BACKGROUND
barley  was domesticated approximately  <dig>  years ago and stands among the four most important cereal crops today  <cit> .
it has received considerable research attention as a model for genetic analyses.
breeding programs around the world are working towards improved varieties with better quality, disease-resistance and agronomic traits  <cit> .
researchers and breeders have increasingly been adopting molecular markers to identify genomic regions influencing traits and to select for desirable phenotypes based on identified marker-trait associations  <cit> .
several barley consensus maps have been built with gel-based marker technologies such as restriction fragment length polymorphism , simple sequence repeats  and amplified restriction fragment length polymorphism   <cit> .
these maps integrate information of markers segregating in different crosses and have provided an important framework for producing and exchanging genetic information among members of the scientific community.
molecular marker technologies, however, are currently undergoing a transition from largely serial technologies based on separating dna fragments according to their size , to highly parallel, hybridization-based technologies that can simultaneously assay hundreds to tens of thousands of markers   <cit> .
this transition is mostly taking place in biomedicine and plant/animal genomics because snp assay development is both time and cost-intensive.
notwithstanding the snp discovery efforts in barley  <cit> , practical spin-offs for barley breeding have yet to be generated.
diversity arrays technology  offers a rapid and dna sequence-independent shortcut to medium-density genome scans of any plant species  <cit> .
a single dart assay simultaneously types hundreds to thousands of snps and insertion/deletion polymorphisms spread across the genome.
barley was one of the first species for which dart markers became available  <cit> .
since then, approximately  <dig>  million data points for  <dig>  lines have been generated for barley breeders and researchers at triticarte p/l.
it is essential to integrate the rapidly growing body of genetic information produced through dart with the existing genetic knowledge generated through other marker technologies.
the key objective of this study, therefore, was to create a "bridge" between dart and other marker systems in the form of a ~ <dig> -locus consensus map that co-locates different types of markers.
this consensus map was built from the combined set of segregation data of ten different populations assayed with dart, most of which were also assayed with ssr, rflp and/or sts markers .
a part of the dna samples stored in 96-well microtiter plates got cross-contaminated during shipment as a result of insufficient sealing of the plates, and the corresponding dart assays, with an excess of '1' scores, had to be discarded.
in the course of constructing this consensus map it became clear that the performance of available software for building consensus maps was insufficient for our high-density dataset.
as a consequence, this study has a second, methodological component, in which we identify some insufficiencies of existing mapping software and explore the performance of alternative map construction strategies in order to highlight the need for software improvements in this area.
RESULTS
software performance with high-density linkage data
joinmap  <dig>  is one of the most commonly used programs for constructing linkage maps for plant populations.
importantly, it appears to be the only software option for building a consensus map from the integrated dataset of multiple populations derived from independent crosses between different pairs of parents  <cit> .
we found, however, that this program reproducibly generated erroneous results with our high-density datasets.
problems with using joinmap to analyze high-density datasets have apparently been encountered by others as well  <cit> .
inspection of graphical genotypes for the locus order generated by the program revealed considerable numbers of misplaced loci and inversions of blocks of loci, which introduced artificial crossovers and inflated maps.
as an example, we built individual maps for the high-density dart datasets of three populations and evaluated map quality by computing the sum of adjacent recombination fractions , a sensitive quantifier of map expansion caused by a suboptimal locus order  <cit> .
compared to a similar set of maps constructed with a well-performing locus-ordering algorithm   <cit> , the linkage groups built with joinmap at the preset default settings of the program were inflated by  <dig> ± 76%.
the program furthermore failed to incorporate  <dig> ± 14% of loci into linkage groups, although some of these loci co-segregated with other loci that were incorporated .
a all values reported are means ± sd across  <dig> linkage groups  or a subset of them in case there was insufficient linkage information for map construction.
the populations used for this test were clipper/sahara, dayton/zhepi <dig> and steptoe/morex.
only dart markers were used, in order to disentangle the potential effect of  dna sample tracking errors from software performance.
the lod threshold was varied, while the other program settings were held constant .
decreasing the rec threshold had a similar effect as increasing the lod threshold .
b map inflation values are the percent increase in sarf for the locus order reported by joinmap compared to the order reported by record.
if joinmap did not incorporate all loci into a map, only the set of incorporated loci were analyzed with record.
for unknown reasons, heavily inflated maps with a large number of misplaced loci were often reported by joinmap to be shorter than maps constructed from the same datasets using joinmap settings that produced a close-to-optimum locus order.
increasing the stringency of the program's settings marginally improved the results.
more stringent thresholds, however, also made it necessary to split linkage groups into subgroups if the remaining linkage information was insufficient to construct a map .
in any case, the relationship between the program's settings and the degree of map expansion appeared to vary across different linkage groups, thus requiring a separate optimization of the settings for each linkage group .
given the large datasets of this study such a case-by-case optimization would not have been feasible because the computation time of the program is proportional to the forth power of the number of markers  <cit> .
we tested an alternative way of improving the performance of joinmap: collapsing co-segregating markers into 'bins'  with a purpose-built perl script and only using a single representative marker per bin for map construction.
this approach almost eliminated the problem of non-incorporated markers and reduced the degree of map expansion to some extent .
a combination of binning and more stringent program settings reduced the degree of map expansion to a negligible level , but had the side effect of breaking up linkage groups .
among the  <dig> linkage-group comparisons, we did not find any case where joinmap produced a shorter map than record.
component maps of individual populations
having established the superior performance of record, we used a combination of record and a purpose-built perl script to construct pilot maps of individual populations.
the graphical genotypes of these maps were then investigated to identify 'singletons'  pointing to potential genotyping errors  <cit> .
we did not replace individual singletons with missing calls because of the possibility of introducing a bias towards a particular  locus order and because recombination events in recombinant inbred lines   could be close to each other as a result of multiple rounds of meioses.
instead we completely removed a limited number of less reliable markers  and lines  that had an excessive number of singletons .
we then re-constructed component maps from seven quality-filtered datasets that had sufficient numbers of lines and markers to build a reliable linkage map.
the datasets contained between  <dig>  and  <dig>  loci  and between  <dig> and  <dig> lines .
the lengths of the resulting maps varied between  <dig> and  <dig>  cm .
the order of loci that were common among the maps was very similar .
the method of doubled haploid  production appeared to have a significant effect on the length of the resulting component maps .
populations produced with the hordeum bulbosum method, which are derived from meioses that lead to female gametes, tended to produce longer maps than anther culture-derived populations, the products of male meioses.
this trend does not coincide with a previous study in which anther-derived populations showed higher recombination rates in distal chromosome areas  <cit> .
it is possible that suppression of recombination in wide crosses was a more important influencing factor because map lengths were negatively correlated  with the number of 'bpb' dart markers scored in different populations, an approximate measure of the genetic distance between parents.
aabbreviations of maps: b/c, barque-73/cpi71284-48; c/s, clipper/sahara; d/z, dayton/zhepi2; f/c, foster/ci4196; s/m, steptoe/morex; t/f, tx9425/franklin; y/f, yerong/franklin.
b co-segregating loci were collapsed into bins  at the population-level, i.e.
without concatenating segregation signatures across populations.
a consensus map from the combined datasets
we built an initial draft of a consensus map with joinmap using a limited set of quality-filtered markers under conditions that were likely to minimize the number of misplaced loci .
we selected the set of  <dig>  'bpb' dart markers for this purpose.
this set of markers was assayed across all populations and contained many good-quality anchors bridging populations.
almost three quarters of the markers  segregated in two or more crosses and more than half of them  in three or more .
within the populations in which they were polymorphic, the vast majority of the 'bpb' markers  were scored with a call rate of >90%.
only lower-quality markers, which tended to have a smaller difference in hybridization intensity between the two allelic states, had lower call rates .
building an initial 'skeleton' map from the 'bpb' dart markers also minimized the chance of human error impacting on map quality for the following reasons:  a single aliquot of each genomic dna sample was simultaneously assayed for the whole set of 'bpb' dart markers, which eliminated the possibility of  dna sample tracking errors impacting the integrity of data assembled from separate marker assays; and  the capture of segregation data for dart markers from microarray images was fully automated, thus eliminating the risk of human errors when linking segregation data to marker names.
to minimize software-induced map inflation , we collapsed the  <dig>  'bpb' markers into  <dig> bins based on their segregation signatures concatenated across populations and used moderately stringent program settings to assemble a dart skeleton bin map with joinmap .
the program nevertheless failed to incorporate between  <dig> and 20% of the loci of each chromosome, although virtually all of them were later confirmed to perfectly fit into the consensus map.
the program also misplaced a significant number of loci.
we therefore improved the locus order manually by inspecting graphical genotypes .
ssr, rflp and sts markers, as well as a second set of 'bpt' dart markers from a different genomic representation, were predominantly assayed in one or two populations only .
these markers were incorporated into the dart consensus framework using a purpose-built perl script.
subsequently, we computed map distances and refined the locus order with other purpose-built perl scripts .
additional file  <dig> contains the segregation data of all loci arranged in the order of the final consensus map.
consensus map features
the consensus map comprised  <dig>  markers mapped to a total of  <dig>  loci  .
this number is considerably larger than the number of markers in previously published consensus maps   <cit> .
only a simultaneously developed ssr consensus map will contain a similar number of markers .
because of the high multiplexing level of dart, the production of the more than half a million data points underlying our consensus map has taken only a fraction of the effort that would be required to generate a comparable ssr dataset.
currently, a single operator can produce such a dataset within two weeks; an improved assay format under development is going to reduce this time to four days or less.
on the average, each chromosome contained  <dig> dart and  <dig> non-dart loci.
the number of dart loci per chromosome ranged from  <dig>  to  <dig> .
the number of non-dart loci ranged from  <dig>  to  <dig>  .
the number of dart loci per chromosome probably reflects the distribution of dna polymorphism across chromosomes more accurately than the numbers of non-dart loci, because researchers may have targeted particular genomic regions of interest using selected ssr or rflp markers.
coverage
the consensus map spanned a total length of  <dig>  cm.
chromosome sizes ranged from  <dig>  cm  to  <dig>  cm  .
the 'bpb' dart markers alone spanned  <dig> % of the total length of the consensus map.
addition of a second set of dart markers  increased coverage to  <dig> %.
the combination of all non-dart markers resulted in a coverage of  <dig> % .
a single 'bpb' dart assay, therefore, provides slightly greater genome coverage than the set of  <dig> ssr and rflp markers included in this study.
the map had no gap larger than  <dig> cm and only nine gaps between  <dig> and  <dig> cm .
the dart subset of markers generated a consensus map with a single gap between  <dig> and  <dig> cm  and five gaps between  <dig> and  <dig> cm on chromosomes 3h, 4h and 5h .
chromosome 4h has previously been noted by others to be less polymorphic than the others  <cit> .
non-dart markers on their own resulted in a map with one gap between  <dig> and  <dig> cm  and two gaps between  <dig> and  <dig> cm on chromosome 6h .
the smaller number of 10– <dig> cm gaps in the non-dart dataset may reflect targeted efforts to fill gaps in component maps with selected ssr or rflp markers.
resolution
the average resolution of the consensus map was evaluated by collapsing co-segregating loci into bins and calculating the average distances between adjacent bins.
the  <dig>  loci of the whole dataset could be distributed into  <dig>  bins with an average inter-bin distance of  <dig>  ±  <dig>  cm .
this resolution was only moderately greater than the resolution obtained with dart loci alone .
the set of 'bpb' dart markers, which were simultaneously assayed in a single reaction, provided a resolution of  <dig>  ±  <dig>  cm .
non-dart markers on their own produced a map with a resolution of  <dig>  ±  <dig>  cm .
dart marker redundancy
the dart markers were originally obtained by cloning random fragments of genomic representations  <cit> , a process that introduces some degree of marker redundancy.
the  <dig>  'bpb' dart loci could be collapsed into  <dig> bins, suggesting a redundancy level of 38%.
co-segregating 'bpb' dart markers, however, were not necessarily multiple copies of a single marker because more than  <dig> of the 'bpb' dart bins contained markers that were in the opposite allelic phase in some crosses .
on the other hand, a small number of genotyping errors may have prevented multiple copies of single markers from being collapsed into bins.
therefore, it may not be surprising that the redundancy estimate obtained from marker segregation analysis was quite similar to the preliminary estimate obtained by clustering the dna sequences of dart markers .
marker redundancy is a transient feature of dart array development, which proceeds by consolidating the most informative clones in new arrays of increasing information content  <cit> .
during this process redundant markers are excluded from the final genotyping array.
marker-dense regions
markers sometimes tend to cluster, either as a consequence of an uneven distribution of recombination events along chromosomes  <cit>  or because they preferentially survey dna polymorphism that is unevenly distributed along chromosomes  <cit> .
regions of the consensus map with high marker densities were visualized by plotting local averages of inter-bin distances and the number of loci per bin along chromosomes .
both dart and non-dart loci showed a moderate tendency to cluster around centromeres as can be deduced from the shorter inter-bin distances and the larger numbers of loci per bin in the vicinity of centromeres.
this clustering tendency, however, was nowhere near as pronounced as, for example, for aflp markers based on methylation-insensitive restriction enzymes  <cit> .
given the different polymorphism-detection principles of dart, ssr and rflp markers, we suggest that the centromeric clustering largely reflects centromeric suppression of recombination  <cit> .
centromeric clustering, however, was less pronounced in chromosome 5h, a feature that was previously noted by others  <cit> .
in some chromosomes the density of dart markers also appeared to be higher in distal regions .
this pattern may reflect a moderate bias of psti-based dart markers towards gene-rich, hypomethylated areas in telomeric chromosome regions  <cit> , a pattern we also observed in wheat  <cit> .
a preliminary analysis of the dna sequences of the 'bpb' dart markers indeed suggests that the majority of them are derived from the genespace .
multi-locus markers
markers mapping to more than one locus, if not recognized, can be a confounding factor in the process of building a consensus map.
among the  <dig>  mapped 'bpb' dart markers, only  <dig>  mapped to two different loci in different populations and one  mapped to three different loci .
the loci of multi-locus dart markers were usually located on different chromosomes; two of them, however, mapped to loci within a single chromosome .
multi-locus markers were more common among other marker types.
the set of  <dig> non-dart markers contained  <dig> markers  that mapped to  <dig> to  <dig> different loci each .
the difference in frequency of multi-locus markers between dart and other marker types reflects the fact that as a hybridization-based method dart inherently selects against multi-locus markers.
the hybridization intensities measured for such markers are a difficult-to-resolve mixture of the contributions of several loci, which makes them appear as 'monomorphic'.
residual singletons
a good indicator of the quality of a linkage map constructed from dh populations is the frequency of singletons , which are often due to genotyping errors  <cit> .
the frequency of singletons was calculated from the  <dig> -bin dataset containing all types of markers by using a purpose-built perl script.
approximately  <dig> % of all calls for non-dart loci were singletons.
dart loci generated singletons at a rate of approximately  <dig> % .
the majority of the loci with singletons introduced less than one singleton per one hundred calls .
not surprisingly, high-quality dart markers, which tend to have allelic states with more contrasting hybridization intensities, generated fewer singletons: the correlation between the across-population average of the marker quality parameter and the percentage of singletons in the concatenated segregation signature was - <dig> .
the frederickson/stander population, and to a lesser extent the igri/atlas <dig> population, contained larger numbers of singletons, presumably because some of the dna samples got cross-contaminated during shipment as a result of insufficient sealing of microtiter plates .
singletons presumably were not only the result of genotyping errors.
the comparatively large distances between adjacent loci on chromosome 4h, true double crossovers events in the ril populations , unstable methylation patterns  <cit>  and possibly gene conversion events  <cit>  may have introduced some singletons.
the reported singleton rates, therefore, almost certainly overestimate the error rates of marker assays.
the overestimation of genotyping error rates, however, was to a degree offset by having removed low-quality markers during the construction of pilot maps .
the frequency of dart singletons, therefore, is in good agreement with the previous  <dig> % estimate of the error rate of dart assays  <cit> .
comparison with component maps
an alternative way to evaluate the quality of a consensus map is to compare the locus arrangement of the consensus map  with the arrangement of loci in the component maps .
we selected seven populations with sufficient numbers of lines and loci for this comparison .
to quantify the consistency of locus order between the two different types of maps, unique loci of each of the seven datasets were alternatively arranged according to the consensus or the component map to compute two alternative sets of locus positions per dataset.
the correlation coefficients for the alternative sets of locus positions ranged from  <dig>  ±  <dig>   to  <dig>  ±  <dig>   .
we conclude that the order of loci in the consensus map properly reflects the arrangement of loci in the individual component maps.
as a separate indicator of the quality of the consensus locus order, we also quantified the degree to which component maps expanded if their loci were arranged according to their order in the consensus map.
chromosome lengths computed with the algorithm of lalouel  <cit>   hardly showed any expansion:  <dig>  ±  <dig> % .
the sum of adjacent recombination fractions , a more sensitive indicator of map expansion caused by suboptimal marker positioning, revealed a minor degree of expansion of  <dig>  ±  <dig> % .
this is not surprising because some residual genotyping errors can cause an incorrect locus order to appear superior to the correct order, which can happen more easily if only the segregation data of a single population are taken into account.
both the indicator of locus order consistency and the degree of map expansion were closely associated with the fraction of dart loci in the component datasets.
datasets dominated by dart markers showed more favourable values .
these trends probably reflect two factors.
first, non-dart markers were, on the average, assayed in fewer populations than dart markers .
their positions on the consensus map, therefore, were more ambiguous, particularly if they were located in regions where component maps differed in length.
second, the initial draft of the consensus map was built from the 'bpb' dart markers only.
any  error in dna sample tracking between dart and non-dart marker assays would have introduced artificial crossovers which may have differentially impacted on the accuracy of locus ordering in component maps and the consensus map .
comparison with a synthetic map constructed from component maps
consensus maps typically are constructed using one of the following two alternative strategies.
in strategy i  the segregation data from several populations are simultaneously considered to compute the optimum order of loci.
in strategy ii, various subsets of loci typed for different populations are separately ordered to construct component maps.
subsequently, a 'synthetic' map is constructed by merging information on locus positions from component maps.
we investigated the relative efficacy of the two approaches by quantifying how similar a synthetic map was to the consensus map built using a combination of joinmap and purpose-built perl scripts.
we built three alternative synthetic maps with phenomap software  to determine the impact of the program settings on the results.
pairwise correlation coefficients for locus positions of the three alternative maps varied between  <dig>   and  <dig>  .
the synthetic map obtained using the map with the largest number of loci  as a 'base'  map, was most similar to the joinmap/perl consensus map, although there were notable differences in chromosome lengths .
a the base  maps selected by phenomap were: clipper/sahara , yerong/franklin , clipper/sahara , dayton/zhepi <dig> , barque-73/cpi71284- <dig>  and dayton/zhepi <dig> .
b the values reported are averages across chromosomes.
eighty-five loci not assayed in the seven populations used to construct the synthetic maps  were removed from the joinmap/perl consensus map before the comparison.
we selected the steptoe/morex-based synthetic map for a quality comparison with the joinmap/perl consensus map.
as for the joinmap/perl consensus map, we quantified the similarity of locus positions between synthetic map and component maps.
the resulting correlation coefficients ranged from  <dig>  ±  <dig>   to  <dig>  ±  <dig>   , a marginally lower range of values compared to the joinmap/perl consensus map .
the locus order of the synthetic map was only slightly less optimal than the locus order of the consensus map: the sarf index indicated a map inflation of  <dig>  ±  <dig> % compared to  <dig>  ±  <dig> % for the consensus map .
we conclude that the synthetic map built with phenomap is reasonably consistent with the consensus map in terms of locus order .
the distances between loci, however, appeared to be somewhat less accurate .
the marginally lower quality of the locus order and the less precise map distances, however, are more than offset by the ease and speed of map construction  compared to the alternative joinmap/perl method .
associations between dart markers and agricultural traits
we sampled non-dart markers from the consensus map that previously had been reported as linked to traits of agricultural relevance .
on the average there were  <dig> ±  <dig> dart markers within  <dig> cm on either side of these loci tagged by non-dart markers .
approximately 95%  of the loci had at least three and more than half of the loci  had more than ten dart markers in their vicinity .
only the β-amylase locus on 4hs tagged by ssr marker hvm <dig> was more than  <dig> cm  from the closest dart marker.
the average number of dart markers around trait-influencing loci varied among chromosomes from  <dig>  to  <dig>  .
additional files  <dig> and 12contain a tabular and a graphical summary of dart markers in the vicinity of loci affecting agricultural traits.
the genetic knowledge encapsulated in this table provides a framework for validating and implementing dart genome profiles to assemble a combination of favorable alleles into improved lines.
the collocation of dart and ssr markers on the consensus map also enables barley breeders to quickly identify target loci through whole-genome dart scans and then use ssr markers from the same regions for marker-assisted selection.
utility of the consensus map for breeding and genomics applications
the consensus map provides a reference for rapidly profiling the genetic diversity within and among the genomes of cultivars, parental lines and new germplasm.
it facilitates identification of co-ancestral or genetically distinct genomic regions and enables the detection of genome rearrangements such as translocations .
the positional information attached to dart markers is going to aid the introgression of novel alleles from wild relatives and to increase the precision with which introgressed fragments can be manipulated   <cit> .
whole-genome selection in the context of improving complex traits and ideotype-breeding strategies is also going to benefit from the consensus map  <cit> .
the marker density of the consensus map would allow tighter marker-trait associations than the resolution levels achieved in typical qtl studies.
depending on population history, association mapping approaches could potentially have a higher resolution  <cit>  and are going to benefit from a consensus map with many precisely ordered loci.
different marker applications, however, require different marker densities.
the dna sequences of dart clones could be used to convert dart markers to single-marker assay formats for applications in breeding programs.
the number of loci targeted by marker-assisted selection, however, is on the rise in many barley breeding programs.
a single assay covering a 'standard' set of agriculturally important loci may soon be more cost-effective than 'mixing and matching' single-marker assays.
we are therefore developing a medium-plex assay format to cost-effectively deploy dart markers from approximately  <dig> key loci in marker-assisted foreground selection programs.
higher densities, on the other hand, could be achieved for chromosome-landing  <cit>  and map-based cloning approaches by simply pyramiding dart markers from several genomic representations.
in this context, we are also working towards integrating dart markers with other high-throughput marker technologies such as snp  <cit> .
CONCLUSIONS
the consensus map built in this study co-locates dart markers with previously mapped ssr, rflp and sts markers and loci influencing agricultural traits.
it provides a framework for deploying dart markers in molecular breeding schemes, for transferring genetic information between different marker systems and for integrating dart markers with other genomic resources.
the study has also highlighted an increasing mismatch between our ability to rapidly genotype a large number of mapping populations and the performance of available software tools to construct a consensus map.
while from a statistical point of view it is preferable to build a consensus map de novo from the integrated set of segregation data, it currently appears preferable to build a synthetic map from separately constructed component maps instead; at least until improved or alternative software options become available  <cit> .

 barley was domesticated approximately years ago and stands among the four most important cereal crops today. it has received considerable research attention as a model for genetic analyses. breeding programs around the world are working towards improved varieties with better quality, disease-resistance and agronomic traits. researchers and breeders have increasingly been adopting molecular markers to identify genomic regions influencing traits and to select for desirable phenotypes based on identified marker-trait associations. several barley consensus maps have been built with gel-based marker technologies such as restriction fragment length polymorphism, simple sequence repeats and amplified restriction fragment length polymorphism. these maps integrate information of markers segregating in different crosses and have provided an important framework for producing and exchanging genetic information among members of the scientific community. molecular marker technologies, however, are currently undergoing a transition from largely serial technologies based on separating dna fragments according to their size, to highly parallel, hybridization-based technologies that can simultaneously assay hundreds to tens of thousands of markers. this transition is mostly taking place in biomedicine and plant/animal genomics because snp assay development is both time and cost-intensive. notwithstanding the snp discovery efforts in barley, practical spin-offs for barley breeding have yet to be generated. diversity arrays technology offers a rapid and dna sequence-independent shortcut to medium-density genome scans of any plant species. a single dart assay simultaneously types hundreds to thousands of snps and insertion/deletion polymorphisms spread across the genome. barley was one of the first species for which dart markers became available. since then, approximately million data points for lines have been generated for barley breeders and researchers at triticarte p/l. it is essential to integrate the rapidly growing body of genetic information produced through dart with the existing genetic knowledge generated through other marker technologies. the key objective of this study, therefore, was to create a "bridge" between dart and other marker systems in the form of a ~ -locus consensus map that co-locates different types of markers. this consensus map was built from the combined set of segregation data of ten different populations assayed with dart, most of which were also assayed with ssr, rflp and/or sts markers. a part of the dna samples stored in 96-well microtiter plates got cross-contaminated during shipment as a result of insufficient sealing of the plates, and the corresponding dart assays, with an excess of'1' scores, had to be discarded. in the course of constructing this consensus map it became clear that the performance of available software for building consensus maps was insufficient for our high-density dataset. as a consequence, this study has a second, methodological component, in which we identify some insufficiencies of existing mapping software and explore the performance of alternative map construction strategies in order to highlight the need for software improvements in this area.  software performance with high-density linkage data joinmap is one of the most commonly used programs for constructing linkage maps for plant populations. importantly, it appears to be the only software option for building a consensus map from the integrated dataset of multiple populations derived from independent crosses between different pairs of parents. we found, however, that this program reproducibly generated erroneous results with our high-density datasets. problems with using joinmap to analyze high-density datasets have apparently been encountered by others as well. inspection of graphical genotypes for the locus order generated by the program revealed considerable numbers of misplaced loci and inversions of blocks of loci, which introduced artificial crossovers and inflated maps. as an example, we built individual maps for the high-density dart datasets of three populations and evaluated map quality by computing the sum of adjacent recombination fractions, a sensitive quantifier of map expansion caused by a suboptimal locus order. compared to a similar set of maps constructed with a well-performing locus-ordering algorithm, the linkage groups built with joinmap at the preset default settings of the program were inflated by ± 76%. the program furthermore failed to incorporate ± 14% of loci into linkage groups, although some of these loci co-segregated with other loci that were incorporated. a all values reported are means ± sd across linkage groups or a subset of them in case there was insufficient linkage information for map construction. the populations used for this test were clipper/sahara, dayton/zhepi and steptoe/morex. only dart markers were used, in order to disentangle the potential effect of dna sample tracking errors from software performance. the lod threshold was varied, while the other program settings were held constant. decreasing the rec threshold had a similar effect as increasing the lod threshold. b map inflation values are the percent increase in sarf for the locus order reported by joinmap compared to the order reported by record. if joinmap did not incorporate all loci into a map, only the set of incorporated loci were analyzed with record. for unknown reasons, heavily inflated maps with a large number of misplaced loci were often reported by joinmap to be shorter than maps constructed from the same datasets using joinmap settings that produced a close-to-optimum locus order. increasing the stringency of the program's settings marginally improved the results. more stringent thresholds, however, also made it necessary to split linkage groups into subgroups if the remaining linkage information was insufficient to construct a map. in any case, the relationship between the program's settings and the degree of map expansion appeared to vary across different linkage groups, thus requiring a separate optimization of the settings for each linkage group. given the large datasets of this study such a case-by-case optimization would not have been feasible because the computation time of the program is proportional to the forth power of the number of markers. we tested an alternative way of improving the performance of joinmap: collapsing co-segregating markers into'bins' with a purpose-built perl script and only using a single representative marker per bin for map construction. this approach almost eliminated the problem of non-incorporated markers and reduced the degree of map expansion to some extent. a combination of binning and more stringent program settings reduced the degree of map expansion to a negligible level, but had the side effect of breaking up linkage groups. among the linkage-group comparisons, we did not find any case where joinmap produced a shorter map than record. component maps of individual populations having established the superior performance of record, we used a combination of record and a purpose-built perl script to construct pilot maps of individual populations. the graphical genotypes of these maps were then investigated to identify'singletons' pointing to potential genotyping errors. we did not replace individual singletons with missing calls because of the possibility of introducing a bias towards a particular locus order and because recombination events in recombinant inbred lines could be close to each other as a result of multiple rounds of meioses. instead we completely removed a limited number of less reliable markers and lines that had an excessive number of singletons. we then re-constructed component maps from seven quality-filtered datasets that had sufficient numbers of lines and markers to build a reliable linkage map. the datasets contained between and loci and between and lines. the lengths of the resulting maps varied between and cm. the order of loci that were common among the maps was very similar. the method of doubled haploid production appeared to have a significant effect on the length of the resulting component maps. populations produced with the hordeum bulbosum method, which are derived from meioses that lead to female gametes, tended to produce longer maps than anther culture-derived populations, the products of male meioses. this trend does not coincide with a previous study in which anther-derived populations showed higher recombination rates in distal chromosome areas. it is possible that suppression of recombination in wide crosses was a more important influencing factor because map lengths were negatively correlated with the number of'bpb' dart markers scored in different populations, an approximate measure of the genetic distance between parents. aabbreviations of maps: b/c, barque-73/cpi71284-48; c/s, clipper/sahara; d/z, dayton/zhepi2; f/c, foster/ci4196; s/m, steptoe/morex; t/f, tx9425/franklin; y/f, yerong/franklin. b co-segregating loci were collapsed into bins at the population-level, i.e. without concatenating segregation signatures across populations. a consensus map from the combined datasets we built an initial draft of a consensus map with joinmap using a limited set of quality-filtered markers under conditions that were likely to minimize the number of misplaced loci. we selected the set of'bpb' dart markers for this purpose. this set of markers was assayed across all populations and contained many good-quality anchors bridging populations. almost three quarters of the markers segregated in two or more crosses and more than half of them in three or more. within the populations in which they were polymorphic, the vast majority of the'bpb' markers were scored with a call rate of >90%. only lower-quality markers, which tended to have a smaller difference in hybridization intensity between the two allelic states, had lower call rates. building an initial'skeleton' map from the'bpb' dart markers also minimized the chance of human error impacting on map quality for the following reasons: a single aliquot of each genomic dna sample was simultaneously assayed for the whole set of'bpb' dart markers, which eliminated the possibility of dna sample tracking errors impacting the integrity of data assembled from separate marker assays; and the capture of segregation data for dart markers from microarray images was fully automated, thus eliminating the risk of human errors when linking segregation data to marker names. to minimize software-induced map inflation, we collapsed the'bpb' markers into bins based on their segregation signatures concatenated across populations and used moderately stringent program settings to assemble a dart skeleton bin map with joinmap. the program nevertheless failed to incorporate between and 20% of the loci of each chromosome, although virtually all of them were later confirmed to perfectly fit into the consensus map. the program also misplaced a significant number of loci. we therefore improved the locus order manually by inspecting graphical genotypes. ssr, rflp and sts markers, as well as a second set of'bpt' dart markers from a different genomic representation, were predominantly assayed in one or two populations only. these markers were incorporated into the dart consensus framework using a purpose-built perl script. subsequently, we computed map distances and refined the locus order with other purpose-built perl scripts. additional file contains the segregation data of all loci arranged in the order of the final consensus map. consensus map features the consensus map comprised markers mapped to a total of loci. this number is considerably larger than the number of markers in previously published consensus maps. only a simultaneously developed ssr consensus map will contain a similar number of markers. because of the high multiplexing level of dart, the production of the more than half a million data points underlying our consensus map has taken only a fraction of the effort that would be required to generate a comparable ssr dataset. currently, a single operator can produce such a dataset within two weeks; an improved assay format under development is going to reduce this time to four days or less. on the average, each chromosome contained dart and non-dart loci. the number of dart loci per chromosome ranged from to. the number of non-dart loci ranged from to. the number of dart loci per chromosome probably reflects the distribution of dna polymorphism across chromosomes more accurately than the numbers of non-dart loci, because researchers may have targeted particular genomic regions of interest using selected ssr or rflp markers. coverage the consensus map spanned a total length of cm. chromosome sizes ranged from cm to cm. the'bpb' dart markers alone spanned % of the total length of the consensus map. addition of a second set of dart markers increased coverage to %. the combination of all non-dart markers resulted in a coverage of %. a single'bpb' dart assay, therefore, provides slightly greater genome coverage than the set of ssr and rflp markers included in this study. the map had no gap larger than cm and only nine gaps between and cm. the dart subset of markers generated a consensus map with a single gap between and cm and five gaps between and cm on chromosomes 3h, 4h and 5h. chromosome 4h has previously been noted by others to be less polymorphic than the others. non-dart markers on their own resulted in a map with one gap between and cm and two gaps between and cm on chromosome 6h. the smaller number of 10– cm gaps in the non-dart dataset may reflect targeted efforts to fill gaps in component maps with selected ssr or rflp markers. resolution the average resolution of the consensus map was evaluated by collapsing co-segregating loci into bins and calculating the average distances between adjacent bins. the loci of the whole dataset could be distributed into bins with an average inter-bin distance of ± cm. this resolution was only moderately greater than the resolution obtained with dart loci alone. the set of'bpb' dart markers, which were simultaneously assayed in a single reaction, provided a resolution of ± cm. non-dart markers on their own produced a map with a resolution of ± cm. dart marker redundancy the dart markers were originally obtained by cloning random fragments of genomic representations, a process that introduces some degree of marker redundancy. the'bpb' dart loci could be collapsed into bins, suggesting a redundancy level of 38%. co-segregating'bpb' dart markers, however, were not necessarily multiple copies of a single marker because more than of the'bpb' dart bins contained markers that were in the opposite allelic phase in some crosses. on the other hand, a small number of genotyping errors may have prevented multiple copies of single markers from being collapsed into bins. therefore, it may not be surprising that the redundancy estimate obtained from marker segregation analysis was quite similar to the preliminary estimate obtained by clustering the dna sequences of dart markers. marker redundancy is a transient feature of dart array development, which proceeds by consolidating the most informative clones in new arrays of increasing information content. during this process redundant markers are excluded from the final genotyping array. marker-dense regions markers sometimes tend to cluster, either as a consequence of an uneven distribution of recombination events along chromosomes or because they preferentially survey dna polymorphism that is unevenly distributed along chromosomes. regions of the consensus map with high marker densities were visualized by plotting local averages of inter-bin distances and the number of loci per bin along chromosomes. both dart and non-dart loci showed a moderate tendency to cluster around centromeres as can be deduced from the shorter inter-bin distances and the larger numbers of loci per bin in the vicinity of centromeres. this clustering tendency, however, was nowhere near as pronounced as, for example, for aflp markers based on methylation-insensitive restriction enzymes. given the different polymorphism-detection principles of dart, ssr and rflp markers, we suggest that the centromeric clustering largely reflects centromeric suppression of recombination. centromeric clustering, however, was less pronounced in chromosome 5h, a feature that was previously noted by others. in some chromosomes the density of dart markers also appeared to be higher in distal regions. this pattern may reflect a moderate bias of psti-based dart markers towards gene-rich, hypomethylated areas in telomeric chromosome regions, a pattern we also observed in wheat. a preliminary analysis of the dna sequences of the'bpb' dart markers indeed suggests that the majority of them are derived from the genespace. multi-locus markers markers mapping to more than one locus, if not recognized, can be a confounding factor in the process of building a consensus map. among the mapped'bpb' dart markers, only mapped to two different loci in different populations and one mapped to three different loci. the loci of multi-locus dart markers were usually located on different chromosomes; two of them, however, mapped to loci within a single chromosome. multi-locus markers were more common among other marker types. the set of non-dart markers contained markers that mapped to to different loci each. the difference in frequency of multi-locus markers between dart and other marker types reflects the fact that as a hybridization-based method dart inherently selects against multi-locus markers. the hybridization intensities measured for such markers are a difficult-to-resolve mixture of the contributions of several loci, which makes them appear as'monomorphic'. residual singletons a good indicator of the quality of a linkage map constructed from dh populations is the frequency of singletons, which are often due to genotyping errors. the frequency of singletons was calculated from the -bin dataset containing all types of markers by using a purpose-built perl script. approximately % of all calls for non-dart loci were singletons. dart loci generated singletons at a rate of approximately %. the majority of the loci with singletons introduced less than one singleton per one hundred calls. not surprisingly, high-quality dart markers, which tend to have allelic states with more contrasting hybridization intensities, generated fewer singletons: the correlation between the across-population average of the marker quality parameter and the percentage of singletons in the concatenated segregation signature was -. the frederickson/stander population, and to a lesser extent the igri/atlas population, contained larger numbers of singletons, presumably because some of the dna samples got cross-contaminated during shipment as a result of insufficient sealing of microtiter plates. singletons presumably were not only the result of genotyping errors. the comparatively large distances between adjacent loci on chromosome 4h, true double crossovers events in the ril populations, unstable methylation patterns and possibly gene conversion events may have introduced some singletons. the reported singleton rates, therefore, almost certainly overestimate the error rates of marker assays. the overestimation of genotyping error rates, however, was to a degree offset by having removed low-quality markers during the construction of pilot maps. the frequency of dart singletons, therefore, is in good agreement with the previous % estimate of the error rate of dart assays. comparison with component maps an alternative way to evaluate the quality of a consensus map is to compare the locus arrangement of the consensus map with the arrangement of loci in the component maps. we selected seven populations with sufficient numbers of lines and loci for this comparison. to quantify the consistency of locus order between the two different types of maps, unique loci of each of the seven datasets were alternatively arranged according to the consensus or the component map to compute two alternative sets of locus positions per dataset. the correlation coefficients for the alternative sets of locus positions ranged from ± to ±. we conclude that the order of loci in the consensus map properly reflects the arrangement of loci in the individual component maps. as a separate indicator of the quality of the consensus locus order, we also quantified the degree to which component maps expanded if their loci were arranged according to their order in the consensus map. chromosome lengths computed with the algorithm of lalouel hardly showed any expansion: ± %. the sum of adjacent recombination fractions, a more sensitive indicator of map expansion caused by suboptimal marker positioning, revealed a minor degree of expansion of ± %. this is not surprising because some residual genotyping errors can cause an incorrect locus order to appear superior to the correct order, which can happen more easily if only the segregation data of a single population are taken into account. both the indicator of locus order consistency and the degree of map expansion were closely associated with the fraction of dart loci in the component datasets. datasets dominated by dart markers showed more favourable values. these trends probably reflect two factors. first, non-dart markers were, on the average, assayed in fewer populations than dart markers. their positions on the consensus map, therefore, were more ambiguous, particularly if they were located in regions where component maps differed in length. second, the initial draft of the consensus map was built from the'bpb' dart markers only. any error in dna sample tracking between dart and non-dart marker assays would have introduced artificial crossovers which may have differentially impacted on the accuracy of locus ordering in component maps and the consensus map. comparison with a synthetic map constructed from component maps consensus maps typically are constructed using one of the following two alternative strategies. in strategy i the segregation data from several populations are simultaneously considered to compute the optimum order of loci. in strategy ii, various subsets of loci typed for different populations are separately ordered to construct component maps. subsequently, a'synthetic' map is constructed by merging information on locus positions from component maps. we investigated the relative efficacy of the two approaches by quantifying how similar a synthetic map was to the consensus map built using a combination of joinmap and purpose-built perl scripts. we built three alternative synthetic maps with phenomap software to determine the impact of the program settings on the results. pairwise correlation coefficients for locus positions of the three alternative maps varied between and. the synthetic map obtained using the map with the largest number of loci as a'base' map, was most similar to the joinmap/perl consensus map, although there were notable differences in chromosome lengths. a the base maps selected by phenomap were: clipper/sahara, yerong/franklin, clipper/sahara, dayton/zhepi, barque-73/cpi71284- and dayton/zhepi. b the values reported are averages across chromosomes. eighty-five loci not assayed in the seven populations used to construct the synthetic maps were removed from the joinmap/perl consensus map before the comparison. we selected the steptoe/morex-based synthetic map for a quality comparison with the joinmap/perl consensus map. as for the joinmap/perl consensus map, we quantified the similarity of locus positions between synthetic map and component maps. the resulting correlation coefficients ranged from ± to ±, a marginally lower range of values compared to the joinmap/perl consensus map. the locus order of the synthetic map was only slightly less optimal than the locus order of the consensus map: the sarf index indicated a map inflation of ± % compared to ± % for the consensus map. we conclude that the synthetic map built with phenomap is reasonably consistent with the consensus map in terms of locus order. the distances between loci, however, appeared to be somewhat less accurate. the marginally lower quality of the locus order and the less precise map distances, however, are more than offset by the ease and speed of map construction compared to the alternative joinmap/perl method. associations between dart markers and agricultural traits we sampled non-dart markers from the consensus map that previously had been reported as linked to traits of agricultural relevance. on the average there were ± dart markers within cm on either side of these loci tagged by non-dart markers. approximately 95% of the loci had at least three and more than half of the loci had more than ten dart markers in their vicinity. only the β-amylase locus on 4hs tagged by ssr marker hvm was more than cm from the closest dart marker. the average number of dart markers around trait-influencing loci varied among chromosomes from to. additional files and 12contain a tabular and a graphical summary of dart markers in the vicinity of loci affecting agricultural traits. the genetic knowledge encapsulated in this table provides a framework for validating and implementing dart genome profiles to assemble a combination of favorable alleles into improved lines. the collocation of dart and ssr markers on the consensus map also enables barley breeders to quickly identify target loci through whole-genome dart scans and then use ssr markers from the same regions for marker-assisted selection. utility of the consensus map for breeding and genomics applications the consensus map provides a reference for rapidly profiling the genetic diversity within and among the genomes of cultivars, parental lines and new germplasm. it facilitates identification of co-ancestral or genetically distinct genomic regions and enables the detection of genome rearrangements such as translocations. the positional information attached to dart markers is going to aid the introgression of novel alleles from wild relatives and to increase the precision with which introgressed fragments can be manipulated. whole-genome selection in the context of improving complex traits and ideotype-breeding strategies is also going to benefit from the consensus map. the marker density of the consensus map would allow tighter marker-trait associations than the resolution levels achieved in typical qtl studies. depending on population history, association mapping approaches could potentially have a higher resolution and are going to benefit from a consensus map with many precisely ordered loci. different marker applications, however, require different marker densities. the dna sequences of dart clones could be used to convert dart markers to single-marker assay formats for applications in breeding programs. the number of loci targeted by marker-assisted selection, however, is on the rise in many barley breeding programs. a single assay covering a'standard' set of agriculturally important loci may soon be more cost-effective than'mixing and matching' single-marker assays. we are therefore developing a medium-plex assay format to cost-effectively deploy dart markers from approximately key loci in marker-assisted foreground selection programs. higher densities, on the other hand, could be achieved for chromosome-landing and map-based cloning approaches by simply pyramiding dart markers from several genomic representations. in this context, we are also working towards integrating dart markers with other high-throughput marker technologies such as snp.  the consensus map built in this study co-locates dart markers with previously mapped ssr, rflp and sts markers and loci influencing agricultural traits. it provides a framework for deploying dart markers in molecular breeding schemes, for transferring genetic information between different marker systems and for integrating dart markers with other genomic resources. the study has also highlighted an increasing mismatch between our ability to rapidly genotype a large number of mapping populations and the performance of available software tools to construct a consensus map. while from a statistical point of view it is preferable to build a consensus map de novo from the integrated set of segregation data, it currently appears preferable to build a synthetic map from separately constructed component maps instead; at least until improved or alternative software options become available. 
the locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. the arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. molecular marker technologies are undergoing a transition from largely serial assays measuring dna fragment sizes to hybridization-based technologies with high multiplexing levels. there is a need to integrate the information generated by dart with previous data produced with gel-based marker technologies. diversity arrays technology is a hybridization-based technology that is increasingly being adopted by barley researchers. the consensus map, built using a combination of joinmap software and several purpose-built perl scripts, comprised loci and spanned cm. our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying dart markers in molecular breeding schemes. dart markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. it contained a total of'bins', with an average inter-bin distance of ± cm. the study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations. on the average, ± dart loci were identified within cm on either side of ssr, rflp or sts loci previously identified as linked to agricultural traits. more than 98% of the map could be covered with a single dart assay.
 molecular marker technologies are undergoing a transition from largely serial assays measuring dna fragment sizes to hybridization-based technologies with high multiplexing levels. diversity arrays technology is a hybridization-based technology that is increasingly being adopted by barley researchers. there is a need to integrate the information generated by dart with previous data produced with gel-based marker technologies. the goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with dart and simple sequence repeat, restriction enzyme fragment polymorphism and/or sequence tagged site markers.  the consensus map, built using a combination of joinmap software and several purpose-built perl scripts, comprised loci and spanned cm. it contained a total of'bins', with an average inter-bin distance of ± cm. more than 98% of the map could be covered with a single dart assay. the arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. the locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. the distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5h. dart markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. on the average, ± dart loci were identified within cm on either side of ssr, rflp or sts loci previously identified as linked to agricultural traits.  our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying dart markers in molecular breeding schemes. the study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations. 