BACKGROUND
triticum monococcum , generally known as einkorn wheat, is an ancient diploid species domesticated in the fertile crescent ~ <dig>  years ago  is closely related to t. urartu , the donor of the a-genome of hexaploid wheat  <cit> .
recently, t. monococcum has gradually been recognised as an attractive diploid model for exploitation of useful traits, discovery of novel genes and variant alleles, and functional genomics.
many traits have been examined in t. monococcum which can be useful for modern wheat breeding  <cit> .
t. monococcum has also been successfully used for gene discovery in a subgenome map-based cloning approach, as exemplified by the cloning of the leaf rust resistance gene lr <dig>  <cit> , the vernalisation genes vrn <dig> and vrn <dig>  <cit> , the domestication locus q  <cit> , and a member of the senescence and nutrient remobilisation controlling nac gene family  <cit> .
natural and artificially mutagenised t. monococcum populations have been made available and were used to identify and map genes of agronomic importance  <cit> .
furthermore, tilling  and vigs  platforms for functional genomics are under development in several laboratories .
thus, in the foreseeable future t. monococcum is expected to play an important role in wheat genetic and genomic studies.
globally, thousands of accessions of t. monococcum have been collected and retained in major germplasm stock centres.
in order to use t. monococcum resources efficiently in programmes on genetic improvement of hexaploid wheat, it is necessary to assess diversity of this species at the genome level.
for this, high-throughput molecular marker technologies are needed.
t. monococcum has been shown to possess a high level of polymorphism at dna marker loci <cit> .
rflp  and aflp  markers have been developed and used for generating genetic linkage maps and for mutation mapping, map-based cloning, and genome synteny comparisons  <cit> .
these markers have also been used to resolve the site of the t. monococcum domestication  <cit> .
microsatellite markers  have been tested in t. monococcum in comparison with hexaploid wheat and its a-genome donor diploid species t. urartu.
this information has been used to produce a genetic map integrating rflps and ssrs  <cit> .
however, the aforementioned markers are primarily gel-based and sequence-dependent.
the cost per data point, labour-intensive assay procedures and the limitation of polymorphism of the current marker technologies restricts their application to whole-genome scan approaches such as large-scale genotyping of germplasm collections, association mapping, pedigree analysis and qtl  mapping as you go  <cit> .
diversity arrays technology  has been developed as a sequence-independent and micro-array hybridisation-based marker system  <cit> .
dart generates medium-density genome scans by scoring the presence versus absence of dna fragments in representations of genomic dna samples.
it simultaneously determines hundreds to thousands of polymorphic loci in a single assay  <cit> .
since its initial development in rice, dart has been employed in genetic mapping, genotyping and diversity assessment in barley  <cit> , arabidopsis  <cit> , cassava  <cit> , sorghum  <cit> , hexaploid and durum wheat  <cit> , and approximately  <dig> other plant species .
dart has also been used to study pan-genomic evolution in non-model organisms  <cit>  because of its high-throughput and cost-effective nature.
we report here the results of a study aimed to  develop a t. monococcum diversity array  for high-throughput genome-wide genotyping,  assess the utility of the dart technology for analysis of genetic diversity in a representative collection of t. monococcum accessions,  compare the relationships between the am-genome and other triticum genomes using dart markers,  produce a genetic linkage map for t. monococcum integrating dart and ssr markers, and  refine the genome locations of two morphological trait loci.
RESULTS
array composition
a total of  <dig> dart clones were developed from a psti/taqi representation generated from a mixture of dna of two t. monococcum accessions mdr <dig> and mdr <dig>  <cit> .
these two accessions were the parents for a large mapping population, which had previously been genotyped with ssr markers and used for mapping several agronomically important traits  <cit> .
in a parallel project, a substantial number of dart clones were developed from genomic representations of other triticum species with different ploidy levels .
we combined clones from all projects to assemble a custom-designed array containing  <dig> clones derived from the two t. monococcum accessions,  <dig> clones derived from hexaploid wheats ,  <dig> clones derived from tetraploid durum wheat , and  <dig> clones derived from  <dig> iranian accessions of t. boeoticum boiss., which is the wild relative of t. monococcum .
genetic diversity amongst t. monococcum accessions revealed by dart
the composite array was used to assess the genetic diversity amongst sixteen t. monococcum accessions listed in table  <dig>  they were pre-selected based on their geographical origins, useful traits, available molecular and genetic tools, as well as genetic relationships assessed using ssr fingerprinting  <cit> .
1selection from a cross between t. monococcum and t. sinskajae   <cit> .
2t.
monococcum dv92; provided by jorge dubcovsky, uc davies, usa.
3t.
monococcum pi  <dig> from usda, ars, usa.
4t.
monococcum aus16273-2; provided by dr. yuri shavrukov, acpfg, australia.
5t.
monococcum l118; provided by benjamin killian, mpi, cologne, germany.
6john innes centre, norwich, united kingdom.
7n.
i. vavilov institute of plant industry, st. petersburg, russian federation.
8united states department of agriculture, agricultural research service, aberdeen, id, usa.
9university of california, davis, ca, usa.
10australian centre for plant functional genomics, canberra, australia.
11max planck institute, cologne, germany.
12huw jones, rothamsted research, harpenden, uk, personal communication.
13kay denyer, john innes centre, uk, personal communication.
in total,  <dig> dart markers were identified as polymorphic amongst the  <dig> accessions.
polymorphism information content  values for these markers were relatively low, with only  <dig> % of darts having pic values of  <dig> - <dig>  and  <dig> % with pic values <  <dig>  .
the average mean pic value was  <dig> .
when the quality of the dart markers  was analysed against their performance, which is determined through call rate and pic values, more than half of the polymorphic dart markers  were in the 90-100% quality category with an average pic value of  <dig>  and call rate of  <dig> %, respectively .
the average pic value decreased with the average quality value.
pic values were >  <dig>  when the marker quality was > 80%.
however, pic values were reduced to  <dig> ,  <dig>  and  <dig>  for markers with the quality values of 70-80%, 60-70% and 50-60%, respectively.
this set of polymorphic dart markers was used to assess the genetic diversity of the  <dig> t. monococcum accessions.
a jaccard similarity matrix was generated and used to construct a principal coordinate plot deciphering the genetic relationships among the accessions .
the first two principal coordinates derived from the scores jointly explained  <dig> % of the total data variance.
there was a clear separation for most of the t. monococcum accessions and partial correlations between genetic relationships and geographic origins.
the accession mdr <dig>  of iranian origin was fairly distantly related to other accessions including most of the accessions of european origin.
this accession is unique in its ability to produce fertile f <dig> hybrids with hexaploid wheat  <cit> .
mdr <dig> is also a known source of resistance to leaf rust  <cit>  and provides resistance to a range of other wheat pathogens .
our previous diversity study using ssr fingerprinting has shown that another iranian accession mdr <dig> also formed an independent clade  <cit> .
furthermore, in the current study mdr <dig> and mdr <dig> were clustered close to each other and were distant to mdr <dig>  amongst the  <dig> analysed accessions there were two accessions with unusual pedigrees.
mdr <dig> is an inbred line derived from a cross between t. monococcum and triticum sinskajae  <cit> , whereas mdr <dig>  is a recombinant inbred line generated by several rounds of crosses and backcrosses between triticum boeoticum and triticum urartu .
relationships between the genomes of t. monococcum and related triticum spp
as described above, the custom-designed dart array contained clones developed from the t. monococcum genome and clones derived from the genomes of other triticum species.
this allowed us to assess  the degree of genetic similarity/diversity between different t. monococcum accessions, and  the relationships between the genome of t. monococcum and the a-genomes of other closely related triticum species.
analysis of the  <dig> t. monococcum accessions revealed that  <dig> of the  <dig> of dart markers  developed from t. monococcum accessions mdr <dig> and mdr <dig> were polymorphic .
in this set of t. monococcum accessions ~10% of dart markers derived from the genomes of hexaploid and tetraploid wheat were polymorphic, whereas only ~ <dig> % of darts derived from t. boeoticum were polymorphic.
the latter markers also displayed the lowest pic values.
a similar trend was observed for the polymorphic dart markers in the f <dig> mapping population derived from mdr <dig> × mdr <dig> cross .
overall, the dart markers developed from t. monococcum genome were found to be more informative than those developed from the genomes of other related triticum species.
t. boeoticum
t. monococcum
to test the power of the dart markers of different origins in resolving the t. monococcum relationships, the  <dig> polymorphic dart markers were split according to their genome origins and principal coordinate analyses were carried out using the four subset data.
the percentages of total data variance explained by the first two coordinates were  <dig> % in hexaploid wheat,  <dig> % in tetraploid wheat,  <dig> % in genomes of t. boeoticum, and  <dig> % in t. monococcum, respectively .
these principal coordinate similarity matrices had a correlation coefficient between  <dig> - <dig>  in describing the relationships among these t. monococcum accessions .
the mantel test  <cit>  indicated that the matrices are highly significantly associated .
t. monococcum
t. boeoticum
numbers between parentheses are the p-values for testing the null hypothesis of no association by mantel test.
to further analyse the relationships of the genomes of t. monococcum and those of hexaploid wheat, we simultaneously hybridised the genomes of nine t. monococcum accessions and  <dig> hexaploid wheat varieties of european, american and chinese origins to a customised triticarte dart array .
the ploidy levels of the genomes did influence the scoring and hence the raw data was analysed to identify dart markers which were not affected by this genome context-dependent scoring.
as shown in table  <dig>   <dig> dart markers were reliably scored, of which  <dig> markers were polymorphic in hexaploid wheat genomes but were commonly present or absent in t. monococcum genomes; whilst  <dig> markers were polymorphic in t. monococcum genomes but were monomorphic in hexaploid genomes.
however, there were  <dig> dart markers which were polymorphic in the genomes of both ploidy.
the principal coordinate plot constructed using these dart markers showed that 38% of the variation was explained by the two-dimensional analysis .
the plot showed a good separation of the genomes with different ploidy levels and the genomes within each ploidy level.
genetic mapping of dart and microsatellite  loci
to confirm that dart markers were inherited in a mendelian manner, we constructed a linkage map for a cross between t. monococcum accessions mdr <dig> and mdr <dig>  <cit> .
hybridising the array with a total of  <dig> f <dig> progeny from this cross as well as the two parental accessions identified  <dig> polymorphic dart markers:  <dig>   <dig>   <dig> and  <dig> were derived from genomes of t. monococcum, hexaploid wheat, tetraploid wheat, and t. boeoticum, respectively .
the same t. monococcum mapping population was also genotyped using microsatellite markers mapped to the a sub-genome of hexaploid wheat  <cit> .
out of  <dig> microsatellite markers analysed,  <dig>  were polymorphic between the two parental t. monococcum accessions mdr <dig> and mdr <dig>  rates of polymorphism for different types of microsatellite markers were as follows: wmc -  <dig> % , cfa and cfd -  <dig> % , barc markers -  <dig> % , gdm and wms -  <dig> % , and dupw -  <dig> % .
the data for dart and microsatellite markers were merged for construction of a genetic linkage map for t. monococcum.
map length and genome coverage
in total,  <dig>  molecular markers were mapped and formed nine linkage groups .
some dart and microsatellite markers were removed from the data during the map construction due to a lack of linked anchor markers.
the two morphological traits, namely awns colour and leaf hairiness, were found to segregate in a 1: <dig> ratio in the mdr <dig> × mdr <dig> t. monococcum mapping population.
therefore, these traits are thought to be controlled by single genes ba  and hl  and were also included in the linkage analysis .
the linkage map derived from the combined data set spanned  <dig>  cm, with an average length of  <dig>  cm per chromosome and an average density of one marker per  <dig>  cm.
each of the seven chromosomes contained both dart and ssr markers.
six of the linkage groups corresponded to six t. monococcum chromosomes, but the chromosome 4am was formed by three linkage groups.
marker distribution amongst chromosomes
various numbers of dart and ssr markers were mapped to individual chromosome linkage groups .
chromosome  <dig> am contained the lowest numbers  of the molecular markers, while the highest numbers  were found on chromosome  <dig> am.
kolmogorov-smirnov tests were performed to compare nonparametrically the equality of the distributions of the dart and ssr markers along individual chromosomes.
the results showed that chromosomes 1am, 3am, 5am, 6am and 7am had p-values smaller than  <dig>  but chromosomes 2am and 4am had larger p-values.
these results suggest that dart and ssr tended to form independent clusters on chromosomes in t. monococcum.
distribution of dart markers derived from genomes of different triticum species
the mapped  <dig> polymorphic dart markers were derived from t. aestivum, t. durum, t. monococcum and t. boeoticum.
figure  <dig> shows the numbers of the four category dart markers and ssr markers on the seven chromosomes.
chi-square goodness-of-fit test of associations between origins of dart markers and t. monococcum linkage groups showed that the distribution of dart markers arising from different genomes and ssr markers was at random across chromosomes .
however, in some cases, dart markers of certain origins were either over- or under-numbered.
for instance, the related a genome dart markers were under-represented on chromosomes 3am, but over-represented on 7am.
fifty-eight of these markers are with known locations in genomes of hexaploid and tetraploid wheat ; only  <dig> are of a-genome origins , suggesting that b and d genomes of other triticum species could provide substantial polymorphic information in the am genome of t. monococcum.
each of the seven t. monococcum chromosomes carried loci of the dart markers that were previously mapped to homoeologous chromosomes in the b- or d-genomes of triticum species .
also, the map positions of some of the a-genome derived darts determined in our study disagreed with those obtained in previous studies.
for instance, some of the markers thought to map to 4a and 7a in hexaploid and tetraploid wheat were mapped to the chromosome 2am in t. monococcum, whereas some other darts thought to map to 7a in hexaploid and tetraploid wheat were mapped to either 4am or 5am in our study .
segregation distortion
in total,  <dig> markers were significantly distorted from the expected mendelian segregation ratios .
these were more or less equally distributed across the genome .
the chromosomes 1am, 2am, 3am, 4am, 5am, 6am and 7am contained  <dig> % ,  <dig> % ,  <dig> % ,  <dig> % ,  <dig> % ,  <dig> %  and  <dig> %  of markers displaying strong allelic frequency distortion, respectively.
for some markers the segregation distortion was in favour of alleles originating from the male parent mdr <dig>  whereas for other markers the segregation distortion was in favour of alleles originating from the female parent mdr <dig> .
the predominant alleles on the chromosomes 1am and 2am were from mdr <dig>  whereas those on 4am were from mdr <dig>  chromosomes 3am, 4am, 5am, 6am and 7am contained marker alleles originating from either male of female parents, displaying frequencies skewed from their mendelian expectations.
fine-mapping of morphological trait loci
in t. monococcum, black awns and hairy leaves are two morphological traits known to be controlled by single dominant genes ba and hl, respectively.
our previous genetic study revealed a linkage between ba and the ssr locus xwmc <dig> on 1am, and between hl and the ssr locus xcfd <dig> on 5am  <cit> .
these two traits segregated in mdr <dig> × mdr <dig> f <dig> mapping population as single independent loci and therefore it was possible to refine the positions of ba and hl in t. monococcum genome.
previous estimations showed that the genetic distance was  <dig>  cm between ba and xwmc <dig>  and  <dig>  cm between hl and xcfd <dig>  linkage analysis combining ssr and dart markers confirmed that ba was located at  <dig>  cm on 1am between the dart marker locus wpt- <dig> at  <dig>  cm and the ssr locus xgwmd <dig> at  <dig>  cm, and hl was located at  <dig>  cm on 5am between the dart marker loci  <dig> at  <dig>  cm and  <dig> at  <dig>  cm.
discussion
the availability of a high density genetic linkage map for a species is essential for identifying qtls of interest, isolation of genes by map-based cloning, comparative mapping, and genome organisation and evolution studies  <cit> .
we have developed polymorphic dart markers from genome representations of two t. monococcum accessions,  <dig> t. boeoticum, and hexaploid and tetraploid triticum species.
the genetic linkage map of t. monococcum constructed in this study is the first map integrating dart and ssr markers.
determining genetic relationships between t. monococcum accessions and with hexaploid wheat varieties using dart marker fingerprinting
dart markers are typically developed from a representation which is generated from a pool of dna samples from a number of accessions, cultivars or breeding lines which as a group cover the genetic diversity within a species or a group of closely related species  <cit> .
in the current study, dart markers were initially generated from genomic representations of two t. monococcum accessions, parents of an f <dig> mapping population.
in addition, this study took advantage of the existence of a large number of dart markers previously developed from hexaploid, tetraploid and other diploid triticum species.
the inclusion of dart clones from various genomes meant the average pic value of the data set  was lower than in comparable studies.
for example, for barley, sorghum and cassava the values obtained were  <dig> ,  <dig>  and  <dig> , respectively  <cit> .
however, the data provided useful information for comparison of triticum genomes of different ploidy levels.
in the genetic diversity assessment of  <dig> t. monococcum accessions,  <dig> %  of the dart markers developed from the two t. monococcum accessions were polymorphic.
dart markers developed from genomes of other triticum species also displayed good polymorphism frequencies  in t. monococcum.
in hexaploid wheat,  <dig> %  of dart markers were found to be polymorphic when assessing the genetic diversity of  <dig> australian cultivars  <cit> .
similar studies in durum wheat and barley revealed only  <dig> % and  <dig> % of polymorphic darts, respectively  <cit> .
thus, the group of  <dig> t. monococcum accession genotyped in this study appeared to be more genetically diverse than the bread and durum wheat collections assayed in those studies.
furthermore, most of the dart markers whose genome locations have been determined in previous studies were located on homoeologous chromosomes of t. monococcum.
thus, dart markers from related genomes were also useful in probing genetic diversity in t. monococcum.
the custom-designed dart array developed here can be used in studies focusing on comparison of the t. monococcum genome with genomes of other triticum species.
principal coordinate analysis of the  <dig> t. monococcum accession revealed that the site of collection is only partially correlated with genetic diversity.
most accessions used in this study have been genotyped with ssr markers in a previous study, and a similar pattern had been observed  <cit> .
thus, dart fingerprints are as useful for resolving the genetic relationships in t. monococcum as non-dart based markers.
one of the accessions, mdr <dig>  was found to be genetically distantly related to other t. monococcum accessions, which raised a question whether it is a true t. monococcum.
however, mdr <dig>  possesses the three major traits of the domesticated einkorn wheat: larger and plumper seeds, a tough rachis preventing spikelets falling apart at maturity, and relatively easy threshing.
these traits are absent in the wild species t. boeoticum.
we have crossed mdr <dig>  with many other t. monococcum accessions and discovered that the crossability could reach 100% .
furthermore, mdr <dig>  did not cluster with hexaploid wheat and was closer to the t. monococcum cluster.
thus, we still consider mdr <dig>  an accession of t. monococcum.
the relationships between the am-genome of t. monococcum and those of hexaploid wheat t. aestivum and its sub-genome donor, t. urartu have been studied in different context.
for instance, chromosome pairing and recombination were studied between homoeologous chromosomes 1a and 1am, 3a and 3am as well as 5a and 5am and were shown to be collinear and differentiated at sub-structural level  <cit> .
good micro-colinearity/colinearity at particular genetic loci has also been reported  <cit> .
other studies as well as the present one demonstrate that many a-genome molecular markers could also be used for genetic studies in t. monococcum  <cit> .
however, their genome-wide use was not previously reported.
the use of dart markers allowed us to assess the relationships in a new perspective.
we showed here that most of the dart markers which are polymorphic in t. monococcum were either conserved or absent in hexaploid genomes, and vice versa, indicative of divergence of the am-genome from the a-genome  <cit> .
on the other hand, many dart markers originated from b- and d-genomes of hexaploid wheat could hybridise to the t. monococcum genomes and provide the polymorphism information in t. monococcum.
thus, at the homoeologous genomes level, there is a complex relationship between t. monococcum and hexaploid wheat.
t. monococcum genetic linkage maps
a t. monococcum genetic map has been previously constructed using mdr <dig>  as one of parental accessions by dubcovsky et al   <cit> .
this map contains  <dig> markers, including rflps, isozymes, seed storage proteins, rrna, and various morphological loci.
the total length of this map is  <dig>  cm, the average chromosome length is  <dig>  cm, and the average marker density is one marker per  <dig>  cm.
more recently, singh et al   <cit>  used a t. boeoticum × t. monococcum ril population to construct another genetic linkage map integrating  <dig> ssr and rflp markers, and two morphological trait loci.
the total length of this map is  <dig> cm, the average chromosome length is  <dig>  cm, and the average marker density is one marker per  <dig>  cm.
in comparison, our current genetic map derived from linkage analysis of an f <dig> population from a cross between mdr <dig> and mdr <dig> integrates  <dig> dart markers,  <dig> ssr markers and two morphological trait loci.
this map spans over  <dig>  cm, with six chromosomes represented by single linkage groups and chromosome 4am by three groups of linked markers.
the average chromosome length is  <dig>  cm, and the average marker density in the genome is one marker per  <dig>  cm.
observed differences in lengths and marker densities for the three t. monococcum linkage maps may well be related to differences in the mapping populations, the types and error-rates of the genetic marker systems used, and/or the algorithms and mapping functions used to compute genetic distances.
for example, singh et al   <cit>  used the haldane's mapping function, whereas the kosambi's mapping function was used in other cases.
the current genetic linkage is also very similar in length and in marker densities to the a-genome maps of hexaploid wheat constructed using the kosambi's map function.
in one case,  <dig> ssr markers were mapped onto a  <dig> cm a-genome with the marker density of one marker per  <dig>  cm  <cit> .
in another study,  <dig> ssr markers were mapped onto a  <dig> cm a-genome with the marker density of one marker per  <dig>  cm  <cit> .
recently dart markers have also been developed and integrated with conventional markers i.e.
ssrs, rflps and aflps in hexaploid wheat  <cit>  and tetraploid wheat  <cit> .
genome organisation and segregation distortion
the am-genome of t. monococcum is closely related to the au-genome of t. urartu and to the a-genome of the hexaploid wheat  <cit> .
this is reflected by the high transferability of ssr markers from hexaploid wheat to t. monococcum and the overall good colinearity of ssr arrangement along the chromosomes  <cit> .
gaps are frequently observed in hexaploid wheat genetic linkage maps making use of ssr, rflp and aflp markers  <cit> .
similarly, singh et al   <cit>  observed four large gaps on linkage maps of chromosomes 2am, 4am and 7am in t. monococcum.
in contrast, in our current linkage map for t. monococcum there was only two gaps on chromosome 4am.
this suggests that dart markers can be used to fill the gaps and help generate higher resolution genetic linkage maps.
strong segregation distortion was observed during construction of our current genetic linkage map for t. monococcum.
over one third of the marker loci across the seven chromosomes displayed allele frequencies skewed from their mendelian expectations.
this was observed for both dart and ssr markers, with preferences for both parental alleles.
segregation distortion has also been observed by dubcovsky et al   <cit>  and singh et al   <cit>  during construction of einkorn wheat genetic linkage maps, regardless of the type of populations used.
in one study   <cit>  15% of the marker loci displayed segregation distortion and were clustered on chromosomes 1am and 7am, while in other study   <cit>  two major distorted regions were detected on chromosome 2am.
strong segregation distortion has also been noted during the construction of durum wheat and bread wheat linkage maps integrating ssr and dart markers  <cit> .
utility of dart markers and the genetic linkage map in t. monococcum
the two parental t. monococcum lines, mdr <dig> and mdr <dig>  used for developing the f <dig> mapping population had a number of contrasting traits including awn colour, leaf pubescence, grain hardness, salt tolerance, as well as resistance to the fungus mycosphaerella graminicola and to soil-borne cereal mosaic viruses  <cit> .
we demonstrated here that the constructed genetic linkage map helped refine the chromosome regions spanning the ba and hl trait loci.
also, this map was recently used to refine the tmstb <dig> locus conferring resistance to m. graminicola isolate ipo <dig> on chromosome 7am, and to identify and map new qtls conferring salt tolerance .
mdr <dig> , one of the parental lines of the f <dig> mapping population used in this study, has previously been used for developing a range of molecular tools including a genetic linkage map integrating rflps, aflps and seed storage proteins  <cit> , a bac library  <cit> , and several populations of chemically- and radiation-induced mutants .
the availability of dart marker information for mdr <dig>  further increases the utility of this accession.
in the near future it should be possible to link various available genetic linkage maps of t. monococcum thereby creating a frame work consensus map for wider applications in molecular genetics and genomics studies.
CONCLUSIONS
dart is a rapid and efficient approach to develop many new molecular markers for genetic studies in t. monococcum.
the constructed genetic linkage map will facilitate localisation and map-based cloning of genes of interest, comparative mapping as well as genome organisation and evolution studies between this ancient diploid species and other crops.

 triticum monococcum, generally known as einkorn wheat, is an ancient diploid species domesticated in the fertile crescent ~ years ago is closely related to t. urartu, the donor of the a-genome of hexaploid wheat. recently, t. monococcum has gradually been recognised as an attractive diploid model for exploitation of useful traits, discovery of novel genes and variant alleles, and functional genomics. many traits have been examined in t. monococcum which can be useful for modern wheat breeding. t. monococcum has also been successfully used for gene discovery in a subgenome map-based cloning approach, as exemplified by the cloning of the leaf rust resistance gene lr, the vernalisation genes vrn and vrn, the domestication locus q, and a member of the senescence and nutrient remobilisation controlling nac gene family. natural and artificially mutagenised t. monococcum populations have been made available and were used to identify and map genes of agronomic importance. furthermore, tilling and vigs platforms for functional genomics are under development in several laboratories. thus, in the foreseeable future t. monococcum is expected to play an important role in wheat genetic and genomic studies. globally, thousands of accessions of t. monococcum have been collected and retained in major germplasm stock centres. in order to use t. monococcum resources efficiently in programmes on genetic improvement of hexaploid wheat, it is necessary to assess diversity of this species at the genome level. for this, high-throughput molecular marker technologies are needed. t. monococcum has been shown to possess a high level of polymorphism at dna marker loci. rflp and aflp markers have been developed and used for generating genetic linkage maps and for mutation mapping, map-based cloning, and genome synteny comparisons. these markers have also been used to resolve the site of the t. monococcum domestication. microsatellite markers have been tested in t. monococcum in comparison with hexaploid wheat and its a-genome donor diploid species t. urartu. this information has been used to produce a genetic map integrating rflps and ssrs. however, the aforementioned markers are primarily gel-based and sequence-dependent. the cost per data point, labour-intensive assay procedures and the limitation of polymorphism of the current marker technologies restricts their application to whole-genome scan approaches such as large-scale genotyping of germplasm collections, association mapping, pedigree analysis and qtl mapping as you go. diversity arrays technology has been developed as a sequence-independent and micro-array hybridisation-based marker system. dart generates medium-density genome scans by scoring the presence versus absence of dna fragments in representations of genomic dna samples. it simultaneously determines hundreds to thousands of polymorphic loci in a single assay. since its initial development in rice, dart has been employed in genetic mapping, genotyping and diversity assessment in barley, arabidopsis, cassava, sorghum, hexaploid and durum wheat, and approximately other plant species. dart has also been used to study pan-genomic evolution in non-model organisms because of its high-throughput and cost-effective nature. we report here the results of a study aimed to develop a t. monococcum diversity array for high-throughput genome-wide genotyping, assess the utility of the dart technology for analysis of genetic diversity in a representative collection of t. monococcum accessions, compare the relationships between the am-genome and other triticum genomes using dart markers, produce a genetic linkage map for t. monococcum integrating dart and ssr markers, and refine the genome locations of two morphological trait loci.  array composition a total of dart clones were developed from a psti/taqi representation generated from a mixture of dna of two t. monococcum accessions mdr and mdr. these two accessions were the parents for a large mapping population, which had previously been genotyped with ssr markers and used for mapping several agronomically important traits. in a parallel project, a substantial number of dart clones were developed from genomic representations of other triticum species with different ploidy levels. we combined clones from all projects to assemble a custom-designed array containing clones derived from the two t. monococcum accessions, clones derived from hexaploid wheats, clones derived from tetraploid durum wheat, and clones derived from iranian accessions of t. boeoticum boiss., which is the wild relative of t. monococcum. genetic diversity amongst t. monococcum accessions revealed by dart the composite array was used to assess the genetic diversity amongst sixteen t. monococcum accessions listed in table they were pre-selected based on their geographical origins, useful traits, available molecular and genetic tools, as well as genetic relationships assessed using ssr fingerprinting. 1selection from a cross between t. monococcum and t. sinskajae. 2t. monococcum dv92; provided by jorge dubcovsky, uc davies, usa. 3t. monococcum pi from usda, ars, usa. 4t. monococcum aus16273-2; provided by dr. yuri shavrukov, acpfg, australia. 5t. monococcum l118; provided by benjamin killian, mpi, cologne, germany. 6john innes centre, norwich, united kingdom. 7n. i. vavilov institute of plant industry, st. petersburg, russian federation. 8united states department of agriculture, agricultural research service, aberdeen, id, usa. 9university of california, davis, ca, usa. 10australian centre for plant functional genomics, canberra, australia. 11max planck institute, cologne, germany. 12huw jones, rothamsted research, harpenden, uk, personal communication. 13kay denyer, john innes centre, uk, personal communication. in total, dart markers were identified as polymorphic amongst the accessions. polymorphism information content values for these markers were relatively low, with only % of darts having pic values of - and % with pic values <. the average mean pic value was. when the quality of the dart markers was analysed against their performance, which is determined through call rate and pic values, more than half of the polymorphic dart markers were in the 90-100% quality category with an average pic value of and call rate of %, respectively. the average pic value decreased with the average quality value. pic values were > when the marker quality was > 80%. however, pic values were reduced to, and for markers with the quality values of 70-80%, 60-70% and 50-60%, respectively. this set of polymorphic dart markers was used to assess the genetic diversity of the t. monococcum accessions. a jaccard similarity matrix was generated and used to construct a principal coordinate plot deciphering the genetic relationships among the accessions. the first two principal coordinates derived from the scores jointly explained % of the total data variance. there was a clear separation for most of the t. monococcum accessions and partial correlations between genetic relationships and geographic origins. the accession mdr of iranian origin was fairly distantly related to other accessions including most of the accessions of european origin. this accession is unique in its ability to produce fertile f hybrids with hexaploid wheat. mdr is also a known source of resistance to leaf rust and provides resistance to a range of other wheat pathogens. our previous diversity study using ssr fingerprinting has shown that another iranian accession mdr also formed an independent clade. furthermore, in the current study mdr and mdr were clustered close to each other and were distant to mdr amongst the analysed accessions there were two accessions with unusual pedigrees. mdr is an inbred line derived from a cross between t. monococcum and triticum sinskajae, whereas mdr is a recombinant inbred line generated by several rounds of crosses and backcrosses between triticum boeoticum and triticum urartu. relationships between the genomes of t. monococcum and related triticum spp as described above, the custom-designed dart array contained clones developed from the t. monococcum genome and clones derived from the genomes of other triticum species. this allowed us to assess the degree of genetic similarity/diversity between different t. monococcum accessions, and the relationships between the genome of t. monococcum and the a-genomes of other closely related triticum species. analysis of the t. monococcum accessions revealed that of the of dart markers developed from t. monococcum accessions mdr and mdr were polymorphic. in this set of t. monococcum accessions ~10% of dart markers derived from the genomes of hexaploid and tetraploid wheat were polymorphic, whereas only ~ % of darts derived from t. boeoticum were polymorphic. the latter markers also displayed the lowest pic values. a similar trend was observed for the polymorphic dart markers in the f mapping population derived from mdr × mdr cross. overall, the dart markers developed from t. monococcum genome were found to be more informative than those developed from the genomes of other related triticum species. t. boeoticum t. monococcum to test the power of the dart markers of different origins in resolving the t. monococcum relationships, the polymorphic dart markers were split according to their genome origins and principal coordinate analyses were carried out using the four subset data. the percentages of total data variance explained by the first two coordinates were % in hexaploid wheat, % in tetraploid wheat, % in genomes of t. boeoticum, and % in t. monococcum, respectively. these principal coordinate similarity matrices had a correlation coefficient between - in describing the relationships among these t. monococcum accessions. the mantel test indicated that the matrices are highly significantly associated. t. monococcum t. boeoticum numbers between parentheses are the p-values for testing the null hypothesis of no association by mantel test. to further analyse the relationships of the genomes of t. monococcum and those of hexaploid wheat, we simultaneously hybridised the genomes of nine t. monococcum accessions and hexaploid wheat varieties of european, american and chinese origins to a customised triticarte dart array. the ploidy levels of the genomes did influence the scoring and hence the raw data was analysed to identify dart markers which were not affected by this genome context-dependent scoring. as shown in table dart markers were reliably scored, of which markers were polymorphic in hexaploid wheat genomes but were commonly present or absent in t. monococcum genomes; whilst markers were polymorphic in t. monococcum genomes but were monomorphic in hexaploid genomes. however, there were dart markers which were polymorphic in the genomes of both ploidy. the principal coordinate plot constructed using these dart markers showed that 38% of the variation was explained by the two-dimensional analysis. the plot showed a good separation of the genomes with different ploidy levels and the genomes within each ploidy level. genetic mapping of dart and microsatellite loci to confirm that dart markers were inherited in a mendelian manner, we constructed a linkage map for a cross between t. monococcum accessions mdr and mdr. hybridising the array with a total of f progeny from this cross as well as the two parental accessions identified polymorphic dart markers: and were derived from genomes of t. monococcum, hexaploid wheat, tetraploid wheat, and t. boeoticum, respectively. the same t. monococcum mapping population was also genotyped using microsatellite markers mapped to the a sub-genome of hexaploid wheat. out of microsatellite markers analysed, were polymorphic between the two parental t. monococcum accessions mdr and mdr rates of polymorphism for different types of microsatellite markers were as follows: wmc - %, cfa and cfd - %, barc markers - %, gdm and wms - %, and dupw - %. the data for dart and microsatellite markers were merged for construction of a genetic linkage map for t. monococcum. map length and genome coverage in total, molecular markers were mapped and formed nine linkage groups. some dart and microsatellite markers were removed from the data during the map construction due to a lack of linked anchor markers. the two morphological traits, namely awns colour and leaf hairiness, were found to segregate in a 1: ratio in the mdr × mdr t. monococcum mapping population. therefore, these traits are thought to be controlled by single genes ba and hl and were also included in the linkage analysis. the linkage map derived from the combined data set spanned cm, with an average length of cm per chromosome and an average density of one marker per cm. each of the seven chromosomes contained both dart and ssr markers. six of the linkage groups corresponded to six t. monococcum chromosomes, but the chromosome 4am was formed by three linkage groups. marker distribution amongst chromosomes various numbers of dart and ssr markers were mapped to individual chromosome linkage groups. chromosome am contained the lowest numbers of the molecular markers, while the highest numbers were found on chromosome am. kolmogorov-smirnov tests were performed to compare nonparametrically the equality of the distributions of the dart and ssr markers along individual chromosomes. the results showed that chromosomes 1am, 3am, 5am, 6am and 7am had p-values smaller than but chromosomes 2am and 4am had larger p-values. these results suggest that dart and ssr tended to form independent clusters on chromosomes in t. monococcum. distribution of dart markers derived from genomes of different triticum species the mapped polymorphic dart markers were derived from t. aestivum, t. durum, t. monococcum and t. boeoticum. figure shows the numbers of the four category dart markers and ssr markers on the seven chromosomes. chi-square goodness-of-fit test of associations between origins of dart markers and t. monococcum linkage groups showed that the distribution of dart markers arising from different genomes and ssr markers was at random across chromosomes. however, in some cases, dart markers of certain origins were either over- or under-numbered. for instance, the related a genome dart markers were under-represented on chromosomes 3am, but over-represented on 7am. fifty-eight of these markers are with known locations in genomes of hexaploid and tetraploid wheat; only are of a-genome origins, suggesting that b and d genomes of other triticum species could provide substantial polymorphic information in the am genome of t. monococcum. each of the seven t. monococcum chromosomes carried loci of the dart markers that were previously mapped to homoeologous chromosomes in the b- or d-genomes of triticum species. also, the map positions of some of the a-genome derived darts determined in our study disagreed with those obtained in previous studies. for instance, some of the markers thought to map to 4a and 7a in hexaploid and tetraploid wheat were mapped to the chromosome 2am in t. monococcum, whereas some other darts thought to map to 7a in hexaploid and tetraploid wheat were mapped to either 4am or 5am in our study. segregation distortion in total, markers were significantly distorted from the expected mendelian segregation ratios. these were more or less equally distributed across the genome. the chromosomes 1am, 2am, 3am, 4am, 5am, 6am and 7am contained %, %, %, %, %, % and % of markers displaying strong allelic frequency distortion, respectively. for some markers the segregation distortion was in favour of alleles originating from the male parent mdr whereas for other markers the segregation distortion was in favour of alleles originating from the female parent mdr. the predominant alleles on the chromosomes 1am and 2am were from mdr whereas those on 4am were from mdr chromosomes 3am, 4am, 5am, 6am and 7am contained marker alleles originating from either male of female parents, displaying frequencies skewed from their mendelian expectations. fine-mapping of morphological trait loci in t. monococcum, black awns and hairy leaves are two morphological traits known to be controlled by single dominant genes ba and hl, respectively. our previous genetic study revealed a linkage between ba and the ssr locus xwmc on 1am, and between hl and the ssr locus xcfd on 5am. these two traits segregated in mdr × mdr f mapping population as single independent loci and therefore it was possible to refine the positions of ba and hl in t. monococcum genome. previous estimations showed that the genetic distance was cm between ba and xwmc and cm between hl and xcfd linkage analysis combining ssr and dart markers confirmed that ba was located at cm on 1am between the dart marker locus wpt- at cm and the ssr locus xgwmd at cm, and hl was located at cm on 5am between the dart marker loci at cm and at cm. discussion the availability of a high density genetic linkage map for a species is essential for identifying qtls of interest, isolation of genes by map-based cloning, comparative mapping, and genome organisation and evolution studies. we have developed polymorphic dart markers from genome representations of two t. monococcum accessions, t. boeoticum, and hexaploid and tetraploid triticum species. the genetic linkage map of t. monococcum constructed in this study is the first map integrating dart and ssr markers. determining genetic relationships between t. monococcum accessions and with hexaploid wheat varieties using dart marker fingerprinting dart markers are typically developed from a representation which is generated from a pool of dna samples from a number of accessions, cultivars or breeding lines which as a group cover the genetic diversity within a species or a group of closely related species. in the current study, dart markers were initially generated from genomic representations of two t. monococcum accessions, parents of an f mapping population. in addition, this study took advantage of the existence of a large number of dart markers previously developed from hexaploid, tetraploid and other diploid triticum species. the inclusion of dart clones from various genomes meant the average pic value of the data set was lower than in comparable studies. for example, for barley, sorghum and cassava the values obtained were, and, respectively. however, the data provided useful information for comparison of triticum genomes of different ploidy levels. in the genetic diversity assessment of t. monococcum accessions, % of the dart markers developed from the two t. monococcum accessions were polymorphic. dart markers developed from genomes of other triticum species also displayed good polymorphism frequencies in t. monococcum. in hexaploid wheat, % of dart markers were found to be polymorphic when assessing the genetic diversity of australian cultivars. similar studies in durum wheat and barley revealed only % and % of polymorphic darts, respectively. thus, the group of t. monococcum accession genotyped in this study appeared to be more genetically diverse than the bread and durum wheat collections assayed in those studies. furthermore, most of the dart markers whose genome locations have been determined in previous studies were located on homoeologous chromosomes of t. monococcum. thus, dart markers from related genomes were also useful in probing genetic diversity in t. monococcum. the custom-designed dart array developed here can be used in studies focusing on comparison of the t. monococcum genome with genomes of other triticum species. principal coordinate analysis of the t. monococcum accession revealed that the site of collection is only partially correlated with genetic diversity. most accessions used in this study have been genotyped with ssr markers in a previous study, and a similar pattern had been observed. thus, dart fingerprints are as useful for resolving the genetic relationships in t. monococcum as non-dart based markers. one of the accessions, mdr was found to be genetically distantly related to other t. monococcum accessions, which raised a question whether it is a true t. monococcum. however, mdr possesses the three major traits of the domesticated einkorn wheat: larger and plumper seeds, a tough rachis preventing spikelets falling apart at maturity, and relatively easy threshing. these traits are absent in the wild species t. boeoticum. we have crossed mdr with many other t. monococcum accessions and discovered that the crossability could reach 100%. furthermore, mdr did not cluster with hexaploid wheat and was closer to the t. monococcum cluster. thus, we still consider mdr an accession of t. monococcum. the relationships between the am-genome of t. monococcum and those of hexaploid wheat t. aestivum and its sub-genome donor, t. urartu have been studied in different context. for instance, chromosome pairing and recombination were studied between homoeologous chromosomes 1a and 1am, 3a and 3am as well as 5a and 5am and were shown to be collinear and differentiated at sub-structural level. good micro-colinearity/colinearity at particular genetic loci has also been reported. other studies as well as the present one demonstrate that many a-genome molecular markers could also be used for genetic studies in t. monococcum. however, their genome-wide use was not previously reported. the use of dart markers allowed us to assess the relationships in a new perspective. we showed here that most of the dart markers which are polymorphic in t. monococcum were either conserved or absent in hexaploid genomes, and vice versa, indicative of divergence of the am-genome from the a-genome. on the other hand, many dart markers originated from b- and d-genomes of hexaploid wheat could hybridise to the t. monococcum genomes and provide the polymorphism information in t. monococcum. thus, at the homoeologous genomes level, there is a complex relationship between t. monococcum and hexaploid wheat. t. monococcum genetic linkage maps a t. monococcum genetic map has been previously constructed using mdr as one of parental accessions by dubcovsky et al. this map contains markers, including rflps, isozymes, seed storage proteins, rrna, and various morphological loci. the total length of this map is cm, the average chromosome length is cm, and the average marker density is one marker per cm. more recently, singh et al used a t. boeoticum × t. monococcum ril population to construct another genetic linkage map integrating ssr and rflp markers, and two morphological trait loci. the total length of this map is cm, the average chromosome length is cm, and the average marker density is one marker per cm. in comparison, our current genetic map derived from linkage analysis of an f population from a cross between mdr and mdr integrates dart markers, ssr markers and two morphological trait loci. this map spans over cm, with six chromosomes represented by single linkage groups and chromosome 4am by three groups of linked markers. the average chromosome length is cm, and the average marker density in the genome is one marker per cm. observed differences in lengths and marker densities for the three t. monococcum linkage maps may well be related to differences in the mapping populations, the types and error-rates of the genetic marker systems used, and/or the algorithms and mapping functions used to compute genetic distances. for example, singh et al used the haldane's mapping function, whereas the kosambi's mapping function was used in other cases. the current genetic linkage is also very similar in length and in marker densities to the a-genome maps of hexaploid wheat constructed using the kosambi's map function. in one case, ssr markers were mapped onto a cm a-genome with the marker density of one marker per cm. in another study, ssr markers were mapped onto a cm a-genome with the marker density of one marker per cm. recently dart markers have also been developed and integrated with conventional markers i.e. ssrs, rflps and aflps in hexaploid wheat and tetraploid wheat. genome organisation and segregation distortion the am-genome of t. monococcum is closely related to the au-genome of t. urartu and to the a-genome of the hexaploid wheat. this is reflected by the high transferability of ssr markers from hexaploid wheat to t. monococcum and the overall good colinearity of ssr arrangement along the chromosomes. gaps are frequently observed in hexaploid wheat genetic linkage maps making use of ssr, rflp and aflp markers. similarly, singh et al observed four large gaps on linkage maps of chromosomes 2am, 4am and 7am in t. monococcum. in contrast, in our current linkage map for t. monococcum there was only two gaps on chromosome 4am. this suggests that dart markers can be used to fill the gaps and help generate higher resolution genetic linkage maps. strong segregation distortion was observed during construction of our current genetic linkage map for t. monococcum. over one third of the marker loci across the seven chromosomes displayed allele frequencies skewed from their mendelian expectations. this was observed for both dart and ssr markers, with preferences for both parental alleles. segregation distortion has also been observed by dubcovsky et al and singh et al during construction of einkorn wheat genetic linkage maps, regardless of the type of populations used. in one study 15% of the marker loci displayed segregation distortion and were clustered on chromosomes 1am and 7am, while in other study two major distorted regions were detected on chromosome 2am. strong segregation distortion has also been noted during the construction of durum wheat and bread wheat linkage maps integrating ssr and dart markers. utility of dart markers and the genetic linkage map in t. monococcum the two parental t. monococcum lines, mdr and mdr used for developing the f mapping population had a number of contrasting traits including awn colour, leaf pubescence, grain hardness, salt tolerance, as well as resistance to the fungus mycosphaerella graminicola and to soil-borne cereal mosaic viruses. we demonstrated here that the constructed genetic linkage map helped refine the chromosome regions spanning the ba and hl trait loci. also, this map was recently used to refine the tmstb locus conferring resistance to m. graminicola isolate ipo on chromosome 7am, and to identify and map new qtls conferring salt tolerance. mdr, one of the parental lines of the f mapping population used in this study, has previously been used for developing a range of molecular tools including a genetic linkage map integrating rflps, aflps and seed storage proteins, a bac library, and several populations of chemically- and radiation-induced mutants. the availability of dart marker information for mdr further increases the utility of this accession. in the near future it should be possible to link various available genetic linkage maps of t. monococcum thereby creating a frame work consensus map for wider applications in molecular genetics and genomics studies.  dart is a rapid and efficient approach to develop many new molecular markers for genetic studies in t. monococcum. the constructed genetic linkage map will facilitate localisation and map-based cloning of genes of interest, comparative mapping as well as genome organisation and evolution studies between this ancient diploid species and other crops. 
dart markers were developed for t. monococcum to assess genetic diversity, compare relationships with hexaploid genomes, and construct a genetic linkage map integrating dart and microsatellite markers. the constructed genetic linkage map will facilitate localisation and map-based cloning of genes of interest, comparative mapping as well as genome organisation and evolution studies between this ancient diploid species and other crops. dart is a rapid and efficient approach to develop many new molecular markers for genetic studies in t. monococcum. for the first time, dart markers were integrated with simple sequence repeat and two morphological trait loci in a genetic map spanning cm in t. monococcum. a dart array, consisting of hexaploid wheat, tetraploid wheat, t. monococcum as well as t. boeoticum representative genomic clones, was used to fingerprint t. monococcum accessions of diverse geographical origins. the dart and ssr genetic loci tended to form independent clusters along the chromosomes. segregation distortion was observed for one third of the dart loci. triticum monococcum is an ancient diploid wheat with many useful traits and is used as a model for wheat gene discovery. the fingerprinting data indicated that the geographic origin of t. monococcum accessions was partially correlated with their genetic variation. in total, polymorphic dart markers were identified, of which were of t. monococcum origin, of hexaploid, of tetraploid, and of t. boeoticum genomes. dart markers could also well distinguish the genetic differences amongst a panel of hexaploid wheat and nine t. monococcum genomes. six chromosomes were represented by single linkage groups, and chromosome 4am was formed by three linkage groups.
 triticum monococcum is an ancient diploid wheat with many useful traits and is used as a model for wheat gene discovery. dart employs a hybridisation-based approach to type thousands of genomic loci in parallel. dart markers were developed for t. monococcum to assess genetic diversity, compare relationships with hexaploid genomes, and construct a genetic linkage map integrating dart and microsatellite markers.  a dart array, consisting of hexaploid wheat, tetraploid wheat, t. monococcum as well as t. boeoticum representative genomic clones, was used to fingerprint t. monococcum accessions of diverse geographical origins. in total, polymorphic dart markers were identified, of which were of t. monococcum origin, of hexaploid, of tetraploid, and of t. boeoticum genomes. the fingerprinting data indicated that the geographic origin of t. monococcum accessions was partially correlated with their genetic variation. dart markers could also well distinguish the genetic differences amongst a panel of hexaploid wheat and nine t. monococcum genomes. for the first time, dart markers were integrated with simple sequence repeat and two morphological trait loci in a genetic map spanning cm in t. monococcum. six chromosomes were represented by single linkage groups, and chromosome 4am was formed by three linkage groups. the dart and ssr genetic loci tended to form independent clusters along the chromosomes. segregation distortion was observed for one third of the dart loci. the ba locus was refined to a cm region between the dart marker locus wpt- and the microsatellite locus xgwmd on 1am; and the hl locus to a cm region between dart loci and on 5am.  dart is a rapid and efficient approach to develop many new molecular markers for genetic studies in t. monococcum. the constructed genetic linkage map will facilitate localisation and map-based cloning of genes of interest, comparative mapping as well as genome organisation and evolution studies between this ancient diploid species and other crops. 