BACKGROUND
bread wheat  is a primary staple crop worldwide, as it accounts for a large portion of calories  consumed by humans.
the complete assembled genome sequence of this important crop holds the key for further guiding improvements in wheat germplasm to meet the needs of an ever increasing world population.
however, the ordering of bac contigs for completing physical maps and assembly of complete genome sequence in large and complex genomes like that of bread wheat, require putting together a highly saturated marker scaffold.
for example, the study reporting the first physical map of a wheat chromosome could anchor only 75 % of the chromosome 3b physical map using  <dig>  markers  <cit>  suggesting that saturated maps with >  <dig>  markers would be needed for complete coverage of the entire wheat genome sequence assembly.
high resolution genome maps also serve as effective tools for many other purposes including molecular breeding and positional gene cloning.
they provide more choice in selecting quality markers in an important chromosome interval for any marker assisted breeding program and to correctly position a gene of interest between close flanking markers in map-based cloning experiments  <cit> .
in plants, map development thus far has been largely limited to recombination based approaches .
however, recombination mapping techniques generally have three main limitations in developing high density molecular maps.
1) the need for polymorphic markers.
low genetic variation in the cultivated pool reduces the number of markers to be mapped by genetic mapping.
this scenario is exacerbated in the case of gene based markers such as ests  <cit> , which are valuable for interspecies comparative studies.
this is due to the selection pressure which tends to remove any non-advantageous allelic polymorphism in the gene space making them highly monomorphic among individuals of the same species.
2) uneven distribution of recombination along the length of the chromosomes  <cit> .
saintenac et al.
<cit>  observed >85-fold difference for crossover frequency per physical distance  for centromeric bin  and sub-telomeric bin  on wheat chromosome 3b.
it was also observed that 42 % of the physical map length of chromosome 3b is represented by only  <dig>  % of the genetic map length in the centromeric regions  <cit> .
consequently, genetic maps lead to inaccurate genetic to physical distance estimates.
more importantly, it becomes a difficult  task, to develop high density marker scaffolds for sequence assembly and to clone genes present in low recombination regions when only relying upon genetic mapping.
3) the need for larger population size to achieve higher resolution.
the physical and practical considerations of exceptionally large populations can quickly render them unreasonable.
the above challenges are worsened in case of the wheat d-genome, which harbors positive alleles for several important traits .
since the d-genome is considered a recent evolutionary addition to the hexaploid wheat genome , there has been limited gene flow from ae.
tauschii  <cit>  and, due to this fact, the wheat varieties show limited marker polymorphism among the d-genome loci  <cit> .
thus, the challenge to develop a saturated d-genome genetic map is intrinsically limited by a dearth of mappable polymorphic markers.
an alternative to recombination-based mapping is radiation hybrid  mapping which uses radiation-induced chromosomal breaks and co-retention pattern of the markers to map them onto chromosomes with the principle that the likelihood of separation of two adjacent markers due to radiation breakage increases with the increase in physical distance  <cit> .
by estimating the frequency of breakage/retention between markers, their relative distance and order can be calculated.
because radiations are expected to cause independent chromosomal breakages, rh mapping offers more uniform resolution across the length of the chromosome in comparison to genetic mapping.
it also offers much higher resolution with relatively small number of rh lines  <cit> .
recently, a rh map constructed using  <dig> lines showed on average 10x higher resolution than the genetic map   <cit> .
moreover, rh is based upon presence  vs. absence  assay, allowing the mapping of monomorphic markers as well.
rh mapping, in combination with recombination mapping, has contributed enormously towards whole genome mapping  and sequence assembly of human and animal genomes .
however, there have been relatively few examples of rh mapping reported in plants .
recent advances in next generation sequencing have made it possible to generate huge amount of sequence data in a short amount of time and at a very low cost.
wheat has also benefited from these advances, as evidenced by the recent reports on sequencing and analysis of the whole genome  <cit> , draft sequences of the a-genome progenitor t. urartu  <cit>  and the d-genome progenitor ae.
tauschii  <cit> .
however, due to the complex nature of the wheat genome, all these studies reported the development of thousands of smaller contigs/scaffolds which ultimately need to be ordered to generate high quality sequence assembly.
this clearly shows the need to develop high density genome maps capable of anchoring and ordering all those thousands of contigs/scaffolds.
to this end, a rh panel  for the d-genome of ae.
tauschii accession ‘al8/78’  <cit>  was developed to complement ongoing efforts by the members of international wheat genome sequencing consortium  toward development of a complete physical map  of ae.
tauschii accession al8/ <dig> and ultimately bread wheat.
here, an al8/78-dgrh <dig> panel was used to develop the first high resolution rh maps for the d-genome of ae.
tauschii accession al8/ <dig>  which were then used for the anchoring of unassigned sequence scaffolds.
this study demonstrates how rh mapping can facilitate the complete sequence assembly of the large and complex plant genomes.
RESULTS
selected rh panel for d-genome of ae.
tauschii accession al8/78
recently a panel of  <dig>  rh lines for mapping of the d-genome of ae.
tauschii accession al8/ <dig>  was developed and characterized  <cit> .
using  <dig> ssr markers from across the whole d-genome, a total of  <dig> rh <dig> lines  were identified, with individual lines showing deletions for at least one of these ssrs.
in the present study,  <dig> informative and one non-informative line  were further characterized using the wheat dart array .
dart analysis resulted in identification of  <dig> dart markers specific to the d-genome  and showed consistent results on four replicates of each of the parental genotypes, i.e.
sw <dig> and ldn.
analysis of the  <dig> line dgrh <dig> panel with  <dig> dart markers showed an average marker loss of  <dig>  %.
the average marker loss based on the  <dig> ssr markers was  <dig>  % for these  <dig> lines.
a simple explanation for this difference in deletion frequency could be that ssrs used in this study were mostly associated with qtl/genes and potentially represent gene rich regions.
the loss of these regions could lead to reduction in vitality.
darts span both the genic and non-genic regions of the genome.
this could result in reduced detectable deletion frequency for ssrs as compared to darts.
in our earlier study  <cit> , it was observed that the average marker loss was significantly higher for repeat junction markers , which are present throughout the genome , compared to ssrs  and ests , which were not significantly different from each other.
overall, with  <dig> markers , an average loss of  <dig>  % was observed for the whole d-genome.
for individual chromosomes, the average marker loss varied from  <dig>  %  to  <dig>  %  and was found to be homogeneous among the seven d-genome chromosomes .
however, the average marker loss for individual chromosomes was negatively correlated  with chromosome size  <cit> .table  <dig> summary of al8/ <dig> d-genome-rh panel characterization
anumber in parenthesis is the total number of obligate breaks in a panel of  <dig> rh lines
bnumber in parenthesis is the total number of deletions in a panel of  <dig> rh lines
cnumber in parenthesis is the range of deletion size
the deletion frequency for individual markers varied from  <dig>  to  <dig>  % and was heterogeneous across the whole genome.
however, χ <dig> homogeneity tests showed that the deletion frequency of individual markers in a given chromosome was homogeneous  for chromosomes 1d, 2d, 4d, 5d and 6d, while it was heterogeneous for chromosomes 3d and 7d.
in case of chromosome 3d, heterogeneity was mainly due to few markers present together on the long arm and less than expected deletion frequency, while in case of chromosome 7d, the heterogeneity was mainly caused by few markers present on the short arm and higher than expected deletion frequency.
the average marker loss for individual dgrh <dig> lines, based on  <dig> markers, ranged from  <dig> to 90 %.
a high correlation  was observed between the deletion frequencies of the rh lines based on characterization with  <dig>  and 641markers .
in the  <dig> dgrh <dig> line panel,  <dig> ssrs detected deletions for only one chromosome in about three fourth  of the lines, while the remaining lines showed deletions for two or three chromosomes only.
however, characterization of the same panel with  <dig> markers  showed that majority of these lines have deletions for multiple chromosomes .
about one third  of the lines showed deletions for all seven d-genome chromosomes and >84 % lines showed deletions for two or more chromosomes.fig.
<dig> characterization of the rh panel with two different set of markers
rh maps for d-genome of aegilops tauschii
the  <dig> dart and  <dig> ssr marker data  <cit>  was used to generate rh maps for the d-genome.
in total,  <dig> markers  could be mapped to  <dig> unique positions on the seven chromosomes .
the number of markers mapped on individual chromosomes ranged from  <dig>  to as many as  <dig> .
the  <dig> co-segregating markers belong to six chromosomes ; chromosome 7d had the highest number  and chromosome 5d had no such markers.table  <dig> statistics on rh maps of all seven d-genome chromosomes of aegilops tauschii accession al8/78
cm/cra
agenetic distances based on huang et al.
<cit>
bphysical sizes based on doležel et al.
<cit>
fig.
<dig> radiation hybrid maps of d-genome of aegilops tauschii accession al8/ <dig>  the chromosome 7d has also one contiguous rh map but due to larger size, it has been split into two parts
the  <dig> markers  covered a total map length of  <dig> .
<dig> cr  with individual chromosome lengths ranging from  <dig> .
<dig> cr  to  <dig> .
<dig> cr  .
the average distance between any two marker loci was  <dig>  cr, which corresponds to  <dig>  cm  <cit>  or  <dig>  mb  <cit> .
the highest marker density was observed for chromosome 7d, which has an average distance of  <dig>  cr between any two marker loci, while the lowest markers density was observed for chromosome 2d, with an average distance of  <dig>  cr between any two marker loci.
resolution of the al8/78-rh maps
one of the most important features in any map is the mapping resolution.
in rh mapping, the resolution may be expressed either as kilobases  to centiray  ratio or centimorgan  to centiray  ratio.
so, the rh map distances were compared with the estimated physical sizes of wheat d-genome chromosomes  <cit>  and genetic distances  <cit>  on the d-genome map .
the estimate for mb/cr was  <dig>  for the whole d-genome, with a range of  <dig>   to  <dig>   for individual chromosomes.
similarly, the estimate for cm/cr were  <dig>  for the whole d-genome, with a range of  <dig>   to  <dig>  .
a high correlation  was observed between the resolution  and the number of marker loci mapped on individual chromosomes, meaning that the resolution was higher for chromosomes with more mapped loci.
this is expected for the resolution in rh mapping depends upon the number of deletions, and the present study as well as the past studies  <cit>  clearly show that with few markers only a few deletions are detected in the rh lines.
the increase in marker density leads to the detection of more deletions, thus resulting in higher resolution.
anchoring the al8/78-rh map with bac contig assembly and draft genome sequence
in order to achieve a completely assembled genome sequence for the wheat d-genome, two important studies were recently published.
luo et al.
<cit>  generated a genetic map with  <dig>  markers  and then anchored a total of  <dig>  gb of d-genome bac contigs to the map, while jia et al.
<cit>  sequenced ae.
tauschii genome to ~90x depth of short reads, and assembled them into sequence scaffolds which represented  <dig>  % of the d-genome.
in order to demonstrate the utility of rh maps in anchoring bac contigs or sequence scaffolds, the present rh maps were used as a framework to anchor a subset of the ae.
tauschii d-genome physical maps and draft genome sequence by blastn comparison of the available sequences of  <dig> dart markers against the marker sequences mapped in al8/78 × as <dig> genetic map  <cit>  and draft genome sequence of ae.
tauschii  <cit> .
a total of  <dig> marker sequences showed matches  with the markers mapped in the al8/78 × as <dig> genetic map of luo et al.
<cit>  .
seven contigs each were anchored to chromosomes 3d and 4d, four contigs each were anchored to chromosomes 2d, 5d and 6d, and three contigs were anchored to chromosome 1d, while only two contigs were anchored to chromosome 7d .
of these  <dig> markers,  <dig> markers were mapped to the same chromosome in our rh maps and genetic maps of luo et al.
<cit> , while four markers were mapped to different chromosomes in the two maps.table  <dig> summary of the al8/78-rh map markers anchored to d-genome sequence scaffolds and bac contigs
ajia et al.
<cit> ; bluo et al.
<cit>
a total of  <dig>  dart sequences showed positive hits with the draft genome sequence of ae.
tauschii .
these  <dig> markers were anchored to  <dig> sequence scaffolds .
among them,  <dig> scaffolds were singularly anchored to dart marker, while the remaining  <dig> scaffolds were anchored to multiple  markers.
the number of sequence scaffolds anchored to individual chromosomes ranged from  <dig>  to  <dig> .
among the  <dig> sequence scaffolds which were anchored to al8/78-rh map, only  <dig> scaffolds  were previously anchored and ordered by jia et al.
<cit>  using a population of 490 f <dig> individuals derived from a cross between the ae.
tauschii accessions y <dig> and al8/ <dig>  we were able to anchor and order an additional  <dig> sequence scaffolds  using rh maps.
in general, a good agreement in marker order was observed between al8/78-rh maps and al8/78 × as <dig> or y2280 × al8/ <dig> genetic maps.
comparison of al8/78-rh maps with other published genetic and rh maps
the accuracy of the marker order can be tested by comparing common markers mapped in different maps.
so, the marker order of al8/78-rh maps was tested by comparison with: 1) a dart consensus genetic map  <cit> ; 2) a snp based genetic map with anchored d-genome bac contigs  <cit> ; 3) a snp genetic map anchoring sequence scaffolds of d-genome ; and 4) a rh map of d-genome of chinese spring .
the al8/78-rh maps were directly compared with the consensus maps by huang et al.
<cit>  and the rh maps by tiwari et al.
<cit>  as all these maps have dart markers with the same nomenclature.
in order to identify the common markers mapped on the ae.
tauschii genetic map by luo et al.
<cit> , the available  <dig> dart sequences mapped in al8/78-rh map were used to blast compare against the sequences containing snp markers positioned on the genetic map  <cit> .
in the case of jia et al.
<cit> , the snp markers were used to anchor the sequence scaffolds onto the y2280 × al8/ <dig> genetic map.
so, to identify the common markers, a blast query was performed with the dart sequences  against the mapped sequence scaffolds.
in relation to the al8/78-rh maps, there are  <dig> markers in common with the dart consensus genetic map  <cit> ,  <dig> markers in common with the cs-rh map  <cit> ,  <dig> markers in common with the bac contig-anchored ae.
tauschii genetic map  and  <dig> markers in common with sequence scaffold-anchored ae.
tauschii map  <cit> .
in total, 75 % of dart markers  mapped in the present study were present on at least one of the above four maps.
the remaining  <dig> dart markers mapped in the al8/78-rh map were not present in any of the four previous studies mentioned.
overall, a good consistency in chromosome assignment and marker order was observed in the comparative maps.
all the markers in common between the al8/78-rh maps and the consensus genetic  <cit>  and cs-rh maps  <cit>  showed complete agreement in chromosome assignment of the markers.
among the  <dig> markers common between the al8/78-rh maps and the al8/78 × as <dig> genetic maps  <cit> , only four markers {wpt- <dig> , wpt- <dig> , wpt- <dig> , wpt- <dig> } were assigned to different chromosomes in the two maps.
however, these four markers showing different chromosome assignment in the al8/78 × as <dig> genetic map, when checked in other published maps  <cit> , were found to be mapped on the same chromosomes as that of the present rh maps.
the markers wpt- <dig> and wpt- <dig> were mapped on 3d and 6d respectively by tiwari et al.
<cit> , the marker wpt- <dig> was mapped on 6d by both tiwari et al.
<cit>  and jia et al.
<cit> , on the same chromosomes as the present rh maps.
the marker wpt- <dig>  was not mapped in any other map but did show positive hybridization to chromosome 7d, when hybridized with genomic representations of flow-sorted chromosome arms  of bread wheat , consistent with the al8/78-rh map data.
among the  <dig> markers in common between al8/78-rh maps and y2280 × al8/ <dig> genetic maps, only eight markers {wpt- <dig> , wpt-7697 , wpt-9662 , wpt- <dig> , wpt- <dig> , wpt- <dig> , wpt-731370 , wpt-2325 } showed different chromosome assignments between the two maps.
however, further investigations showed that three {wpt-9662 , wpt- <dig> , wpt-2325 } out of the eight markers were mapped by tiwari et al.
<cit>  and/or huang et al.
<cit>  on the same chromosomes as that of the al8/78-rh maps.
the remaining five markers were not mapped in any other maps.
however, three of the remaining five markers {wpt-4988 , wpt- <dig>  wpt- <dig> } showed positive hybridization to the same chromosome on which they were mapped in our rh maps, when hybridized with the flow-sorted chromosome arms .
the marker order in al8/78-rh maps also showed a good agreement with all the above mentioned maps.
majority of the markers with disparity in order showed minor local flipped rearrangements.
there were only few markers which showed large differences in placement on the chromosomes in comparison with some of the earlier studies.
an agreement in map position can be estimated by calculating the rank order correlations between the map position of al8/78-rh maps and other published maps.
the mean rank order correlations between the map position of each chromosome of al8/78-rh maps with the y2280 × al8/ <dig> genetic map  <cit> , al8/78 × as <dig> genetic map  <cit> , dart consensus genetic map  <cit>  and cs-rh map  <cit>  averaged  <dig>  ,  <dig>  ,  <dig>   and  <dig>  , respectively .
further analysis on the data suggests that the few low values of rank order correlations between map positions of chromosomes are mainly due to a small number of common markers as well as due to several co-segregating markers present in maps other than the al8/78-rh maps.
however, overall these comparisons with multiple studies clearly validate the accuracy of al8/78-rh maps.table  <dig> rank correlation coefficient values between the map position of each chromosome of al8/78-rh maps with four other published maps of d-genome
ajia et al.
<cit> ; bluo et al.
<cit> ; chuang et al.
<cit> ; dtiwari et al.
<cit>
*figures in parenthesis are the probability values; higher p-values in all cases were due to fewer common markers between the compared maps
size of deletions in the al8/78-rh panel
to estimate the number of deletions , all the data belonging to mapped markers was arranged in map order for each line.
an obligate break was scored between two loci  whenever the score for marker loci a = <dig> and for a + 1 =  <dig> or vice versa.
with  <dig> mapped markers, a total of  <dig>  obligate breaks including  <dig> terminal and  <dig>  interstitial breaks were observed across the whole d-genome.
the average number of obligate breaks per line was  <dig>  , with a maximum of  <dig> breaks in a single line for the whole d-genome.
for individual chromosomes, the average number of obligate breaks per line ranged from  <dig>   to  <dig>  .
the number of obligate breaks per chromosome were homogeneous among chromosomes .
one obligate break is required for a terminal deletion, while two obligate breaks are required for an interstitial deletion.
based on  <dig> mapped markers,  <dig>  breaks yielded a total of  <dig>  deletions, including  <dig> terminal and  <dig>  interstitial deletions for the whole d-genome.
the average number of deletions in the whole d-genome for a single line was  <dig>  with a maximum of  <dig> deletions for a single line.
the average number of deletions per line for individual chromosomes was homogeneous  and ranged from  <dig>   to  <dig>  .
the number of obligate breaks/deletions, among other factors, depends on chromosome length and number of markers used for characterization.
in the present study, both the number of markers and the physical length of the chromosomes had a significant correlation with the average number of deletions detected for a particular chromosome.
however, the correlation between the number of markers and number of deletions  was much higher as compared to the correlations between physical length of the chromosome and number of deletions .
to estimate the size of deletions, the physical size of the deletions for individual chromosomes was estimated first in rh map distances  and then converted into physical distances  using the physical sizes of individual chromosomes  <cit> .
individually, the size of deletions ranged from  <dig>  to 748 mb .
however, the average deletion size for the whole d-genome was  <dig>  mb, while for individual chromosomes it varied from  <dig>  mb for chromosome 7d to  <dig>  mb for chromosome 4d .
a very high negative rank order correlation  was observed between the number of markers and the average deletion size for a particular chromosome.
this is expected because with few markers mostly larger deletions are detected.
however, an increase in marker density leads to detection of smaller deletions which ultimately reduces the average size of deletions.
based on  <dig> marker data, the frequency of deletions of different sizes in the rh panel was almost similar for different chromosomes .
deletions of <20 mb form the major group followed by deletions ranging from  <dig> to 40 mb.
together, the deletions of sizes <40 mb represent about 77 % of the total deletions detected for the whole d-genome.
a small proportion  of deletions of sizes larger than 200 mb was also observed.fig.
<dig> distribution of deletion sizes in al8/78-dgrh <dig> panel
discussion
sequencing costs have decreased radically in the past few years.
however, it still is not possible to completely assemble short sequence reads of large and complex plant genomes such as wheat.
so, to achieve the goal of an accurate sequence assembly of wheat, several approaches are being followed.
recently, several studies aimed at achieving the complete genome sequence of the d-genome of wheat were published  <cit> .
luo et al.
<cit>  fingerprinted bac clones which were then assembled into contigs, while jia et al.
<cit>  used a very high depth of short sequence reads to develop sequence contigs/scaffolds.
however, in both cases, a high density genome map is needed to anchor, order or align the large number of contigs/scaffolds onto the chromosomes.
therefore, the availability of a high quality genome map is of utmost importance  in generating a correct genome assembly in species with large and complex genomes.
in this study, we characterized a rh panel in depth and demonstrated how development of a rh map can contribute to the assembly of large and complex genomes.
characterization of radiation induced deletions
the majority of the informative lines when initially selected with few markers  generally showed deletions for a single chromosome  <cit> , but when characterized with more markers  in the present study, deletions for multiple chromosomes were detected .
the reason for this discovery of additional affected chromosomes could be that the majority of the deletions caused by gamma rays are small in size  and are not detected when using only a few widely spaced markers.
however, when more markers are used the deletion detection efficiency across the genome is improved.
this also is supported by a high correlation  observed between the number of detected deletions on any chromosome and density of markers mapped on that particular chromosome.
similar results were previously reported  <cit> .
it indicates that informative lines might have multiple deletions for all chromosomes requiring high density of markers for detection.
based on  <dig> mapped markers, the average deletion size was estimated to be 42 mb at the whole genome level; however, deletion size differed widely amongst the seven d-genome chromosomes.
these differences could be attributed mainly to the different number of markers mapped on each chromosome .
the increase in number of markers used for genotyping leads to the detection of additional smaller deletions, which ultimately results in decreased average deletion size determination.
this observation is evident in a very high rank order correlation  between the number of markers and the average deletion size for a particular chromosome.
this also means that the estimates of average deletion size are likely to decrease further when these rh lines are genotyped with more markers.
when the deletions were grouped based on size, the frequency of deletions of different sizes showed almost similar patterns for all chromosomes .
the majority of deletions on all chromosomes fall into two smallest size groups, 0– <dig> and 20–40 mb.
however, it was interesting to observe that in the 0–20 mb group, the proportion of deletions was positively correlated with the number of marker loci mapped on a particular chromosome.
but, as we move towards larger size deletion groups , a highly significant negative correlation between proportion of deletions and number of marker loci mapped on a particular chromosome was observed.
these observations indicate that an increase in number of markers for genotyping will lead to the detection of additional smaller deletions, which correspondingly means a decrease in the proportion of larger deletions.
these smaller deletions will be the key in developing high resolution genome maps for whole genome sequence assembly.
rh maps of ae.
tauschii genome and mapping resolution
the accession al8/ <dig> was chosen for it has been used extensively for genomic studies including the sequencing of d-genome chromosomes  <cit> .
the rh maps reported here demonstrate the utility of rh methodology in achieving higher resolution across the length of the chromosome, anchoring and ordering bac contigs/sequence scaffold and identification of potential errors in genetic maps.
one of the factors affecting the rh map resolution is the panel size.
the al8/78-rh maps were developed using a total of  <dig> rh lines.
most of the studies in animals, however, have used about  <dig> rh lines .
there were two main reasons to use a larger rh panel when developing high density rh maps in plants.
first, compared to the animal rh panels, most of the plant rh panels have lower deletion frequency as most are represented by viable plants.
the average marker deletion frequency of the rh panel used in this study was  <dig>  %, while most of the animal panels have a reported 70-80 % deletion frequency .
secondly, studies have shown that utilizing panels with larger number of lines yield fewer linkage groups and provide more accurate information  <cit> .
thus, we believe that the use of comparably larger rh panels in plants might be one of the possible solutions towards achieving the resolutions and level of success for complete genome mapping.
in this study, a total of  <dig> markers  were mapped to  <dig> unique loci .
comparatively, a study reporting the consensus maps of the d-genome of wheat based on the genetic data from about  <dig> populations could resolve only  <dig>  % of the dart loci  <cit> .
this shows the power of rh lines in resolving the closely linked markers.
in the present study, the main reason for the co-segregation of ~ <dig>  % of the markers with other loci was some level of redundancy in genomic representation of dart markers.
when the available sequences of  <dig> dart markers were analyzed for redundancy, it was observed that  <dig>  % of the markers were redundant .
a recent study which analyzed sequences of dart markers from the a and b genomes of wheat, also estimated that about  <dig>  % of the markers showed sequence similarity with other markers  <cit> .
this suggests that the present set of rh lines was able to resolve majority  of the unique loci.
the precision of an rh map can be characterized by the resolution, expressed in the kilobase  to centiray  ratio.
the present maps display an average mapping resolution of  <dig>  mb/cr.
in comparison to genetic maps, it was  <dig>  cm/cr.
therefore, our rh map resolution is higher than the resolution reported for chromosome 3b  or resolution  and the number of markers loci mapped on individual chromosome.
accuracy of al8/78-rh maps
the accuracy of the al8/78-rh maps was tested for both chromosome assignment and marker order by comparing with previously published genetic and rh maps  <cit> .
the al8/ <dig> rh maps were in complete agreement in chromosome assignment with the consensus genetic maps by huang et al.
<cit>  and cs-rh maps by tiwari et al.
<cit> .
this observation is expected as each of these studies used the dart platform for genotyping.
only a few markers  showed different chromosome assignments from the genetic maps by luo et al.
<cit>  and jia et al.
<cit> .
a simple reason for these differences in chromosomal assignment could be that the three studies used different genotyping methods and as wheat has a complex and highly repetitive genome, depending on genotyping method different orthologous or paralogous sequences could be amplified resulting in different assignment of marker.
however, the results of al8/ <dig> rh mapping were confirmed when these few discrepancies were further investigated by comparison with other studies  and/or hybridization with the genomic representations of flow-sorted chromosome arms of bread wheat.
the results showed that most of these questionable markers  respect the chromosome assignment of al8/78-rh maps.
this shows the potential of rh mapping approach for correcting genotyping errors.
in terms of marker order, there was a good agreement between the al8/78-rh map and other available maps.
the few differences in marker order and local inversions observed could be due to several reasons including different methods of genotyping, differences in genetic material, presence of repeat elements/paralogues, different analysis algorithms and parameters used in different studies for mapping.
more specifically, the differences observed with dart consensus genetic map could be attributed to the fact that the consensus map is based on the data from about  <dig> bi-parental populations, which may have different structural and recombination frequency variations.
similarly, the differences with cs-rh map could be attributed to differences on d-genome as a result of  <dig>  years of evolution.
the al8/78-rh maps showed highest values of rank order correlation when compared with y2280 × al8/ <dig> and al8/78 × as <dig> genetic maps, which could be expected as both of these genetic maps used a mapping population developed using al8/ <dig> as one of the parent  <cit> .
the few differences observed in marker order could be attributed to different genotyping methods used in these studies.
however, as all the mapping approaches are prone to minor errors, information from several sources including genetic and radiation hybrid maps may help in achieving high quality genome sequence assembly.
rh mapping increases the number of mappable markers
one of the major constraints in developing high density maps using recombination mapping is the dependence upon polymorphism.
in wheat, most of the mapping populations show about 20-40 % genetic polymorphism  <cit> , which further decreases when mapping gene based markers .
the above challenge becomes even worse in the case for the wheat d-genome, since it is the most recent evolutionary addition to the hexaploid wheat, and there has been limited gene flow from ae.
tauschii  <cit> .
due to this fact, the current wheat germplasm show much lower molecular marker polymorphism for the d-genome loci, compared to the a or b genomes  <cit> .
thus, any attempts to develop saturated maps for the wheat d-genome using genetic mapping confront a persistent problem of having limited mappable polymorphic markers.
on the other hand, rh mapping does not rely on allelic polymorphism and uses assays for the presence and absence of marker loci, thus making it possible to map monomorphic loci.
the published genetic mapping studies have reported very few polymorphic markers for the d-genome  <cit> .
using bi-parental genetic populations, a total of  <dig> dart markers by akbari et al.
<cit> , and  <dig> markers by wang et al.
<cit>  were mapped on d-genome of wheat using dart array.
the study by sorrells et al.
<cit>  was able to map  <dig> dart markers on the d-genome of the itmi population as this population was developed using a synthetic wheat with the d-genome of ae.
tauschii.
a recent study which integrated the mapping data from more than  <dig> bi-parental genetic populations to develop a high-density dart consensus map had a total of only  <dig> dart markers placed on d-genome  <cit> .
compared to the above mentioned genetic mapping studies, using only a  <dig> rh line population, we were able to map  <dig> dart markers, while another rh study was able to map  <dig> dart markers using a panel for the d-genome of chinese spring  <cit> .
in total, both these studies were able to map  <dig> dart markers  onto the d-genome of wheat.
this clearly shows that with rh mapping populations, the number of mappable markers can be increased several fold over conventional genetic mapping methods.
this advantage will be important toward obtaining a complete marker scaffold for developing a whole genome assembly in wheat and other complex plant genomes.
radiation hybrids offer more uniform mapping
another critical issue with genetic mapping is the non-uniform distribution of recombination along the length of a chromosome.
it is also well established that a large portion of the chromosomes around the centromere in plants generally show very low recombination or no recombination  <cit>  and this holds true with the d-genome of wheat  <cit> .
luo et al.
<cit>  observed that although the genes were distributed across the entire length of ae.
tauschii chromosomes, the average recombination rate ranged from almost zero in proximal chromosome regions to about  <dig> - <dig>  cm/mb in the distal regions.
therefore, using genetic mapping, it becomes very difficult, if not impossible, to develop high resolution maps for anchoring sequence scaffold/contigs and to clone genes/qtl from the low recombination regions, particularly in the regions near the centromere.
comparatively, rh maps show more uniform and complete coverage of the genome as they use radiation induced deletions rather than genetic recombinations  <cit> .
the results of this study also showed that the deletion frequency for individual markers was homogeneous for majority of the chromosomes and no significant differences in deletion frequency were observed across a given chromosome .
the genetic maps of the d-genome of al8/ <dig> showed that almost no recombination occurred in the proximal regions of all chromosomes  <cit> .
however, the physical maps of al8/ <dig>  <cit>  show that the genes are distributed across the length of the chromosomes and the regions of high gene density are interspersed by regions of low gene density.
a somewhat similar pattern was observed for deletion frequency of markers for all the chromosomes, where regions of high deletion frequency were interspersed by regions of low deletion frequency .
the reason could be that the most of the dart markers represent gene sequences  <cit>  and the irradiated cells with deletions in gene rich regions are less likely to survive the selection during rh plant development.
this could mean that the observed deletion frequency for gene rich regions will be less, while it will be higher for low gene density regions.fig.
<dig> a scattergram of the marker retention frequencies along d-genome chromosomes.
the horizontal line represents the approximate position of the centromeres as estimated by hybridization of dart sequences with the genomic representations of flow-sorted chromosome arms of bread wheat
additional evidence of a more uniform distribution of markers in rh maps was observed when al8/78-rh maps were compared with genetic maps  <cit> .
the analysis shows several blocks of low marker coverage on most of the chromosomes in genetic maps in comparison to our rh maps.
this observation was clearly visible on chromosomes 3d and 7d which have dense rh maps .
however, when al8/78-rh maps are compared with cs-rh maps  <cit> , most of the chromosomes show much better uniform distribution of markers in both the rh maps .
another evidence for a more uniform distribution of markers in rh maps was the number of marker loci mapped on individual chromosomes.
among the d-genome chromosomes, most of the genetic mapping studies were able to map only few markers on chromosomes 4d and 5d compared to other chromosomes due to lack of polymorphism for these chromosomes  <cit> .
comparatively, in the present study, if the chromosomes 3d and 7d which have more markers because the dart array was enriched with these chromosome specific genomic representations  are excluded, the remaining five chromosomes of the al8/78-rh maps have almost similar number of mapped loci.
this clearly shows that rh maps have great potential to complement genetic mapping approach for developing high density genome maps for the purpose of assembly of large plant genomes as well as for cloning genes in low recombination regions.
al8/78-rh maps for sequence assembly
in last few years with the rapid developments in dna sequencing technologies, more than fifty plant genomes have been published  <cit> .
however, still it is not possible to de novo generate one contiguous sequence for a given chromosome using only next generation sequencing data in large and complex genomes.
in wheat, recently published draft sequence assemblies of the a-  <cit>  and d-genome  <cit>  reported the generation of thousands of scaffolds with a n <dig> length of only  <dig>  kb and  <dig>  kb, respectively.
another recent study using bac fingerprinting, generated  <dig>  bac contigs  <cit> .
in both cases, a high resolution genome map is needed to anchor and order these scaffold and contigs for high quality genome assembly.
given the high and uniform resolution of al8/78-rh maps and the fact that a larger panel comprised of  <dig> informative lines  is now available through this project  <cit> , the use of rh might be promising approach to anchor and order most of the sequence scaffolds of the  <dig> -gb d-genome.
CONCLUSIONS
a radiation hybrid panel was used to construct maps for the genome of ae.
tauschii accession al8/ <dig>  the rh maps showed good accuracy when compared with several other independent genetic and rh data sets.
these rh maps showed a mapping resolution of  <dig>  mb and  <dig>  cm per cr.
the rh approach was able to map almost three times more markers compared to the available genetic maps of the d-genome and provided much more uniform distribution of markers across the genome.
the panel of only  <dig> rh lines was able to resolve >97 % of the loci.
finally, the rh maps were used to anchor  <dig> bac contigs  and  <dig> sequence scaffold , more than half of which were not anchored to any previous maps.
clearly, dense al8/78-rh maps have the potential to anchor and order available bac contigs  <cit>  and sequence scaffolds  <cit> , which can ultimately lead to an improved and complete genome assembly of the wheat d-genome.
this study provides a model for developing dense and accurate molecular maps for completing sequence assembly in large and complex plant genomes where de novo sequence assembly is difficult.

 bread wheat is a primary staple crop worldwide, as it accounts for a large portion of calories consumed by humans. the complete assembled genome sequence of this important crop holds the key for further guiding improvements in wheat germplasm to meet the needs of an ever increasing world population. however, the ordering of bac contigs for completing physical maps and assembly of complete genome sequence in large and complex genomes like that of bread wheat, require putting together a highly saturated marker scaffold. for example, the study reporting the first physical map of a wheat chromosome could anchor only 75 % of the chromosome 3b physical map using markers suggesting that saturated maps with >  markers would be needed for complete coverage of the entire wheat genome sequence assembly. high resolution genome maps also serve as effective tools for many other purposes including molecular breeding and positional gene cloning. they provide more choice in selecting quality markers in an important chromosome interval for any marker assisted breeding program and to correctly position a gene of interest between close flanking markers in map-based cloning experiments. in plants, map development thus far has been largely limited to recombination based approaches. however, recombination mapping techniques generally have three main limitations in developing high density molecular maps. 1) the need for polymorphic markers. low genetic variation in the cultivated pool reduces the number of markers to be mapped by genetic mapping. this scenario is exacerbated in the case of gene based markers such as ests, which are valuable for interspecies comparative studies. this is due to the selection pressure which tends to remove any non-advantageous allelic polymorphism in the gene space making them highly monomorphic among individuals of the same species. 2) uneven distribution of recombination along the length of the chromosomes. saintenac et al. observed >85-fold difference for crossover frequency per physical distance for centromeric bin and sub-telomeric bin on wheat chromosome 3b. it was also observed that 42 % of the physical map length of chromosome 3b is represented by only  % of the genetic map length in the centromeric regions. consequently, genetic maps lead to inaccurate genetic to physical distance estimates. more importantly, it becomes a difficult task, to develop high density marker scaffolds for sequence assembly and to clone genes present in low recombination regions when only relying upon genetic mapping. 3) the need for larger population size to achieve higher resolution. the physical and practical considerations of exceptionally large populations can quickly render them unreasonable. the above challenges are worsened in case of the wheat d-genome, which harbors positive alleles for several important traits. since the d-genome is considered a recent evolutionary addition to the hexaploid wheat genome, there has been limited gene flow from ae. tauschii and, due to this fact, the wheat varieties show limited marker polymorphism among the d-genome loci. thus, the challenge to develop a saturated d-genome genetic map is intrinsically limited by a dearth of mappable polymorphic markers. an alternative to recombination-based mapping is radiation hybrid mapping which uses radiation-induced chromosomal breaks and co-retention pattern of the markers to map them onto chromosomes with the principle that the likelihood of separation of two adjacent markers due to radiation breakage increases with the increase in physical distance. by estimating the frequency of breakage/retention between markers, their relative distance and order can be calculated. because radiations are expected to cause independent chromosomal breakages, rh mapping offers more uniform resolution across the length of the chromosome in comparison to genetic mapping. it also offers much higher resolution with relatively small number of rh lines. recently, a rh map constructed using lines showed on average 10x higher resolution than the genetic map. moreover, rh is based upon presence vs. absence assay, allowing the mapping of monomorphic markers as well. rh mapping, in combination with recombination mapping, has contributed enormously towards whole genome mapping and sequence assembly of human and animal genomes. however, there have been relatively few examples of rh mapping reported in plants. recent advances in next generation sequencing have made it possible to generate huge amount of sequence data in a short amount of time and at a very low cost. wheat has also benefited from these advances, as evidenced by the recent reports on sequencing and analysis of the whole genome, draft sequences of the a-genome progenitor t. urartu and the d-genome progenitor ae. tauschii. however, due to the complex nature of the wheat genome, all these studies reported the development of thousands of smaller contigs/scaffolds which ultimately need to be ordered to generate high quality sequence assembly. this clearly shows the need to develop high density genome maps capable of anchoring and ordering all those thousands of contigs/scaffolds. to this end, a rh panel for the d-genome of ae. tauschii accession ‘al8/78’ was developed to complement ongoing efforts by the members of international wheat genome sequencing consortium toward development of a complete physical map of ae. tauschii accession al8/ and ultimately bread wheat. here, an al8/78-dgrh panel was used to develop the first high resolution rh maps for the d-genome of ae. tauschii accession al8/ which were then used for the anchoring of unassigned sequence scaffolds. this study demonstrates how rh mapping can facilitate the complete sequence assembly of the large and complex plant genomes.  selected rh panel for d-genome of ae. tauschii accession al8/78 recently a panel of rh lines for mapping of the d-genome of ae. tauschii accession al8/ was developed and characterized. using ssr markers from across the whole d-genome, a total of rh lines were identified, with individual lines showing deletions for at least one of these ssrs. in the present study, informative and one non-informative line were further characterized using the wheat dart array. dart analysis resulted in identification of dart markers specific to the d-genome and showed consistent results on four replicates of each of the parental genotypes, i.e. sw and ldn. analysis of the line dgrh panel with dart markers showed an average marker loss of  %. the average marker loss based on the ssr markers was  % for these lines. a simple explanation for this difference in deletion frequency could be that ssrs used in this study were mostly associated with qtl/genes and potentially represent gene rich regions. the loss of these regions could lead to reduction in vitality. darts span both the genic and non-genic regions of the genome. this could result in reduced detectable deletion frequency for ssrs as compared to darts. in our earlier study, it was observed that the average marker loss was significantly higher for repeat junction markers, which are present throughout the genome, compared to ssrs and ests, which were not significantly different from each other. overall, with markers, an average loss of  % was observed for the whole d-genome. for individual chromosomes, the average marker loss varied from  % to  % and was found to be homogeneous among the seven d-genome chromosomes. however, the average marker loss for individual chromosomes was negatively correlated with chromosome size.table summary of al8/ d-genome-rh panel characterization anumber in parenthesis is the total number of obligate breaks in a panel of rh lines bnumber in parenthesis is the total number of deletions in a panel of rh lines cnumber in parenthesis is the range of deletion size the deletion frequency for individual markers varied from to  % and was heterogeneous across the whole genome. however, χ homogeneity tests showed that the deletion frequency of individual markers in a given chromosome was homogeneous for chromosomes 1d, 2d, 4d, 5d and 6d, while it was heterogeneous for chromosomes 3d and 7d. in case of chromosome 3d, heterogeneity was mainly due to few markers present together on the long arm and less than expected deletion frequency, while in case of chromosome 7d, the heterogeneity was mainly caused by few markers present on the short arm and higher than expected deletion frequency. the average marker loss for individual dgrh lines, based on markers, ranged from to 90 %. a high correlation was observed between the deletion frequencies of the rh lines based on characterization with and 641markers. in the dgrh line panel, ssrs detected deletions for only one chromosome in about three fourth of the lines, while the remaining lines showed deletions for two or three chromosomes only. however, characterization of the same panel with markers showed that majority of these lines have deletions for multiple chromosomes. about one third of the lines showed deletions for all seven d-genome chromosomes and >84 % lines showed deletions for two or more chromosomes.fig. characterization of the rh panel with two different set of markers rh maps for d-genome of aegilops tauschii the dart and ssr marker data was used to generate rh maps for the d-genome. in total, markers could be mapped to unique positions on the seven chromosomes. the number of markers mapped on individual chromosomes ranged from to as many as. the co-segregating markers belong to six chromosomes; chromosome 7d had the highest number and chromosome 5d had no such markers.table statistics on rh maps of all seven d-genome chromosomes of aegilops tauschii accession al8/78 cm/cra agenetic distances based on huang et al. bphysical sizes based on doležel et al. fig. radiation hybrid maps of d-genome of aegilops tauschii accession al8/ the chromosome 7d has also one contiguous rh map but due to larger size, it has been split into two parts the markers covered a total map length of. cr with individual chromosome lengths ranging from. cr to. cr. the average distance between any two marker loci was cr, which corresponds to  cm or  mb. the highest marker density was observed for chromosome 7d, which has an average distance of cr between any two marker loci, while the lowest markers density was observed for chromosome 2d, with an average distance of cr between any two marker loci. resolution of the al8/78-rh maps one of the most important features in any map is the mapping resolution. in rh mapping, the resolution may be expressed either as kilobases to centiray ratio or centimorgan to centiray ratio. so, the rh map distances were compared with the estimated physical sizes of wheat d-genome chromosomes and genetic distances on the d-genome map. the estimate for mb/cr was for the whole d-genome, with a range of to for individual chromosomes. similarly, the estimate for cm/cr were for the whole d-genome, with a range of to. a high correlation was observed between the resolution and the number of marker loci mapped on individual chromosomes, meaning that the resolution was higher for chromosomes with more mapped loci. this is expected for the resolution in rh mapping depends upon the number of deletions, and the present study as well as the past studies clearly show that with few markers only a few deletions are detected in the rh lines. the increase in marker density leads to the detection of more deletions, thus resulting in higher resolution. anchoring the al8/78-rh map with bac contig assembly and draft genome sequence in order to achieve a completely assembled genome sequence for the wheat d-genome, two important studies were recently published. luo et al. generated a genetic map with markers and then anchored a total of gb of d-genome bac contigs to the map, while jia et al. sequenced ae. tauschii genome to ~90x depth of short reads, and assembled them into sequence scaffolds which represented  % of the d-genome. in order to demonstrate the utility of rh maps in anchoring bac contigs or sequence scaffolds, the present rh maps were used as a framework to anchor a subset of the ae. tauschii d-genome physical maps and draft genome sequence by blastn comparison of the available sequences of dart markers against the marker sequences mapped in al8/78 × as genetic map and draft genome sequence of ae. tauschii. a total of marker sequences showed matches with the markers mapped in the al8/78 × as genetic map of luo et al.. seven contigs each were anchored to chromosomes 3d and 4d, four contigs each were anchored to chromosomes 2d, 5d and 6d, and three contigs were anchored to chromosome 1d, while only two contigs were anchored to chromosome 7d. of these markers, markers were mapped to the same chromosome in our rh maps and genetic maps of luo et al., while four markers were mapped to different chromosomes in the two maps.table summary of the al8/78-rh map markers anchored to d-genome sequence scaffolds and bac contigs ajia et al.; bluo et al. a total of dart sequences showed positive hits with the draft genome sequence of ae. tauschii. these markers were anchored to sequence scaffolds. among them, scaffolds were singularly anchored to dart marker, while the remaining scaffolds were anchored to multiple markers. the number of sequence scaffolds anchored to individual chromosomes ranged from to. among the sequence scaffolds which were anchored to al8/78-rh map, only scaffolds were previously anchored and ordered by jia et al. using a population of 490 f individuals derived from a cross between the ae. tauschii accessions y and al8/ we were able to anchor and order an additional sequence scaffolds using rh maps. in general, a good agreement in marker order was observed between al8/78-rh maps and al8/78 × as or y2280 × al8/ genetic maps. comparison of al8/78-rh maps with other published genetic and rh maps the accuracy of the marker order can be tested by comparing common markers mapped in different maps. so, the marker order of al8/78-rh maps was tested by comparison with: 1) a dart consensus genetic map; 2) a snp based genetic map with anchored d-genome bac contigs; 3) a snp genetic map anchoring sequence scaffolds of d-genome; and 4) a rh map of d-genome of chinese spring. the al8/78-rh maps were directly compared with the consensus maps by huang et al. and the rh maps by tiwari et al. as all these maps have dart markers with the same nomenclature. in order to identify the common markers mapped on the ae. tauschii genetic map by luo et al., the available dart sequences mapped in al8/78-rh map were used to blast compare against the sequences containing snp markers positioned on the genetic map. in the case of jia et al., the snp markers were used to anchor the sequence scaffolds onto the y2280 × al8/ genetic map. so, to identify the common markers, a blast query was performed with the dart sequences against the mapped sequence scaffolds. in relation to the al8/78-rh maps, there are markers in common with the dart consensus genetic map, markers in common with the cs-rh map, markers in common with the bac contig-anchored ae. tauschii genetic map and markers in common with sequence scaffold-anchored ae. tauschii map. in total, 75 % of dart markers mapped in the present study were present on at least one of the above four maps. the remaining dart markers mapped in the al8/78-rh map were not present in any of the four previous studies mentioned. overall, a good consistency in chromosome assignment and marker order was observed in the comparative maps. all the markers in common between the al8/78-rh maps and the consensus genetic and cs-rh maps showed complete agreement in chromosome assignment of the markers. among the markers common between the al8/78-rh maps and the al8/78 × as genetic maps, only four markers {wpt-, wpt-, wpt-, wpt- } were assigned to different chromosomes in the two maps. however, these four markers showing different chromosome assignment in the al8/78 × as genetic map, when checked in other published maps, were found to be mapped on the same chromosomes as that of the present rh maps. the markers wpt- and wpt- were mapped on 3d and 6d respectively by tiwari et al., the marker wpt- was mapped on 6d by both tiwari et al. and jia et al., on the same chromosomes as the present rh maps. the marker wpt- was not mapped in any other map but did show positive hybridization to chromosome 7d, when hybridized with genomic representations of flow-sorted chromosome arms of bread wheat, consistent with the al8/78-rh map data. among the markers in common between al8/78-rh maps and y2280 × al8/ genetic maps, only eight markers {wpt-, wpt-7697, wpt-9662, wpt-, wpt-, wpt-, wpt-731370, wpt-2325 } showed different chromosome assignments between the two maps. however, further investigations showed that three {wpt-9662, wpt-, wpt-2325 } out of the eight markers were mapped by tiwari et al. and/or huang et al. on the same chromosomes as that of the al8/78-rh maps. the remaining five markers were not mapped in any other maps. however, three of the remaining five markers {wpt-4988, wpt- wpt- } showed positive hybridization to the same chromosome on which they were mapped in our rh maps, when hybridized with the flow-sorted chromosome arms. the marker order in al8/78-rh maps also showed a good agreement with all the above mentioned maps. majority of the markers with disparity in order showed minor local flipped rearrangements. there were only few markers which showed large differences in placement on the chromosomes in comparison with some of the earlier studies. an agreement in map position can be estimated by calculating the rank order correlations between the map position of al8/78-rh maps and other published maps. the mean rank order correlations between the map position of each chromosome of al8/78-rh maps with the y2280 × al8/ genetic map, al8/78 × as genetic map, dart consensus genetic map and cs-rh map averaged,, and, respectively. further analysis on the data suggests that the few low values of rank order correlations between map positions of chromosomes are mainly due to a small number of common markers as well as due to several co-segregating markers present in maps other than the al8/78-rh maps. however, overall these comparisons with multiple studies clearly validate the accuracy of al8/78-rh maps.table rank correlation coefficient values between the map position of each chromosome of al8/78-rh maps with four other published maps of d-genome ajia et al.; bluo et al.; chuang et al.; dtiwari et al. *figures in parenthesis are the probability values; higher p-values in all cases were due to fewer common markers between the compared maps size of deletions in the al8/78-rh panel to estimate the number of deletions, all the data belonging to mapped markers was arranged in map order for each line. an obligate break was scored between two loci whenever the score for marker loci a = and for a + 1 =  or vice versa. with mapped markers, a total of obligate breaks including terminal and interstitial breaks were observed across the whole d-genome. the average number of obligate breaks per line was, with a maximum of breaks in a single line for the whole d-genome. for individual chromosomes, the average number of obligate breaks per line ranged from to. the number of obligate breaks per chromosome were homogeneous among chromosomes. one obligate break is required for a terminal deletion, while two obligate breaks are required for an interstitial deletion. based on mapped markers, breaks yielded a total of deletions, including terminal and interstitial deletions for the whole d-genome. the average number of deletions in the whole d-genome for a single line was with a maximum of deletions for a single line. the average number of deletions per line for individual chromosomes was homogeneous and ranged from to. the number of obligate breaks/deletions, among other factors, depends on chromosome length and number of markers used for characterization. in the present study, both the number of markers and the physical length of the chromosomes had a significant correlation with the average number of deletions detected for a particular chromosome. however, the correlation between the number of markers and number of deletions was much higher as compared to the correlations between physical length of the chromosome and number of deletions. to estimate the size of deletions, the physical size of the deletions for individual chromosomes was estimated first in rh map distances and then converted into physical distances using the physical sizes of individual chromosomes. individually, the size of deletions ranged from to 748 mb. however, the average deletion size for the whole d-genome was  mb, while for individual chromosomes it varied from  mb for chromosome 7d to  mb for chromosome 4d. a very high negative rank order correlation was observed between the number of markers and the average deletion size for a particular chromosome. this is expected because with few markers mostly larger deletions are detected. however, an increase in marker density leads to detection of smaller deletions which ultimately reduces the average size of deletions. based on marker data, the frequency of deletions of different sizes in the rh panel was almost similar for different chromosomes. deletions of <20 mb form the major group followed by deletions ranging from to 40 mb. together, the deletions of sizes <40 mb represent about 77 % of the total deletions detected for the whole d-genome. a small proportion of deletions of sizes larger than 200 mb was also observed.fig. distribution of deletion sizes in al8/78-dgrh panel discussion sequencing costs have decreased radically in the past few years. however, it still is not possible to completely assemble short sequence reads of large and complex plant genomes such as wheat. so, to achieve the goal of an accurate sequence assembly of wheat, several approaches are being followed. recently, several studies aimed at achieving the complete genome sequence of the d-genome of wheat were published. luo et al. fingerprinted bac clones which were then assembled into contigs, while jia et al. used a very high depth of short sequence reads to develop sequence contigs/scaffolds. however, in both cases, a high density genome map is needed to anchor, order or align the large number of contigs/scaffolds onto the chromosomes. therefore, the availability of a high quality genome map is of utmost importance in generating a correct genome assembly in species with large and complex genomes. in this study, we characterized a rh panel in depth and demonstrated how development of a rh map can contribute to the assembly of large and complex genomes. characterization of radiation induced deletions the majority of the informative lines when initially selected with few markers generally showed deletions for a single chromosome, but when characterized with more markers in the present study, deletions for multiple chromosomes were detected. the reason for this discovery of additional affected chromosomes could be that the majority of the deletions caused by gamma rays are small in size and are not detected when using only a few widely spaced markers. however, when more markers are used the deletion detection efficiency across the genome is improved. this also is supported by a high correlation observed between the number of detected deletions on any chromosome and density of markers mapped on that particular chromosome. similar results were previously reported. it indicates that informative lines might have multiple deletions for all chromosomes requiring high density of markers for detection. based on mapped markers, the average deletion size was estimated to be 42 mb at the whole genome level; however, deletion size differed widely amongst the seven d-genome chromosomes. these differences could be attributed mainly to the different number of markers mapped on each chromosome. the increase in number of markers used for genotyping leads to the detection of additional smaller deletions, which ultimately results in decreased average deletion size determination. this observation is evident in a very high rank order correlation between the number of markers and the average deletion size for a particular chromosome. this also means that the estimates of average deletion size are likely to decrease further when these rh lines are genotyped with more markers. when the deletions were grouped based on size, the frequency of deletions of different sizes showed almost similar patterns for all chromosomes. the majority of deletions on all chromosomes fall into two smallest size groups, 0– and 20–40 mb. however, it was interesting to observe that in the 0–20 mb group, the proportion of deletions was positively correlated with the number of marker loci mapped on a particular chromosome. but, as we move towards larger size deletion groups, a highly significant negative correlation between proportion of deletions and number of marker loci mapped on a particular chromosome was observed. these observations indicate that an increase in number of markers for genotyping will lead to the detection of additional smaller deletions, which correspondingly means a decrease in the proportion of larger deletions. these smaller deletions will be the key in developing high resolution genome maps for whole genome sequence assembly. rh maps of ae. tauschii genome and mapping resolution the accession al8/ was chosen for it has been used extensively for genomic studies including the sequencing of d-genome chromosomes. the rh maps reported here demonstrate the utility of rh methodology in achieving higher resolution across the length of the chromosome, anchoring and ordering bac contigs/sequence scaffold and identification of potential errors in genetic maps. one of the factors affecting the rh map resolution is the panel size. the al8/78-rh maps were developed using a total of rh lines. most of the studies in animals, however, have used about rh lines. there were two main reasons to use a larger rh panel when developing high density rh maps in plants. first, compared to the animal rh panels, most of the plant rh panels have lower deletion frequency as most are represented by viable plants. the average marker deletion frequency of the rh panel used in this study was  %, while most of the animal panels have a reported 70-80 % deletion frequency. secondly, studies have shown that utilizing panels with larger number of lines yield fewer linkage groups and provide more accurate information. thus, we believe that the use of comparably larger rh panels in plants might be one of the possible solutions towards achieving the resolutions and level of success for complete genome mapping. in this study, a total of markers were mapped to unique loci. comparatively, a study reporting the consensus maps of the d-genome of wheat based on the genetic data from about populations could resolve only  % of the dart loci. this shows the power of rh lines in resolving the closely linked markers. in the present study, the main reason for the co-segregation of ~  % of the markers with other loci was some level of redundancy in genomic representation of dart markers. when the available sequences of dart markers were analyzed for redundancy, it was observed that  % of the markers were redundant. a recent study which analyzed sequences of dart markers from the a and b genomes of wheat, also estimated that about  % of the markers showed sequence similarity with other markers. this suggests that the present set of rh lines was able to resolve majority of the unique loci. the precision of an rh map can be characterized by the resolution, expressed in the kilobase to centiray ratio. the present maps display an average mapping resolution of  mb/cr. in comparison to genetic maps, it was  cm/cr. therefore, our rh map resolution is higher than the resolution reported for chromosome 3b or resolution and the number of markers loci mapped on individual chromosome. accuracy of al8/78-rh maps the accuracy of the al8/78-rh maps was tested for both chromosome assignment and marker order by comparing with previously published genetic and rh maps. the al8/ rh maps were in complete agreement in chromosome assignment with the consensus genetic maps by huang et al. and cs-rh maps by tiwari et al.. this observation is expected as each of these studies used the dart platform for genotyping. only a few markers showed different chromosome assignments from the genetic maps by luo et al. and jia et al.. a simple reason for these differences in chromosomal assignment could be that the three studies used different genotyping methods and as wheat has a complex and highly repetitive genome, depending on genotyping method different orthologous or paralogous sequences could be amplified resulting in different assignment of marker. however, the results of al8/ rh mapping were confirmed when these few discrepancies were further investigated by comparison with other studies and/or hybridization with the genomic representations of flow-sorted chromosome arms of bread wheat. the results showed that most of these questionable markers respect the chromosome assignment of al8/78-rh maps. this shows the potential of rh mapping approach for correcting genotyping errors. in terms of marker order, there was a good agreement between the al8/78-rh map and other available maps. the few differences in marker order and local inversions observed could be due to several reasons including different methods of genotyping, differences in genetic material, presence of repeat elements/paralogues, different analysis algorithms and parameters used in different studies for mapping. more specifically, the differences observed with dart consensus genetic map could be attributed to the fact that the consensus map is based on the data from about bi-parental populations, which may have different structural and recombination frequency variations. similarly, the differences with cs-rh map could be attributed to differences on d-genome as a result of  years of evolution. the al8/78-rh maps showed highest values of rank order correlation when compared with y2280 × al8/ and al8/78 × as genetic maps, which could be expected as both of these genetic maps used a mapping population developed using al8/ as one of the parent. the few differences observed in marker order could be attributed to different genotyping methods used in these studies. however, as all the mapping approaches are prone to minor errors, information from several sources including genetic and radiation hybrid maps may help in achieving high quality genome sequence assembly. rh mapping increases the number of mappable markers one of the major constraints in developing high density maps using recombination mapping is the dependence upon polymorphism. in wheat, most of the mapping populations show about 20-40 % genetic polymorphism, which further decreases when mapping gene based markers. the above challenge becomes even worse in the case for the wheat d-genome, since it is the most recent evolutionary addition to the hexaploid wheat, and there has been limited gene flow from ae. tauschii. due to this fact, the current wheat germplasm show much lower molecular marker polymorphism for the d-genome loci, compared to the a or b genomes. thus, any attempts to develop saturated maps for the wheat d-genome using genetic mapping confront a persistent problem of having limited mappable polymorphic markers. on the other hand, rh mapping does not rely on allelic polymorphism and uses assays for the presence and absence of marker loci, thus making it possible to map monomorphic loci. the published genetic mapping studies have reported very few polymorphic markers for the d-genome. using bi-parental genetic populations, a total of dart markers by akbari et al., and markers by wang et al. were mapped on d-genome of wheat using dart array. the study by sorrells et al. was able to map dart markers on the d-genome of the itmi population as this population was developed using a synthetic wheat with the d-genome of ae. tauschii. a recent study which integrated the mapping data from more than bi-parental genetic populations to develop a high-density dart consensus map had a total of only dart markers placed on d-genome. compared to the above mentioned genetic mapping studies, using only a rh line population, we were able to map dart markers, while another rh study was able to map dart markers using a panel for the d-genome of chinese spring. in total, both these studies were able to map dart markers onto the d-genome of wheat. this clearly shows that with rh mapping populations, the number of mappable markers can be increased several fold over conventional genetic mapping methods. this advantage will be important toward obtaining a complete marker scaffold for developing a whole genome assembly in wheat and other complex plant genomes. radiation hybrids offer more uniform mapping another critical issue with genetic mapping is the non-uniform distribution of recombination along the length of a chromosome. it is also well established that a large portion of the chromosomes around the centromere in plants generally show very low recombination or no recombination and this holds true with the d-genome of wheat. luo et al. observed that although the genes were distributed across the entire length of ae. tauschii chromosomes, the average recombination rate ranged from almost zero in proximal chromosome regions to about -  cm/mb in the distal regions. therefore, using genetic mapping, it becomes very difficult, if not impossible, to develop high resolution maps for anchoring sequence scaffold/contigs and to clone genes/qtl from the low recombination regions, particularly in the regions near the centromere. comparatively, rh maps show more uniform and complete coverage of the genome as they use radiation induced deletions rather than genetic recombinations. the results of this study also showed that the deletion frequency for individual markers was homogeneous for majority of the chromosomes and no significant differences in deletion frequency were observed across a given chromosome. the genetic maps of the d-genome of al8/ showed that almost no recombination occurred in the proximal regions of all chromosomes. however, the physical maps of al8/ show that the genes are distributed across the length of the chromosomes and the regions of high gene density are interspersed by regions of low gene density. a somewhat similar pattern was observed for deletion frequency of markers for all the chromosomes, where regions of high deletion frequency were interspersed by regions of low deletion frequency. the reason could be that the most of the dart markers represent gene sequences and the irradiated cells with deletions in gene rich regions are less likely to survive the selection during rh plant development. this could mean that the observed deletion frequency for gene rich regions will be less, while it will be higher for low gene density regions.fig. a scattergram of the marker retention frequencies along d-genome chromosomes. the horizontal line represents the approximate position of the centromeres as estimated by hybridization of dart sequences with the genomic representations of flow-sorted chromosome arms of bread wheat additional evidence of a more uniform distribution of markers in rh maps was observed when al8/78-rh maps were compared with genetic maps. the analysis shows several blocks of low marker coverage on most of the chromosomes in genetic maps in comparison to our rh maps. this observation was clearly visible on chromosomes 3d and 7d which have dense rh maps. however, when al8/78-rh maps are compared with cs-rh maps, most of the chromosomes show much better uniform distribution of markers in both the rh maps. another evidence for a more uniform distribution of markers in rh maps was the number of marker loci mapped on individual chromosomes. among the d-genome chromosomes, most of the genetic mapping studies were able to map only few markers on chromosomes 4d and 5d compared to other chromosomes due to lack of polymorphism for these chromosomes. comparatively, in the present study, if the chromosomes 3d and 7d which have more markers because the dart array was enriched with these chromosome specific genomic representations are excluded, the remaining five chromosomes of the al8/78-rh maps have almost similar number of mapped loci. this clearly shows that rh maps have great potential to complement genetic mapping approach for developing high density genome maps for the purpose of assembly of large plant genomes as well as for cloning genes in low recombination regions. al8/78-rh maps for sequence assembly in last few years with the rapid developments in dna sequencing technologies, more than fifty plant genomes have been published. however, still it is not possible to de novo generate one contiguous sequence for a given chromosome using only next generation sequencing data in large and complex genomes. in wheat, recently published draft sequence assemblies of the a- and d-genome reported the generation of thousands of scaffolds with a n length of only  kb and  kb, respectively. another recent study using bac fingerprinting, generated bac contigs. in both cases, a high resolution genome map is needed to anchor and order these scaffold and contigs for high quality genome assembly. given the high and uniform resolution of al8/78-rh maps and the fact that a larger panel comprised of informative lines is now available through this project, the use of rh might be promising approach to anchor and order most of the sequence scaffolds of the -gb d-genome.  a radiation hybrid panel was used to construct maps for the genome of ae. tauschii accession al8/ the rh maps showed good accuracy when compared with several other independent genetic and rh data sets. these rh maps showed a mapping resolution of  mb and  cm per cr. the rh approach was able to map almost three times more markers compared to the available genetic maps of the d-genome and provided much more uniform distribution of markers across the genome. the panel of only rh lines was able to resolve >97 % of the loci. finally, the rh maps were used to anchor bac contigs and sequence scaffold, more than half of which were not anchored to any previous maps. clearly, dense al8/78-rh maps have the potential to anchor and order available bac contigs and sequence scaffolds, which can ultimately lead to an improved and complete genome assembly of the wheat d-genome. this study provides a model for developing dense and accurate molecular maps for completing sequence assembly in large and complex plant genomes where de novo sequence assembly is difficult. 
this study demonstrates how rh mapping, which offered high and uniform resolution across the length of the chromosome, can facilitate the complete sequence assembly of the large and complex plant genomes. radiation hybrid mapping has proven to be an excellent tool for the development of such maps for it offers much higher and more uniform marker resolution across the length of the chromosome compared to genetic mapping and does not require marker polymorphism per se, as it is based on presence vs. absence marker assay. tauschii accession al8/ to confirm map order accuracy, the al8/78-rh maps were compared with:1) a dart consensus genetic map constructed using more than bi-parental populations, 2) a rh map of the d-genome of reference hexaploid wheat ’chinese spring’, and 3) two snp-based genetic maps, one with anchored d-genome bac contigs and another with anchored d-genome sequence scaffolds. with mapped markers, a total of deletions for the whole d-genome were detected with an average deletion size of  mb. in this study, a line rh panel was genotyped with ssrs and dart markers to develop the first high resolution rh maps of the entire d-genome of ae. using marker sequences, the rh maps were also anchored with a bac contig based physical map and draft sequence of the d-genome of ae. the rh maps showed almost perfect agreement with several published maps with regard to chromosome assignments of markers. this study reports the development of first high resolution rh maps for the d-genome of ae. the average distance between any two marker loci was cr which corresponds to  cm or  mb. the large and complex genome of bread wheat requires high resolution genome maps with saturated marker scaffolds to anchor and orient bac contigs/ sequence scaffolds for whole genome assembly. aegilops tauschiideletionphysical mappingradiation hybrid mappingsequence assemblywheatissue-copyright-statement© the author 2015 a total of markers were mapped to unique positions on the seven d-genome chromosomes, with a total map length of. tauschii sequence scaffolds, of which were not anchored earlier to the d-genome. tauschii accession al8/ which were then used for the anchoring of unassigned sequence scaffolds. the mean rank correlations between the position of markers on al8/ maps and the four published maps, ranged from to, suggesting a good agreement in marker order.
 the large and complex genome of bread wheat requires high resolution genome maps with saturated marker scaffolds to anchor and orient bac contigs/ sequence scaffolds for whole genome assembly. radiation hybrid mapping has proven to be an excellent tool for the development of such maps for it offers much higher and more uniform marker resolution across the length of the chromosome compared to genetic mapping and does not require marker polymorphism per se, as it is based on presence vs. absence marker assay. methods in this study, a line rh panel was genotyped with ssrs and dart markers to develop the first high resolution rh maps of the entire d-genome of ae. tauschii accession al8/ to confirm map order accuracy, the al8/78-rh maps were compared with:1) a dart consensus genetic map constructed using more than bi-parental populations, 2) a rh map of the d-genome of reference hexaploid wheat ’chinese spring’, and 3) two snp-based genetic maps, one with anchored d-genome bac contigs and another with anchored d-genome sequence scaffolds. using marker sequences, the rh maps were also anchored with a bac contig based physical map and draft sequence of the d-genome of ae. tauschii.  a total of markers were mapped to unique positions on the seven d-genome chromosomes, with a total map length of. cr. the average distance between any two marker loci was cr which corresponds to  cm or  mb. the average mapping resolution across the d-genome was estimated to be  mb or  cm. the rh maps showed almost perfect agreement with several published maps with regard to chromosome assignments of markers. the mean rank correlations between the position of markers on al8/ maps and the four published maps, ranged from to, suggesting a good agreement in marker order. with mapped markers, a total of deletions for the whole d-genome were detected with an average deletion size of  mb. a total of markers were anchored to ae. tauschii sequence scaffolds, of which were not anchored earlier to the d-genome.  this study reports the development of first high resolution rh maps for the d-genome of ae. tauschii accession al8/ which were then used for the anchoring of unassigned sequence scaffolds. this study demonstrates how rh mapping, which offered high and uniform resolution across the length of the chromosome, can facilitate the complete sequence assembly of the large and complex plant genomes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords aegilops tauschiideletionphysical mappingradiation hybrid mappingsequence assemblywheatissue-copyright-statement© the author 2015 