair changes are the most abundant small-scale genetic variation pre- sent between related sequences of DNA [4]. To date, most SNP development efforts in larger, more complex genomes such as barley have focused on “complexity reduction” techniques that aim to sequence a fraction of the genome, such as that represented in EST collections. Once a panel of markers is established from initial SNP discovery, samples from a selected population are then genotyped using oligo-extension or array-based platforms [5]. Both these strategies were used for construction of the current barley SNP-based maps [3,6,7]. The emergence of massively-parallel, next-generation sequencing (NGS) platforms capable of producing mil- lions of short (50-100 bp) DNA sequence reads has reduced the costs of DNA sequencing and offers the tantalizing possibility of making direct, genotyping-by- sequencing (GBS) practical (Reviewed in [8]). Recently, Huang and colleagues [9] have elegantly demonstrated how genotyping using NGS data can facilitate the rapid development of linkage maps in domesticated rice, Oryza sativa. Despite the attractiveness of this approach and availability of next-generation sequencing platforms, at present, GBS methods retain significant limitations. First, current protocols for synthesis of DNA fragment libraries compatible with high-throughput sequencing platforms are laborious, costly and would be impractical for production efforts involving hundreds of samples [10]. Second, sequence-based genotyping is restricted to those species with available, high-quality, pseudomole- cule-sized genome assemblies [9]. While many key economic and scientifically meritorious species will undoubtedly be sequenced as a direct result of the ongoing revolution in NGS technologies, what is Page 2 of 13 required are marker platforms that can provide GBS independent of the status of an assembled genome. Restriction-site Associated DNA (RAD) markers detect genetic variation adjacent to restriction enzyme cleavage sites across a target genome [11]. The first iteration of RAD markers facilitated cloning of mutants isolated from genetic screens in classic model systems [12,13]. More recent efforts have focused on adapting the RAD technique for use in NGS platforms, specifi- cally the Illumina sequencing-by-synthesis method, to enable individual sequence based genotyping of samples [14]. The sequenced RAD marker system enjoys two favourable characteristics for high-throughput GBS. As previously mentioned, the RAD method uses restriction enzymes as a complexity reduction strategy to reduce the sequenced portion of the genome anywhere from 0.01% to 10% [15]. Furthermore, RAD protocols facili- tate the creation of highly multiplexed NGS sequencing formulations containing many tens of samples in a single library, thereby reducing library preparation costs [14]. While previously published RAD studies have explored NGS of limited numbers of individuals or bulked genotyping of pooled populations, the objective of this research was to determine the feasibility of con- structing a RAD marker genetic map in barley. We used the OWB population as a mapping resource in order to directly compare RAD and EST-based SNP maps and to assess the quality and utility of a linkage map built with the two types of data. Results & Discussion Genome Analyzer sequence results, SNP Discovery and DH Genotyping A total of 2,010,583 36-bp sequence reads were obtained for the OWB dominant and recessive inbred genetic stocks (parents of the OWB mapping population), while 27,704,592 sequence reads were obtained for the 93 member DH mapping population (Additional file 1: Table S1). Illumina sequences from the OWB parental lines were first used for identification of SNPs. Putative alleles were mined from the sequence data using several custom PerlScripts and filtering procedures. First, raw 36-bp Illumina sequence reads were partitioned into discrete files using a 5’ multiplex identifier (MID) corre- sponding to each OWB sample and the restriction enzyme site SbfI (TGCAGG). Segregated data from each line was then collapsed into putative RAD sequence clusters comprised of a minimum of eight (8×) redun- dant sequence reads per locus. Sequences not attaining the 8× sequence coverage threshold were excluded from further analysis, as were putative high-copy RAD sequences where the number of sequence occurrences in each cluster was greater than 500 (500×). Homologous