BACKGROUND
common wheat  is an allohexaploid species with three distinct genomes, a, b and d, which have been defined by genome analysis  <cit> .
the wheat genome consists of  <dig> chromosomes, and each of the three subgenomes contributes  <dig> chromosomes  <cit> .
the genome has a total length of  <dig>  gb  <cit> , which is extremely large compared to the sequenced genomes of other grass species: rice , brachypodium distachyon , sorghum , maize  and barley .
most of the wheat genome is occupied by various types of repetitive dna sequences, which account for more than 80 % of the entire genome  <cit> .
although wheat has been widely used in cytogenetics because of the high visibility of its chromosomes, complex features, such as its large size, high repeat content and polyploidy, have hampered molecular approaches to date, particularly whole-genome sequencing.
however, recent technological advances have paved the way for genomic approaches that work for even complex genomes, such as wheat.
for example, whole-genome sequences of the common wheat cultivar ‘chinese spring’ , wild diploid wheat triticum urartu  and wild diploid goat grass aegilops tauschii  were analyzed using next-generation sequencing  technology .
the international wheat genome sequencing consortium  has been coordinating wheat genome sequencing to enhance knowledge of the structure and function of the bread wheat genome  and to provide tools to facilitate the breeding of improved varieties.
the consortium has adapted a strategy that relies on the purification of individual chromosome arms from wheat telosomic lines  <cit>  by flow-cytometric sorting to reduce the sample complexity  <cit>  and obtain chromosome-specific genomic data.
recently, the iwgsc presented a draft sequence of all  <dig> wheat chromosomes  <cit>  in which the dna of flow-sorted fractions was sequenced by ngs.
to obtain a reference genome sequence using bac-by-bac sequencing, physical mapping of the bac contigs  is currently underway for each of the  <dig> chromosomes  <cit> .
to date, bac-based physical maps have been developed for chromosomes 1a, 1b, 3b and 6a .
most recently, during the preparation of this paper, two additional physical maps for chromosome arms 3ds and 5ds were released  <cit> .
our group is responsible for sequencing wheat chromosome 6b  under the framework of iwgsc.
this chromosome is the third largest chromosome in common wheat, with an estimated molecular size of 914 mb: 415 mb representing the short arm  and 498 mb representing the long arm   <cit> .
as a first step, we conducted whole-chromosome shotgun sequencing using dna that had been amplified from the flow-sorted chromosome arms 6bs and 6bl with massively parallel  <dig> pyrosequencing  <cit> .
the survey sequence data were highly informative for determining the genomic composition of chromosome 6b.
within a total of 508 mb of assembled sequence ,  <dig> gene loci were predicted, and more than 70 % of the 6b assembly consisted of repetitive sequences.
functional non-protein-coding rnas, such as micro-rna, transfer rna and ribosomal rna , were also identified.
in particular, the short arm  is characterized by the presence of secondary constriction, the nucleolus organizer region , which features a locus for the rrna genes   <cit> .
the nor is designated as nor-b <dig>  <cit> .
we identified several contigs containing the rdna locus, and they exhibited extremely high read depths, indicating that these contigs were part of the nor on 6bs.
in this study, we established a bac-based physical map of chromosome 6b with the goal of developing a high-quality reference sequence for the wheat genome.
to achieve this goal, two bac libraries were constructed using arm-specific dna samples.
in addition, we used two recently developed genomic tools to physically map all of the contigs onto chromosome 6b.
this process was performed using insertion site-based polymorphism  markers  that were designed from the junction sequences between transposable elements and their flanking sequences  <cit> , and a radiation hybrid  mapping panel  that was derived from a series of chromosome deletion lines generated by γ-ray irradiation  <cit> .
combining the above genomic resources with other wheat-specific data, we were able to develop a robust system to anchor and order contigs onto the high-resolution rh map of chromosome 6b.
the physical map not only allows us to understand key structural features and the evolutionary history of chromosome 6b but will also enable the use of genome sequencing to create a high-quality map-based reference sequence in the near future.
RESULTS
construction of 6b arm-specific bac libraries
chromosome arms 6bs and 6bl were purified by flow-cytometric sorting from the double ditelosomic line 6b of cs  <cit> , as described by tanaka et al.
<cit> .
the average purities in the sorted fractions were  <dig> and 92 % for 6bs and 6bl, respectively.
high-molecular-weight dna prepared from the purified arms was used to construct the bac libraries taacsp6bsha and taacsp6blha, which were specific to 6bs and 6bl, respectively .
the libraries consisted of  <dig>  and  <dig>  bac clones with average insert lengths of  <dig> and 130 kb, representing  <dig>  and  <dig> genome equivalents of the estimated size of 6bs and 6bl, respectively .table  <dig> arm-specific bac libraries on chromosome 6b
construction of a bac-based physical map of wheat chromosome 6b
whole genome profiling   <cit>  was used to fingerprint a total of  <dig>  and  <dig>  bacs from 6bs and 6bl, representing  <dig> times the total physical lengths of the arms, respectively .
after deconvolution for the assignment of high-quality reads, we made several modifications to the standard wgp procedure following philippe et al.
<cit>  to ensure a highly accurate physical map.
we initially excluded bacs with less than 30 %  or more than  <dig>  times  the average number of tags per bac from the analysis based on the average number of tags per bac  .
this filtering resulted in the availability of  <dig>  and  <dig>  bacs for assembly on 6bs and 6bl, representing  <dig>  and  <dig>  times the total physical length, respectively .
assembly of the fingerprints of 6bs and 6bl was performed using fingerprinted contigs  software  <cit> .
a stepwise method was used to improve the quality of the contigs in the assembly, which was initially defined during the physical map construction of chromosome 3b  <cit>  and then modified for the wgp fingerprints  <cit> .
using the 6b-specific parameters at final cut-off values of 1e− <dig> and 1e− <dig>  which were determined based on the balance among the number of contigs, the number of q-contigs and the coverage of the physical map, we constructed the first physical maps of 6bs and 6bl with  <dig> and  <dig> contigs, respectively .fig.
<dig> comparison of the contig assemblies between cut-offs of 1e− <dig> and 1e− <dig> in the chromosome arms of 6bs and 6bl.
a the number of contigs and their cumulative sizes at each cutoff value.
the colors in the bars represent the number of clones comprising one bac contig.
b total number of singleton clones in the physical map at each cut-off value.
c total number of q-contigs containing ≤10 % questionable clones in the physical map at each cut-off value.
d total number of contigs in the physical maps of 6bs and 6bl with an optimal cut-off value.
eight colors in circles represent the number of clones comprising one bac contig.
the sizes of the circles indicate the relative total number of contigs in the physical map of 6bs and 6bl
these physical maps still contained a large number of small contigs , particularly on 6bs .
however, the small contigs might be redundant because of their low-quality fingerprints containing genomic fragments that were already present in the large contigs.
in addition, they had the potential to carry bac clones derived from chromosomes other than 6b due to dna contamination during the chromosome-sorting process .
to avoid any chromosomal redundancy and contamination, we eliminated  <dig> and  <dig> of these small and low-confidence contigs from the above physical map constructed primarily on the two arms of 6bs and 6bl, respectively, based on the results of a homology search of the wgp tag sequences against the whole-genome survey sequence  <cit>  and/or the depth of the bac clones within each contig.
finally, we constructed the bac-based physical map of chromosome arms 6bs and 6bl, which contained  <dig> and  <dig> contigs, respectively .table  <dig> physical maps of chromosome arms 6bs and 6bl
acoverage was calculated based on the estimated size of each arm: 415 mb for 6bs and 498 mb for 6bl
the above process greatly improved our bac-based physical map.
the first physical maps revealed estimated sizes of  <dig> and 495 mb that corresponded to 119 % and 99 % of the arm lengths, n <dig> values of  <dig> and 2422 kb and l <dig> values of  <dig> and  <dig> contigs for 6bs and 6bl, respectively .
however, the final maps showed remarkable increases in the n <dig> values  and a decrease in the l <dig> values , although the total lengths for both arms decreased to  <dig> and 475 mb, covering approximately  <dig> and 95 % of the entire genomic region for 6bs and 6bl, respectively .
the reason for the lower coverage of 6bs remains unclear, but it is likely a consequence of the purity of the sorted chromosome arm dna , the number of wgp tags on each bac  or differences in the genomic structure between 6bs and 6bl .
further overlap analysis of neighboring clones within each contig using the fpc mtp module allowed us to select  <dig> and  <dig> minimal tiling path  clones along the two arms, laying a foundation for the map-based genomic sequencing of the entire chromosome 6b .
anchoring of chromosome 6b-specific markers to determine the physical location of the bac contigs
to obtain a pseudomolecule sequence, which is the final goal of iwgsc, it is essential to map the contigs onto their specific genomic regions on wheat chromosome 6b.
for that purpose, a set of anchors is needed.
as shown in table  <dig>  we first searched through the available marker information in public databases, such as the “graingenes cmap”  and the “national bioresource project -wheat, japan” , and  <dig> ssr and rflp markers  were found to be potentially useful for chromosome 6b.
second, we collected a resource of  <dig> dna markers from the pcr-based landmark unique gene  database .
these markers are known est-pcr markers that were developed by taking advantage of the orthologous gene conservation between rice and wheat  <cit> .
to develop new markers, we designed  <dig> and  <dig> pcr primer pairs from the genic sequences by using the barley genomezipper   <cit>  and the syntenic relationships of rice , b. distachyon  and sorghum , respectively.
finally, using the survey sequences of wheat chromosome 6b  <cit> , we prepared an additional  <dig> isbp pcr primer pairs  from the junction sequences between transposable elements and their flanking sequences .table  <dig> molecular markers used for the physical mapping
genic markersa
agenic markers include the plug marker, genomezipper-based markers and orthologous sequence markers
after validating the quality and effectiveness of the above  <dig> dna markers by pcr, we found that up to  <dig> primer pairs  could amplify a single sharp band from the dna samples of flow-sorted chromosome 6b arms or pooled arm-specific bac libraries.
these primer pairs represent good markers of the chromosomal locations of the contigs .
as shown in fig.
<dig>  using large-scale pcr-screening of two arm-specific bac libraries, we successfully localized the above  <dig> anchor markers onto individual bac clones among a total of  <dig> contigs .
the isbp markers provided more effective anchoring of the contigs compared to the genic markers , supporting previous results .
in addition,  <dig> markers were specific to wheat chromosome 6b and were polymorphic between the two wheat varieties cs and ‘mironovskaya 808’ , which were the parents used to generate the mapping population of recombinant inbred lines   <cit>  that was used for genetic mapping of contigs.fig.
<dig> relative contributions of different types of molecular marker used for anchoring by pcr screening for the physical maps of the chromosome arms of 6bs  and 6bl .
the number of markers anchored on the bac contigs is indicated in parentheses after the marker type.
the number of contigs anchored and their physical size  are indicated for each marker type.
the venn diagram in the square illustrates the relative contribution of each marker type to the number and total size of contigs anchored by one or more marker types in the pcr screening of bac libraries.
single circles in orange or gray represent the number and total size of the newly anchored contigs with mtp-derived markers or unanchored contigs, respectively, which were not anchored by the pcr screening
to anchor the contigs without any of  <dig> 6b-specific anchor marker hits, we developed a point-by-point supplemental set of  <dig> novel 6b-specific markers .
finally, the total number of contigs anchored by the chromosome 6b markers was increased to  <dig>  including  <dig> contigs anchored by  <dig> markers on 6bs and  <dig> contigs anchored by  <dig> markers on 6bl .table  <dig> characteristics of the bac contigs in the physical map of chromosome 6b
acoverage was calculated based on the estimated size of each arm and the entire chromosome: 415 mb for 6bs; 498 mb for 6bl; and 914 mb for 6b
first, we attempted the genetic mapping of contigs on chromosome 6b.
the use of  <dig> genetic markers located on the genetic map generated with the ril population mentioned above led to the chromosomal assignment of  <dig> contigs , encompassing a genomic region of 287 mb, on the two arms, which represents 31 % of chromosome 6b .
however, genetic markers alone were not sufficient for the physical mapping of all contigs that were assembled for wheat chromosome 6b.
to overcome this problem, we used rh mapping in chromosome deletion lines that were produced by γ-ray irradiation to determine the chromosomal locations and orders of all of the anchor dna markers for the 6b contigs.
this strategy was used because rh mapping provides results that are independent of polymorphisms.
thus, it is a powerful tool for the high-resolution mapping of wheat chromosomes  <cit> .
using the rh panel of  <dig> lines and  <dig> chromosome 6b deletion lines made by endo and gill  <cit> , we established an rh map with one linkage group consisting of  <dig> markers , in which a total of  <dig> obligate breaks were identified with an estimated resolution of approximately 622 kb/break .
the total length of the rh map was  <dig>  cr, indicating an average space of  <dig>  cr/loci and an average distance of 509 kb/cr .
based on the rh mapping results,  <dig> fpc-assembled contigs were mapped.
however,  <dig> of these mapped contigs  were judged to be chimeric because the markers anchored to the contig were located on distant, unrelated positions of the rh map.
therefore, we split each of these  <dig> contigs into two distinct contigs based on the fpc data and the anchored marker positions.
finally, we determined the locations and/or directions of  <dig> contigs on wheat chromosome 6b, including the contigs that mapped to the same locus .
these contigs revealed a total physical length of  <dig>  mb , representing approximately 87 % of chromosome 6b .
although  <dig> contigs, one third of the total contigs, still remained unmapped, most of these contigs were assembled using a small number of bac clones with a total physical length of  <dig>  mb and an average size of 172 kb per contig , so they cover only  <dig>  % of chromosome 6b.table  <dig> integrated physical map and map resolution
athe number of obligate breaks in each deletion bin was calculated by using the map region between the distal and proximal markers
fig.
<dig> rh map of chromosome 6b and contig order in three deletion bins of 6bs.
a deletion bin map of chromosome 6b.
twenty deletion bins are illustrated by colored boxes.
b rh map of chromosome 6b.
the map is divided into segments corresponding to deletion bins with the same colors.
the striped segment on 6bs represents the putative nucleolus organizer region .
the segment colored in black represents a boundary between 6bs and 6bl.
c rh map at deletion bins 6bs <dig>  6bs <dig> and 6bs <dig>  the marker name is indicated to the right of the chromosome.
d bac contigs assigned to the rh map.
the name of the bac contigs is displayed in colored boxes, the size of which reflects the relative contig size.
the colors of the boxes correspond to those of the deletion bins.
e corresponding region of rice chromosome  <dig>  each dashed line represents the relationship between a genic marker located in a wheat bac contig and the orthologous rice gene
our rh mapping could offer more refined deletion bins for chromosome 6b than were previously available  <cit> .
the  <dig> breakpoints in the 6b deletion lines and  <dig> markers defined  <dig> chromosomal blocks .
these  <dig> blocks were designated as improved deletion bins .
each deletion bin was denoted by the name of the deletion line and the breakpoint at the proximal end of the bin .
based on the number of obligate breaks and the bin size , the resolution of all  <dig> deletion bins was estimated to range from  <dig>  to  <dig>  mb/break .
five bins  showed a relatively low resolution with >900 kb/break, which may have occurred for the following reasons: 1) the large cumulative size of the contigs with a limited number of obligate breaks ; 2) one locus mapped by a large number of markers ; and 3) a bin mapped with only one large contig .
deletion bins that mapped to regions close to the centromere a remarkably high resolution.
for example, bin c-6bl <dig> had the highest resolution,  <dig>  mb/break .
this result shows that rh mapping is useful for the chromosomal assignment of contigs, even within the centromeric region, where recombination events are extremely rare.
notably, the largest space between markers on the rh map was located between kgs <dig>  and isbp <dig> , which were separated by  <dig>  cr .
clearly, this chromosomal site was associated with the chromosome breakpoint of deletion line 6bs- <dig>  which was located near nor-b <dig>  <cit> .
these results suggest that this space over  <dig> cr corresponds to the nor region .
although  <dig> of the  <dig> contigs  were specifically positioned in order along chromosome 6b ,  <dig> contigs were localized within a total of  <dig> loci , which indicated that multiple contigs resided within the same locus.
for example,  <dig> of the  <dig> loci within the bin of 6bl <dig> carried  <dig> to  <dig> contigs in which the relative order was unknown at this stage.
however,  <dig> of these  <dig> contigs  were anchored by genic markers with orders that were estimated from the syntenic relationships of rice, b. distachyon and sorghum.
these efforts led to the successful determination of the physical locations of a total of  <dig> contigs, representing a total length of  <dig>  mb and covering approximately 83 % of chromosome 6b .
in the present study, we developed an rh map for chromosome 6b and determined the order of the contigs.
our study clearly demonstrates the appropriateness and effectiveness of the rh mapping approach for ordering contigs on wheat chromosome compared to typical strategies, such as genetic mapping and deletion bin mapping.
the rh mapping method has important advantages.
it produces uniform chromosomal breaks, allows for direct localization and does not require marker polymorphisms.
thus, it allows for the determination of the chromosomal locations and order of contigs with high resolution and accuracy.
rh mapping was able to define the order of  <dig> markers based on contig order .
the marker order on the physical rh map provides more detail and a higher resolution compared to previous deletion bin mapping of chromosome 6b .
although the marker density of the 6b rh map  is lower than those of other physical maps , all of our markers are pcr markers, which will be valuable for future map-based gene cloning and marker-assisted selection in breeding compared to the array-based markers used in the previous studies.
fine mapping of the nor-b <dig> region on the short arm of chromosome 6b
the integration of fpc-assembled bac contigs with a high-resolution rh map of chromosome 6b allowed us to characterize several features that are specific to the genomic composition and structure of chromosome 6b.
in common wheat, the majority of the highly repeated rrna genes for 18s,  <dig> s and 25s rnas  are located at the nor loci on chromosome arms 1bs and 6bs, as well as at minor sites on chromosome arms 1as and 5ds, which have been designated nor-b <dig>  nor-b <dig>  nor-a <dig> and nor-d <dig>  respectively  <cit> .
nor-b <dig> is recognized as the secondary constriction on satellite chromosome 6b, which indicates that genes in nor-b <dig> are transcriptionally active  <cit> .
our rh mapping defined a position for nor-b <dig> on chromosome 6bs .
to determine the physical position of nor-b <dig> in detail, we developed one dna marker using the intergenic spacer sequences  between the 18s and 25s rrna genes  <cit> .
screening of our bac libraries with the igs marker revealed that two contigs, ctg <dig>  and ctg <dig> , located in the deletion bin of 6bs <dig>  putatively contained the rrna genes .
contig ctg <dig> was mapped onto a genomic region immediately downstream of the estimated nor-b <dig> site using one isbp marker, isbp <dig>  and contig ctg <dig> had a chromosomal position that was  <dig>  cr downstream of that marker on the rh map.
although three other contigs, ctg <dig> , ctg <dig>  and ctg <dig> , were located between ctg <dig> and ctg <dig> , no mtp clones were detected in these contigs by pcr screening using the igs marker.
however, blast analysis of genomic sequences of mtp bac clones  clearly confirmed the presence of an rdna unit within the bac contigs of not only ctg <dig> and ctg <dig> but also ctg <dig> .fig.
<dig> bac contigs including rrna gene sequences.
the 6bs rh map from a distal locus of kgs <dig> on bin 6bs <dig> to a proximal locus isbp <dig> on bin 6bs <dig> is shown at the top of the figure.
five bac contigs corresponding to this region—ctg <dig>  ctg <dig>  ctg <dig>  ctg <dig> and ctg142—are shown, in which each black line shows the individual bac clone and the red line indicates the mtp clone.
red vertical lines indicate the positions of three rrna genes for 18s,  <dig> s and 25s and the igs marker .
the other black vertical lines indicate the positions of the anchoring markers .
genic markers are shown in italics
previous research has identified approximately  <dig> rrna genes on chromosome 6b  <cit> .
considering that the length of each copy  is approximately 9 kb  <cit> , the complete region encompassing the nor-b <dig> locus could be estimated as approximately  <dig>  mb.
although the five bac contigs showed a total physical length of  <dig>  mb, the copy numbers of the rrna genes detected within them were extremely limited .
therefore, the current bac-based physical map partially covers the border region  of the nor-b <dig> locus, and it does not include the core rdna array region of nor-b <dig>  furthermore, the physically mapped bac contigs in the genomic region on the other side  of the nor-b <dig> locus do not contain the rrna genes.
interestingly, all of the rrna genes discovered on the above contigs that mapped near the nor-b <dig> locus appeared to be interrupted by other types of genomic sequences, including transposons and other genes.
this is in striking contrast to the nor region in rice.
in rice , the rrna genes are distributed in a uniform array throughout the nor, encompassing a region of approximately  <dig>  mb on chromosome  <dig>   <cit> .
moreover, the rdna units have been found to exist as clusters in tandem arrays, even within the border region of the nor, demonstrating a clear boundary between the rdna repeat and its flanking region  <cit> .
in the region flanking the rdnas on rice chromosome  <dig>  ty3/gypsy retrotransposons accumulates at a density twice as high as that of the entire rice genome  <cit> .
regarding chromosome 6b, two isbp markers, isbp <dig> and isbp <dig>  were designed based on the junction sequences, including the gypsy-type retrotransposon , and these mapped close to the positions of the rrna genes on ctg <dig> and ctg <dig>  respectively .
the other five isbp markers mapped to the same region , including other types of repeat sequences, such as dna transposons  and unknown repeat sequences .
we previously found that the dna transposon diffusion is involved in the propagation of micro rna genes and specific transfer rna genes in wheat chromosome 6b based on the analysis of 6b survey sequences  <cit> .
the ambiguity found in the border region of nor-b <dig> might be another example of specific transposons containing rrna genes becoming diffused around the border region of nor-b <dig>  resulting in insertions of the rrna genes into the genomic sequence outside of the rdna array.
we identified three gene loci  that displayed colinearity between wheat and rice , and other mapped genes on ctg <dig> were also found to have conserved gene order relative to rice chromosome  <dig>  these results support the concept that the border region of nor-b <dig> originally possessed a structure similar to that of rice; however, the occurrence of genomic rearrangements due to the transposition of various repeat sequences has resulted in the present ambiguity at the proximal border of nor-b <dig>  this feature may reflect general characteristics of the wheat genome, which contains highly repetitive sequences.
a detailed evaluation of the structure of nor-b <dig> and its relationship to the repetitive sequences will be presented in future sequencing studies.
fine mapping of the centromere on wheat chromosome 6b
in multicellular eukaryotes, the chromosomal region known as the centromere plays an important role in both mitotic and meiotic nuclear divisions.
centromeres in wheat are associated with highly repetitive ty3/gypsy elements.
moreover, the regions surrounding centromeres  are highly heterochromatic.
in the present study, a total of  <dig> contigs were mapped within a genomic region represented by deletion bins of c-6bs <dig>  c-6bl <dig> and 6bl <dig>  where the centromere of wheat chromosome 6b should be located .
a number of centromere-specific sequences have been reported for wheat and its relatives, including  <dig> ty3/gypsy-type centromeric retrotransposons in wheat   <cit> , one centromeric satellite-like repeat  in wheat  <cit> , one ty3/gypsy retrotransposon  in ae.
tauschii  <cit> , and one dna marker sequence  in rye.
using the  <dig> centromere-specific sequences described above, we performed a blast search against the bac-based assembled genomic sequences obtained from all of the above contigs .
a total of  <dig> sequences  were detected within the different mtp bac clones from  <dig> contigs that were physically located within the above three deletion bins.
in particular, eight of these contigs were found to carry at least three ty3/gypsy-type sequences , indicating a significant accumulation of ty3/gypsy retrotransposons in these genomic regions, which is consistent with results reported for other wheat chromosomes  <cit> .
the results obtained from the mapping and sequencing of these contigs clearly indicate that they contain a portion of the centromeric region of wheat chromosome 6b .
contig ctg <dig> , located proximally on the short arm of chromosome 6b, contains eight centromere-specific sequences, whereas six and four centromere-specific sequences were found within the two contigs ctg <dig>  and ctg <dig> , which mapped to the proximal region of the long arm .
many centromeric sequences were detected within other contigs  .
these results suggest that the genomic region from contig ctg <dig> to contig ctg <dig>  with an estimated physical size of  <dig>  mb , might correspond to the centromere of wheat chromosome 6b.
however, we did not find any contigs with dominant satellite tandem repeats  <cit>  in our physical map, although we detected sequences for the centromeric satellite-like repeat in ctg <dig> on 6bs.
a possible explanation for this observation is that the postulated ‘centromere core region’ with dominant satellite tandem repeats was not cloned or assembled because of its highly repetitive sequence composition.fig.
<dig> bac contigs located in the centromeric region of chromosome 6b.
the proximal three bins on chromosome 6b, c-6bs- <dig>  c-6bl- <dig> and 6bl <dig>  are represented in colored segments of the rh map, corresponding to the colors of each bin in fig.
4a.
the black segment represents the boundary between 6bs and 6bl.
the marker name is indicated to the right of the map.
bac contigs assigned to the rh map are represented by colored boxes, the sizes of which reflect the relative contig sizes.
the names of  <dig> centromeric sequences used as queries for the blast search are listed in the table at the bottom of the figure, and each sequence is represented by a letter from ‘a’ to ‘q’.
these letters, shown on the right of the bac contigs, denote the presence of the corresponding centromeric sequences in those contigs.
the corresponding region of rice chromosome  <dig> is aligned on the right side of the figure.
each colored line represents the relationship between a genic marker located in a wheat bac contig and the orthologous rice gene.
black dashed lines indicate the relationships between two wheat centromeric genic markers, tnac <dig> on ctg <dig>  and gz6h <dig> on ctg <dig> , and their orthologous genes in rice
a shift in the position of the centromere since the divergence of wheat and rice has been suggested based on comparisons between wheat chromosome 3b and rice chromosome  <dig>  <cit> .
to dtermine whether the centromeric shift occurred between chromosome 6b and rice chromosome  <dig>  we investigated the relationships of orthologous genes in the centromeric region between wheat 6b and rice  <dig>  a total of  <dig> genic markers were mapped on the contigs at the centromeric deletion bins , of which  <dig> markers were associated with pericentromeric rice genes on chromosome  <dig> .
this result supports a conservation of the centromeric region, as shown in a previous study  <cit> .
the centromeric region, from contig ctg <dig>  to contig ctg <dig> , was interposed between the genic markers tnac <dig>  and gz6h <dig>  .
these two markers aligned to rice genes os02g <dig> and os02g <dig>  respectively, on the short arm and at a position  <dig>  mb distal to the centromere of rice chromosome  <dig> .
this suggests that the shift in the position of the centromere occurred between wheat 6b and rice  <dig>  as observed between wheat 3b and rice  <dig>
distribution of genes along wheat chromosome 6b
genes are not evenly distributed along wheat chromosomes  <cit> .
after the removal of redundant markers based on sequence similarity, we discovered  <dig> markers derived from independent genes , which were able to anchor  <dig> contigs .
to further extend the substantial gene content on chromosome 6b, we applied the gene models from the survey sequence data  <cit> .
the sequence contigs assembled from the 6b survey sequencing were mapped to the corresponding bac contigs using their sequence similarities with the wgp tag sequences by blast.
as a result,  <dig>  and  <dig>  % of 6bs tags and 6bl tags were mapped on  <dig>  iwgsc 6bs sequence contigs  and  <dig>  iwgsc 6bl sequence contigs .
finally, we mapped  <dig> annotated genes on the survey sequence contigs to the physical map.
after removal of redundancy,  <dig> genes were integrated into the analysis based on the survey sequence data.
in total,  <dig> independent genes  were assigned to  <dig> contigs .
based on the chromosomal locations of these genes on the contigs aligned along the rh map , we obtained insight into the gene distribution pattern along chromosome 6b .
the calculation of the numbers of genes and physical sizes of the gene containing contigs revealed an average gene density with similar values of  <dig>  and  <dig>  gene/mb for the two chromosome arms of 6bs and 6bl, respectively.
however, the average gene densities for each contig were different; they ranged from  <dig>  to  <dig>  genes/mb that were observed on the pericentromeric contig ctg <dig> on 6bs and the telomeric contig ctg <dig> on 6bl, respectively.
the gene densities tended to increase from the centromeric to telomeric regions along chromosome 6b .
this result was similar to the gene distribution on chromosomes 1b and 3b based on information obtained for deletion bins or the currently completed genomic sequences  <cit> .fig.
<dig> gene density of each contig on chromosome 6b.
the distribution patterns of gene density on each of the  <dig> contigs on chromosome 6b are indicated in genes/mb.
a the pattern of all  <dig> genes assigned on the physical map.
b the pattern of  <dig> syntenic genes, compared to those of rice chromosome  <dig>  b. distachyon chromosome  <dig> or sorghum chromosome  <dig>  c the pattern of  <dig> nonsyntenic genes
on chromosomes 1b and 3b, the distribution of syntenic genes had no impact on the overall gene-density gradient  <cit> .
we found that  <dig> of  <dig> genes assigned on chromosome 6b had orthologs on rice chromosome  <dig>  b. distachyon chromosome  <dig> or sorghum chromosome  <dig> and the remaining  <dig> genes were nonsyntenic.
the distribution patterns of the nonsyntenic group on 6bs showed a clear gradient of gene density, becoming increasingly dense from centromere to telomere, whereas there was no discernable gradient pattern for the syntenic group .
this shows that the gradient of gene density along 6bs is due to the presence of nonsyntenic genes, which was consistent with the general pattern of gene density on wheat chromosomes 1b and 3b  <cit> .
in contrast, on 6bl, both the nonsyntenic and syntenic genes increased in density from centromere to telomere .
this result suggests that the general pattern discussed above may not apply to 6bl.
however, our result did not completely cover the positions of the genes on the 6b physical map; we used  <dig> genes, representing only half of the  <dig> high-confidence genes annotated on chromosome 6b based on the iwgsc survey sequencing data  <cit> .
completion of the genomic sequencing and gene annotation will reveal the gene organization on chromosome 6b more definitively.
local rearrangements on wheat chromosome 6b
previous comparative studies have demonstrated a syntenic and evolutionary relationship between the wheat homoeologous group  <dig> chromosomes and rice chromosome  <dig> , b. distachyon chromosome  <dig>  or sorghum chromosome  <dig>  .
of the  <dig> genes used in the present study for chromosome 6b,  <dig>   <dig> and  <dig> genes were syntenic with those of os <dig>  bradi <dig> and sb <dig>  respectively, revealing strong genome colinearity between wheat and other grass species .
however, the present study identified several specific genome rearrangements caused by events, such as inversions or translocations, as well as a lack of colinearity resulting from insertions and deletions.fig.
<dig> shared synteny between wheat chromosome 6b and the chromosomes in rice, b. distachyon and sorghum.
wheat chromosome 6b is indicated on the right side of the circle, and each color corresponds to a deletion bin shown in fig.
<dig>  the chromosomes of a rice , b
b. distachyon  and c sorghum  are represented on the left side of the circle.
d comparisons between wheat chromosome 6b  and the homologous chromosomes .
the lines represent the relationships between mapped genes on the 6b physical map and the orthologous genes in rice, b. distachyon and sorghum.
the order of wheat genes in a given deletion bin is predicted by the alignment of contigs along the rh map
two inversions  were detected between wheat 6bs and the three syntenic chromosomes, os <dig>  bradi <dig> and sb <dig> , providing strong evidence for evolutionary events specific to the wheat lineage.
however, between chromosome 6b and rice os <dig>  two additional inversions were observed on the long arm , one of which  was also identified between wheat and b. distachyon .
this finding suggests that the inversion involving chromosomal region  <dig> – <dig>  mb occurred recently, after the divergence of brachypodieae and triticeae.
interestingly, the other inversion on the long arm  shared by wheat chromosome 6b and bradi <dig> but not observed on os <dig> was detected between chromosome 6d of ae.
tauschii and os <dig> , suggesting that this event occurred after the divergence of ehrhartoideae  and pooideae  and before the divergence of pooideae.
a comparison of wheat chromosome 6b with bradi <dig> indicated several complex rearrangements that appeared to have occurred within the genomic region of its long arm ranging from the deletion bin 6bl <dig> to the proximal two-fifth part of 6bl <dig> .
this region included several translocations and inversions , as well as a reciprocal translocation between the two regions on  <dig> – <dig>  and  <dig> – <dig>  mb.
these translocations might be brachypodieae-specific  because the synteny between 6b and os <dig> in this region was well conserved .table  <dig> genomic regions containing chromosome rearrangements between 6b and its homologous chromosomes in other species
athe size of the genomic region on chromosome 6b is estimated based on the cumulative size of the bac contigs positioned in the corresponding rh map region
a comparison of wheat chromosome 6b with sb <dig> led to the discovery of one large inversion  of the long arm 6bl .
because wheat 6b and os <dig> or bradi <dig> share the same inversion relative to sb <dig>  this chromosomal structural change might be unique to the sorghum lineage.
we discovered two reciprocal translocations between wheat 6b and sb <dig> , which corresponded exactly to the two pooideae-specific inversions described above .
these data allow us to propose the following scenarios for rearrangements on the long arm during the evolution of grass species : 1) the chromosomal region corresponding to  <dig> – <dig>  mb on the physical map was inverted after the divergence of panicoideae  and ehrhartoideae/pooideae; 2) the distal part of the first inversion,  <dig> – <dig>  mb, was inverted again after the divergence of ehrhartoideae and pooideae; and 3) the proximal region of the first inversion,  <dig> – <dig>  mb, was recently inverted after the divergence of brachypodieae and triticeae.
these results also indicate that chromosomal rearrangements occurred frequently in the distal regions of the homologous chromosomes, including wheat 6b.fig.
<dig> evolutionary events underlying the chromosomal rearrangements on 6b and the homologous chromosomes in other species.
a phylogenetic tree representing the grass subfamilies panicoideae, ehrhartoideae and pooideae is shown.
pooideae includes the additional subgroups brachypodieae and triticeae.
the estimated time point for each rearrangement event is indicated by a black line on the tree branch.
the chromosomal region of each rearrangement is indicated by its physical length  based on the 6b physical map.
rearrangements on the short and long arms are represented by red and blue characters, respectively.
the chromosomal regions of each inversion are represented by arrows on the left side of the c-banding karyotype of chromosome 6b
disruption of synteny in the proximal region of wheat chromosome 6bl
a large rearrangement in the proximal region  was observed between 6b and the sb <dig>  os <dig>  and bradi <dig> chromosomes; that is, fewer syntenic genes were mapped to this genomic region.
such an interruption of synteny in the proximal region of wheat homoeologous group  <dig> chromosomes relative to rice chromosome os <dig> has been previously described by the mapping of orthologous genes  <cit> .
it has also been documented between chromosome 6d of ae.
tauschii and os <dig> based on high-resolution physical mapping  <cit> .
using the genomic sequences of the mtp bac clones in this region , we estimated the number of genes in these bins that were homologous to the rice genes by a blast analysis.
from  <dig> mtp clones  in the three bins, we identified  <dig> gene sequences homologous to the rice genes.
we found  <dig> and  <dig> gene sequences that were homologous to os <dig> and os <dig>  respectively, and 25– <dig> gene sequences were derived from other chromosomes in rice.
we investigated the colinearity of the  <dig> sequences that were homologous to os <dig> based on their positions on os <dig>  but we failed to detect a certain level of colinearity.
the estimated proportion of nonsyntenic genes in this region was very high  compared with the proportion  in the entire chromosome 6b based on our survey sequence data, as noted previously  <cit> .
furthermore, a recent genome sequencing study reported that the proportion of nonsyntenic genes in the proximal region of chromosome 3b was 28 %, and even in distal regions, the proportions were  <dig> and 53 %  <cit> .
these results suggest that the interruption was unusual; it was not due to the simple integration of chromosomal segments from a rice chromosome other than os <dig> into this region but rather to the abundant and random accumulation of nonsyntenic genes that might disrupt the synteny between wheat chromosome 6b and rice chromosome  <dig>  although we do not currently have any data to explain the mechanisms underlying the disruption in colinearity in this region, it might be due to structural differences in the centromeric region, including the previously described centromere shifting.
large interruptions of approximately 30–40 mb of the genomic region of bradi <dig> and sb <dig> were also found .
the interruption between 6b and bradi <dig> revealed characteristics of the chromosomal structures of bradi <dig>  which was postulated to correspond to three rice chromosomes , and its pericentromeric 10–45 mb region was syntenic with os <dig> and os <dig>  <cit> .
therefore, the interruption between 6b and bradi <dig> resulted from the insertion of other chromosomal segments in bradi <dig>  the cause of the interruption between 6b and sb <dig> remains unclear, although the small number of annotated gene in this 30-mb genomic region corresponding to the proximal region of sb <dig>  <cit>  is estimated to be one of the causes.
in summary, we could infer the structural features of chromosome 6b with high resolution based on the the physical localizations of genic markers obtained by rh mapping .
the results provide information to understand the molecular and biological mechanisms underlying the genomic divergence and chromosomal evolution of wheat.
CONCLUSIONS
here, we provide a bac-based physical map of wheat chromosome 6b with an estimated size of  <dig>  mb that covers 91 % of the chromosome, which allowed us to select mtp bac clones for the map-based genomic sequencing of chromosome 6b.
the development of  <dig> anchor markers and the construction of a high-resolution rh map permitted the successful localization of  <dig> of the assembled contigs to their chromosomal regions, representing 87 %  of the entire chromosome.
the establishment and analysis of the integrated physical map also led to the discovery of several important features of chromosome 6b, including the fine chromosomal localization and organization of nor-b <dig> and the centromere, the gene distribution patterns and the evolutionary history of chromosomal rearrangements among grass species.
furthermore, the use of marker information from syntenic genes led to the identification of chromosomal regions that were highly conserved or frequently rearranged, which is useful for our understanding of the complexity of genome evolution in wheat.

 common wheat is an allohexaploid species with three distinct genomes, a, b and d, which have been defined by genome analysis. the wheat genome consists of chromosomes, and each of the three subgenomes contributes chromosomes. the genome has a total length of gb, which is extremely large compared to the sequenced genomes of other grass species: rice, brachypodium distachyon, sorghum, maize and barley. most of the wheat genome is occupied by various types of repetitive dna sequences, which account for more than 80 % of the entire genome. although wheat has been widely used in cytogenetics because of the high visibility of its chromosomes, complex features, such as its large size, high repeat content and polyploidy, have hampered molecular approaches to date, particularly whole-genome sequencing. however, recent technological advances have paved the way for genomic approaches that work for even complex genomes, such as wheat. for example, whole-genome sequences of the common wheat cultivar ‘chinese spring’, wild diploid wheat triticum urartu and wild diploid goat grass aegilops tauschii were analyzed using next-generation sequencing technology. the international wheat genome sequencing consortium has been coordinating wheat genome sequencing to enhance knowledge of the structure and function of the bread wheat genome and to provide tools to facilitate the breeding of improved varieties. the consortium has adapted a strategy that relies on the purification of individual chromosome arms from wheat telosomic lines by flow-cytometric sorting to reduce the sample complexity and obtain chromosome-specific genomic data. recently, the iwgsc presented a draft sequence of all wheat chromosomes in which the dna of flow-sorted fractions was sequenced by ngs. to obtain a reference genome sequence using bac-by-bac sequencing, physical mapping of the bac contigs is currently underway for each of the chromosomes. to date, bac-based physical maps have been developed for chromosomes 1a, 1b, 3b and 6a. most recently, during the preparation of this paper, two additional physical maps for chromosome arms 3ds and 5ds were released. our group is responsible for sequencing wheat chromosome 6b under the framework of iwgsc. this chromosome is the third largest chromosome in common wheat, with an estimated molecular size of 914 mb: 415 mb representing the short arm and 498 mb representing the long arm. as a first step, we conducted whole-chromosome shotgun sequencing using dna that had been amplified from the flow-sorted chromosome arms 6bs and 6bl with massively parallel pyrosequencing. the survey sequence data were highly informative for determining the genomic composition of chromosome 6b. within a total of 508 mb of assembled sequence, gene loci were predicted, and more than 70 % of the 6b assembly consisted of repetitive sequences. functional non-protein-coding rnas, such as micro-rna, transfer rna and ribosomal rna, were also identified. in particular, the short arm is characterized by the presence of secondary constriction, the nucleolus organizer region, which features a locus for the rrna genes. the nor is designated as nor-b. we identified several contigs containing the rdna locus, and they exhibited extremely high read depths, indicating that these contigs were part of the nor on 6bs. in this study, we established a bac-based physical map of chromosome 6b with the goal of developing a high-quality reference sequence for the wheat genome. to achieve this goal, two bac libraries were constructed using arm-specific dna samples. in addition, we used two recently developed genomic tools to physically map all of the contigs onto chromosome 6b. this process was performed using insertion site-based polymorphism markers that were designed from the junction sequences between transposable elements and their flanking sequences, and a radiation hybrid mapping panel that was derived from a series of chromosome deletion lines generated by γ-ray irradiation. combining the above genomic resources with other wheat-specific data, we were able to develop a robust system to anchor and order contigs onto the high-resolution rh map of chromosome 6b. the physical map not only allows us to understand key structural features and the evolutionary history of chromosome 6b but will also enable the use of genome sequencing to create a high-quality map-based reference sequence in the near future.  construction of 6b arm-specific bac libraries chromosome arms 6bs and 6bl were purified by flow-cytometric sorting from the double ditelosomic line 6b of cs, as described by tanaka et al.. the average purities in the sorted fractions were and 92 % for 6bs and 6bl, respectively. high-molecular-weight dna prepared from the purified arms was used to construct the bac libraries taacsp6bsha and taacsp6blha, which were specific to 6bs and 6bl, respectively. the libraries consisted of and bac clones with average insert lengths of and 130 kb, representing and genome equivalents of the estimated size of 6bs and 6bl, respectively.table arm-specific bac libraries on chromosome 6b construction of a bac-based physical map of wheat chromosome 6b whole genome profiling was used to fingerprint a total of and bacs from 6bs and 6bl, representing times the total physical lengths of the arms, respectively. after deconvolution for the assignment of high-quality reads, we made several modifications to the standard wgp procedure following philippe et al. to ensure a highly accurate physical map. we initially excluded bacs with less than 30 % or more than times the average number of tags per bac from the analysis based on the average number of tags per bac. this filtering resulted in the availability of and bacs for assembly on 6bs and 6bl, representing and times the total physical length, respectively. assembly of the fingerprints of 6bs and 6bl was performed using fingerprinted contigs software. a stepwise method was used to improve the quality of the contigs in the assembly, which was initially defined during the physical map construction of chromosome 3b and then modified for the wgp fingerprints. using the 6b-specific parameters at final cut-off values of 1e− and 1e− which were determined based on the balance among the number of contigs, the number of q-contigs and the coverage of the physical map, we constructed the first physical maps of 6bs and 6bl with and contigs, respectively.fig. comparison of the contig assemblies between cut-offs of 1e− and 1e− in the chromosome arms of 6bs and 6bl. a the number of contigs and their cumulative sizes at each cutoff value. the colors in the bars represent the number of clones comprising one bac contig. b total number of singleton clones in the physical map at each cut-off value. c total number of q-contigs containing ≤10 % questionable clones in the physical map at each cut-off value. d total number of contigs in the physical maps of 6bs and 6bl with an optimal cut-off value. eight colors in circles represent the number of clones comprising one bac contig. the sizes of the circles indicate the relative total number of contigs in the physical map of 6bs and 6bl these physical maps still contained a large number of small contigs, particularly on 6bs. however, the small contigs might be redundant because of their low-quality fingerprints containing genomic fragments that were already present in the large contigs. in addition, they had the potential to carry bac clones derived from chromosomes other than 6b due to dna contamination during the chromosome-sorting process. to avoid any chromosomal redundancy and contamination, we eliminated and of these small and low-confidence contigs from the above physical map constructed primarily on the two arms of 6bs and 6bl, respectively, based on the results of a homology search of the wgp tag sequences against the whole-genome survey sequence and/or the depth of the bac clones within each contig. finally, we constructed the bac-based physical map of chromosome arms 6bs and 6bl, which contained and contigs, respectively.table physical maps of chromosome arms 6bs and 6bl acoverage was calculated based on the estimated size of each arm: 415 mb for 6bs and 498 mb for 6bl the above process greatly improved our bac-based physical map. the first physical maps revealed estimated sizes of and 495 mb that corresponded to 119 % and 99 % of the arm lengths, n values of and 2422 kb and l values of and contigs for 6bs and 6bl, respectively. however, the final maps showed remarkable increases in the n values and a decrease in the l values, although the total lengths for both arms decreased to and 475 mb, covering approximately and 95 % of the entire genomic region for 6bs and 6bl, respectively. the reason for the lower coverage of 6bs remains unclear, but it is likely a consequence of the purity of the sorted chromosome arm dna, the number of wgp tags on each bac or differences in the genomic structure between 6bs and 6bl. further overlap analysis of neighboring clones within each contig using the fpc mtp module allowed us to select and minimal tiling path clones along the two arms, laying a foundation for the map-based genomic sequencing of the entire chromosome 6b. anchoring of chromosome 6b-specific markers to determine the physical location of the bac contigs to obtain a pseudomolecule sequence, which is the final goal of iwgsc, it is essential to map the contigs onto their specific genomic regions on wheat chromosome 6b. for that purpose, a set of anchors is needed. as shown in table  we first searched through the available marker information in public databases, such as the “graingenes cmap” and the “national bioresource project -wheat, japan”, and ssr and rflp markers were found to be potentially useful for chromosome 6b. second, we collected a resource of dna markers from the pcr-based landmark unique gene database. these markers are known est-pcr markers that were developed by taking advantage of the orthologous gene conservation between rice and wheat. to develop new markers, we designed and pcr primer pairs from the genic sequences by using the barley genomezipper and the syntenic relationships of rice, b. distachyon and sorghum, respectively. finally, using the survey sequences of wheat chromosome 6b, we prepared an additional isbp pcr primer pairs from the junction sequences between transposable elements and their flanking sequences.table molecular markers used for the physical mapping genic markersa agenic markers include the plug marker, genomezipper-based markers and orthologous sequence markers after validating the quality and effectiveness of the above dna markers by pcr, we found that up to primer pairs could amplify a single sharp band from the dna samples of flow-sorted chromosome 6b arms or pooled arm-specific bac libraries. these primer pairs represent good markers of the chromosomal locations of the contigs. as shown in fig.  using large-scale pcr-screening of two arm-specific bac libraries, we successfully localized the above anchor markers onto individual bac clones among a total of contigs. the isbp markers provided more effective anchoring of the contigs compared to the genic markers, supporting previous results. in addition, markers were specific to wheat chromosome 6b and were polymorphic between the two wheat varieties cs and ‘mironovskaya 808’, which were the parents used to generate the mapping population of recombinant inbred lines that was used for genetic mapping of contigs.fig. relative contributions of different types of molecular marker used for anchoring by pcr screening for the physical maps of the chromosome arms of 6bs and 6bl. the number of markers anchored on the bac contigs is indicated in parentheses after the marker type. the number of contigs anchored and their physical size are indicated for each marker type. the venn diagram in the square illustrates the relative contribution of each marker type to the number and total size of contigs anchored by one or more marker types in the pcr screening of bac libraries. single circles in orange or gray represent the number and total size of the newly anchored contigs with mtp-derived markers or unanchored contigs, respectively, which were not anchored by the pcr screening to anchor the contigs without any of 6b-specific anchor marker hits, we developed a point-by-point supplemental set of novel 6b-specific markers. finally, the total number of contigs anchored by the chromosome 6b markers was increased to including contigs anchored by markers on 6bs and contigs anchored by markers on 6bl.table characteristics of the bac contigs in the physical map of chromosome 6b acoverage was calculated based on the estimated size of each arm and the entire chromosome: 415 mb for 6bs; 498 mb for 6bl; and 914 mb for 6b first, we attempted the genetic mapping of contigs on chromosome 6b. the use of genetic markers located on the genetic map generated with the ril population mentioned above led to the chromosomal assignment of contigs, encompassing a genomic region of 287 mb, on the two arms, which represents 31 % of chromosome 6b. however, genetic markers alone were not sufficient for the physical mapping of all contigs that were assembled for wheat chromosome 6b. to overcome this problem, we used rh mapping in chromosome deletion lines that were produced by γ-ray irradiation to determine the chromosomal locations and orders of all of the anchor dna markers for the 6b contigs. this strategy was used because rh mapping provides results that are independent of polymorphisms. thus, it is a powerful tool for the high-resolution mapping of wheat chromosomes. using the rh panel of lines and chromosome 6b deletion lines made by endo and gill, we established an rh map with one linkage group consisting of markers, in which a total of obligate breaks were identified with an estimated resolution of approximately 622 kb/break. the total length of the rh map was cr, indicating an average space of cr/loci and an average distance of 509 kb/cr. based on the rh mapping results, fpc-assembled contigs were mapped. however, of these mapped contigs were judged to be chimeric because the markers anchored to the contig were located on distant, unrelated positions of the rh map. therefore, we split each of these contigs into two distinct contigs based on the fpc data and the anchored marker positions. finally, we determined the locations and/or directions of contigs on wheat chromosome 6b, including the contigs that mapped to the same locus. these contigs revealed a total physical length of  mb, representing approximately 87 % of chromosome 6b. although contigs, one third of the total contigs, still remained unmapped, most of these contigs were assembled using a small number of bac clones with a total physical length of  mb and an average size of 172 kb per contig, so they cover only  % of chromosome 6b.table integrated physical map and map resolution athe number of obligate breaks in each deletion bin was calculated by using the map region between the distal and proximal markers fig. rh map of chromosome 6b and contig order in three deletion bins of 6bs. a deletion bin map of chromosome 6b. twenty deletion bins are illustrated by colored boxes. b rh map of chromosome 6b. the map is divided into segments corresponding to deletion bins with the same colors. the striped segment on 6bs represents the putative nucleolus organizer region. the segment colored in black represents a boundary between 6bs and 6bl. c rh map at deletion bins 6bs 6bs and 6bs the marker name is indicated to the right of the chromosome. d bac contigs assigned to the rh map. the name of the bac contigs is displayed in colored boxes, the size of which reflects the relative contig size. the colors of the boxes correspond to those of the deletion bins. e corresponding region of rice chromosome each dashed line represents the relationship between a genic marker located in a wheat bac contig and the orthologous rice gene our rh mapping could offer more refined deletion bins for chromosome 6b than were previously available. the breakpoints in the 6b deletion lines and markers defined chromosomal blocks. these blocks were designated as improved deletion bins. each deletion bin was denoted by the name of the deletion line and the breakpoint at the proximal end of the bin. based on the number of obligate breaks and the bin size, the resolution of all deletion bins was estimated to range from to  mb/break. five bins showed a relatively low resolution with >900 kb/break, which may have occurred for the following reasons: 1) the large cumulative size of the contigs with a limited number of obligate breaks; 2) one locus mapped by a large number of markers; and 3) a bin mapped with only one large contig. deletion bins that mapped to regions close to the centromere a remarkably high resolution. for example, bin c-6bl had the highest resolution,  mb/break. this result shows that rh mapping is useful for the chromosomal assignment of contigs, even within the centromeric region, where recombination events are extremely rare. notably, the largest space between markers on the rh map was located between kgs and isbp, which were separated by cr. clearly, this chromosomal site was associated with the chromosome breakpoint of deletion line 6bs- which was located near nor-b. these results suggest that this space over cr corresponds to the nor region. although of the contigs were specifically positioned in order along chromosome 6b, contigs were localized within a total of loci, which indicated that multiple contigs resided within the same locus. for example, of the loci within the bin of 6bl carried to contigs in which the relative order was unknown at this stage. however, of these contigs were anchored by genic markers with orders that were estimated from the syntenic relationships of rice, b. distachyon and sorghum. these efforts led to the successful determination of the physical locations of a total of contigs, representing a total length of  mb and covering approximately 83 % of chromosome 6b. in the present study, we developed an rh map for chromosome 6b and determined the order of the contigs. our study clearly demonstrates the appropriateness and effectiveness of the rh mapping approach for ordering contigs on wheat chromosome compared to typical strategies, such as genetic mapping and deletion bin mapping. the rh mapping method has important advantages. it produces uniform chromosomal breaks, allows for direct localization and does not require marker polymorphisms. thus, it allows for the determination of the chromosomal locations and order of contigs with high resolution and accuracy. rh mapping was able to define the order of markers based on contig order. the marker order on the physical rh map provides more detail and a higher resolution compared to previous deletion bin mapping of chromosome 6b. although the marker density of the 6b rh map is lower than those of other physical maps, all of our markers are pcr markers, which will be valuable for future map-based gene cloning and marker-assisted selection in breeding compared to the array-based markers used in the previous studies. fine mapping of the nor-b region on the short arm of chromosome 6b the integration of fpc-assembled bac contigs with a high-resolution rh map of chromosome 6b allowed us to characterize several features that are specific to the genomic composition and structure of chromosome 6b. in common wheat, the majority of the highly repeated rrna genes for 18s, s and 25s rnas are located at the nor loci on chromosome arms 1bs and 6bs, as well as at minor sites on chromosome arms 1as and 5ds, which have been designated nor-b nor-b nor-a and nor-d respectively. nor-b is recognized as the secondary constriction on satellite chromosome 6b, which indicates that genes in nor-b are transcriptionally active. our rh mapping defined a position for nor-b on chromosome 6bs. to determine the physical position of nor-b in detail, we developed one dna marker using the intergenic spacer sequences between the 18s and 25s rrna genes. screening of our bac libraries with the igs marker revealed that two contigs, ctg and ctg, located in the deletion bin of 6bs putatively contained the rrna genes. contig ctg was mapped onto a genomic region immediately downstream of the estimated nor-b site using one isbp marker, isbp and contig ctg had a chromosomal position that was cr downstream of that marker on the rh map. although three other contigs, ctg, ctg and ctg, were located between ctg and ctg, no mtp clones were detected in these contigs by pcr screening using the igs marker. however, blast analysis of genomic sequences of mtp bac clones clearly confirmed the presence of an rdna unit within the bac contigs of not only ctg and ctg but also ctg.fig. bac contigs including rrna gene sequences. the 6bs rh map from a distal locus of kgs on bin 6bs to a proximal locus isbp on bin 6bs is shown at the top of the figure. five bac contigs corresponding to this region—ctg ctg ctg ctg and ctg142—are shown, in which each black line shows the individual bac clone and the red line indicates the mtp clone. red vertical lines indicate the positions of three rrna genes for 18s, s and 25s and the igs marker. the other black vertical lines indicate the positions of the anchoring markers. genic markers are shown in italics previous research has identified approximately rrna genes on chromosome 6b. considering that the length of each copy is approximately 9 kb, the complete region encompassing the nor-b locus could be estimated as approximately  mb. although the five bac contigs showed a total physical length of  mb, the copy numbers of the rrna genes detected within them were extremely limited. therefore, the current bac-based physical map partially covers the border region of the nor-b locus, and it does not include the core rdna array region of nor-b furthermore, the physically mapped bac contigs in the genomic region on the other side of the nor-b locus do not contain the rrna genes. interestingly, all of the rrna genes discovered on the above contigs that mapped near the nor-b locus appeared to be interrupted by other types of genomic sequences, including transposons and other genes. this is in striking contrast to the nor region in rice. in rice, the rrna genes are distributed in a uniform array throughout the nor, encompassing a region of approximately  mb on chromosome. moreover, the rdna units have been found to exist as clusters in tandem arrays, even within the border region of the nor, demonstrating a clear boundary between the rdna repeat and its flanking region. in the region flanking the rdnas on rice chromosome ty3/gypsy retrotransposons accumulates at a density twice as high as that of the entire rice genome. regarding chromosome 6b, two isbp markers, isbp and isbp were designed based on the junction sequences, including the gypsy-type retrotransposon, and these mapped close to the positions of the rrna genes on ctg and ctg respectively. the other five isbp markers mapped to the same region, including other types of repeat sequences, such as dna transposons and unknown repeat sequences. we previously found that the dna transposon diffusion is involved in the propagation of micro rna genes and specific transfer rna genes in wheat chromosome 6b based on the analysis of 6b survey sequences. the ambiguity found in the border region of nor-b might be another example of specific transposons containing rrna genes becoming diffused around the border region of nor-b resulting in insertions of the rrna genes into the genomic sequence outside of the rdna array. we identified three gene loci that displayed colinearity between wheat and rice, and other mapped genes on ctg were also found to have conserved gene order relative to rice chromosome these results support the concept that the border region of nor-b originally possessed a structure similar to that of rice; however, the occurrence of genomic rearrangements due to the transposition of various repeat sequences has resulted in the present ambiguity at the proximal border of nor-b this feature may reflect general characteristics of the wheat genome, which contains highly repetitive sequences. a detailed evaluation of the structure of nor-b and its relationship to the repetitive sequences will be presented in future sequencing studies. fine mapping of the centromere on wheat chromosome 6b in multicellular eukaryotes, the chromosomal region known as the centromere plays an important role in both mitotic and meiotic nuclear divisions. centromeres in wheat are associated with highly repetitive ty3/gypsy elements. moreover, the regions surrounding centromeres are highly heterochromatic. in the present study, a total of contigs were mapped within a genomic region represented by deletion bins of c-6bs c-6bl and 6bl where the centromere of wheat chromosome 6b should be located. a number of centromere-specific sequences have been reported for wheat and its relatives, including ty3/gypsy-type centromeric retrotransposons in wheat, one centromeric satellite-like repeat in wheat, one ty3/gypsy retrotransposon in ae. tauschii, and one dna marker sequence in rye. using the centromere-specific sequences described above, we performed a blast search against the bac-based assembled genomic sequences obtained from all of the above contigs. a total of sequences were detected within the different mtp bac clones from contigs that were physically located within the above three deletion bins. in particular, eight of these contigs were found to carry at least three ty3/gypsy-type sequences, indicating a significant accumulation of ty3/gypsy retrotransposons in these genomic regions, which is consistent with results reported for other wheat chromosomes. the results obtained from the mapping and sequencing of these contigs clearly indicate that they contain a portion of the centromeric region of wheat chromosome 6b. contig ctg, located proximally on the short arm of chromosome 6b, contains eight centromere-specific sequences, whereas six and four centromere-specific sequences were found within the two contigs ctg and ctg, which mapped to the proximal region of the long arm. many centromeric sequences were detected within other contigs. these results suggest that the genomic region from contig ctg to contig ctg with an estimated physical size of  mb, might correspond to the centromere of wheat chromosome 6b. however, we did not find any contigs with dominant satellite tandem repeats in our physical map, although we detected sequences for the centromeric satellite-like repeat in ctg on 6bs. a possible explanation for this observation is that the postulated ‘centromere core region’ with dominant satellite tandem repeats was not cloned or assembled because of its highly repetitive sequence composition.fig. bac contigs located in the centromeric region of chromosome 6b. the proximal three bins on chromosome 6b, c-6bs- c-6bl- and 6bl are represented in colored segments of the rh map, corresponding to the colors of each bin in fig. 4a. the black segment represents the boundary between 6bs and 6bl. the marker name is indicated to the right of the map. bac contigs assigned to the rh map are represented by colored boxes, the sizes of which reflect the relative contig sizes. the names of centromeric sequences used as queries for the blast search are listed in the table at the bottom of the figure, and each sequence is represented by a letter from ‘a’ to ‘q’. these letters, shown on the right of the bac contigs, denote the presence of the corresponding centromeric sequences in those contigs. the corresponding region of rice chromosome is aligned on the right side of the figure. each colored line represents the relationship between a genic marker located in a wheat bac contig and the orthologous rice gene. black dashed lines indicate the relationships between two wheat centromeric genic markers, tnac on ctg and gz6h on ctg, and their orthologous genes in rice a shift in the position of the centromere since the divergence of wheat and rice has been suggested based on comparisons between wheat chromosome 3b and rice chromosome. to dtermine whether the centromeric shift occurred between chromosome 6b and rice chromosome we investigated the relationships of orthologous genes in the centromeric region between wheat 6b and rice a total of genic markers were mapped on the contigs at the centromeric deletion bins, of which markers were associated with pericentromeric rice genes on chromosome. this result supports a conservation of the centromeric region, as shown in a previous study. the centromeric region, from contig ctg to contig ctg, was interposed between the genic markers tnac and gz6h. these two markers aligned to rice genes os02g and os02g respectively, on the short arm and at a position  mb distal to the centromere of rice chromosome. this suggests that the shift in the position of the centromere occurred between wheat 6b and rice as observed between wheat 3b and rice distribution of genes along wheat chromosome 6b genes are not evenly distributed along wheat chromosomes. after the removal of redundant markers based on sequence similarity, we discovered markers derived from independent genes, which were able to anchor contigs. to further extend the substantial gene content on chromosome 6b, we applied the gene models from the survey sequence data. the sequence contigs assembled from the 6b survey sequencing were mapped to the corresponding bac contigs using their sequence similarities with the wgp tag sequences by blast. as a result, and  % of 6bs tags and 6bl tags were mapped on iwgsc 6bs sequence contigs and iwgsc 6bl sequence contigs. finally, we mapped annotated genes on the survey sequence contigs to the physical map. after removal of redundancy, genes were integrated into the analysis based on the survey sequence data. in total, independent genes were assigned to contigs. based on the chromosomal locations of these genes on the contigs aligned along the rh map, we obtained insight into the gene distribution pattern along chromosome 6b. the calculation of the numbers of genes and physical sizes of the gene containing contigs revealed an average gene density with similar values of and gene/mb for the two chromosome arms of 6bs and 6bl, respectively. however, the average gene densities for each contig were different; they ranged from to genes/mb that were observed on the pericentromeric contig ctg on 6bs and the telomeric contig ctg on 6bl, respectively. the gene densities tended to increase from the centromeric to telomeric regions along chromosome 6b. this result was similar to the gene distribution on chromosomes 1b and 3b based on information obtained for deletion bins or the currently completed genomic sequences.fig. gene density of each contig on chromosome 6b. the distribution patterns of gene density on each of the contigs on chromosome 6b are indicated in genes/mb. a the pattern of all genes assigned on the physical map. b the pattern of syntenic genes, compared to those of rice chromosome b. distachyon chromosome or sorghum chromosome c the pattern of nonsyntenic genes on chromosomes 1b and 3b, the distribution of syntenic genes had no impact on the overall gene-density gradient. we found that of genes assigned on chromosome 6b had orthologs on rice chromosome b. distachyon chromosome or sorghum chromosome and the remaining genes were nonsyntenic. the distribution patterns of the nonsyntenic group on 6bs showed a clear gradient of gene density, becoming increasingly dense from centromere to telomere, whereas there was no discernable gradient pattern for the syntenic group. this shows that the gradient of gene density along 6bs is due to the presence of nonsyntenic genes, which was consistent with the general pattern of gene density on wheat chromosomes 1b and 3b. in contrast, on 6bl, both the nonsyntenic and syntenic genes increased in density from centromere to telomere. this result suggests that the general pattern discussed above may not apply to 6bl. however, our result did not completely cover the positions of the genes on the 6b physical map; we used genes, representing only half of the high-confidence genes annotated on chromosome 6b based on the iwgsc survey sequencing data. completion of the genomic sequencing and gene annotation will reveal the gene organization on chromosome 6b more definitively. local rearrangements on wheat chromosome 6b previous comparative studies have demonstrated a syntenic and evolutionary relationship between the wheat homoeologous group chromosomes and rice chromosome, b. distachyon chromosome or sorghum chromosome. of the genes used in the present study for chromosome 6b, and genes were syntenic with those of os bradi and sb respectively, revealing strong genome colinearity between wheat and other grass species. however, the present study identified several specific genome rearrangements caused by events, such as inversions or translocations, as well as a lack of colinearity resulting from insertions and deletions.fig. shared synteny between wheat chromosome 6b and the chromosomes in rice, b. distachyon and sorghum. wheat chromosome 6b is indicated on the right side of the circle, and each color corresponds to a deletion bin shown in fig.  the chromosomes of a rice, b b. distachyon and c sorghum are represented on the left side of the circle. d comparisons between wheat chromosome 6b and the homologous chromosomes. the lines represent the relationships between mapped genes on the 6b physical map and the orthologous genes in rice, b. distachyon and sorghum. the order of wheat genes in a given deletion bin is predicted by the alignment of contigs along the rh map two inversions were detected between wheat 6bs and the three syntenic chromosomes, os bradi and sb, providing strong evidence for evolutionary events specific to the wheat lineage. however, between chromosome 6b and rice os two additional inversions were observed on the long arm, one of which was also identified between wheat and b. distachyon. this finding suggests that the inversion involving chromosomal region –  mb occurred recently, after the divergence of brachypodieae and triticeae. interestingly, the other inversion on the long arm shared by wheat chromosome 6b and bradi but not observed on os was detected between chromosome 6d of ae. tauschii and os, suggesting that this event occurred after the divergence of ehrhartoideae and pooideae and before the divergence of pooideae. a comparison of wheat chromosome 6b with bradi indicated several complex rearrangements that appeared to have occurred within the genomic region of its long arm ranging from the deletion bin 6bl to the proximal two-fifth part of 6bl. this region included several translocations and inversions, as well as a reciprocal translocation between the two regions on – and –  mb. these translocations might be brachypodieae-specific because the synteny between 6b and os in this region was well conserved.table genomic regions containing chromosome rearrangements between 6b and its homologous chromosomes in other species athe size of the genomic region on chromosome 6b is estimated based on the cumulative size of the bac contigs positioned in the corresponding rh map region a comparison of wheat chromosome 6b with sb led to the discovery of one large inversion of the long arm 6bl. because wheat 6b and os or bradi share the same inversion relative to sb this chromosomal structural change might be unique to the sorghum lineage. we discovered two reciprocal translocations between wheat 6b and sb, which corresponded exactly to the two pooideae-specific inversions described above. these data allow us to propose the following scenarios for rearrangements on the long arm during the evolution of grass species: 1) the chromosomal region corresponding to –  mb on the physical map was inverted after the divergence of panicoideae and ehrhartoideae/pooideae; 2) the distal part of the first inversion, –  mb, was inverted again after the divergence of ehrhartoideae and pooideae; and 3) the proximal region of the first inversion, –  mb, was recently inverted after the divergence of brachypodieae and triticeae. these results also indicate that chromosomal rearrangements occurred frequently in the distal regions of the homologous chromosomes, including wheat 6b.fig. evolutionary events underlying the chromosomal rearrangements on 6b and the homologous chromosomes in other species. a phylogenetic tree representing the grass subfamilies panicoideae, ehrhartoideae and pooideae is shown. pooideae includes the additional subgroups brachypodieae and triticeae. the estimated time point for each rearrangement event is indicated by a black line on the tree branch. the chromosomal region of each rearrangement is indicated by its physical length based on the 6b physical map. rearrangements on the short and long arms are represented by red and blue characters, respectively. the chromosomal regions of each inversion are represented by arrows on the left side of the c-banding karyotype of chromosome 6b disruption of synteny in the proximal region of wheat chromosome 6bl a large rearrangement in the proximal region was observed between 6b and the sb os and bradi chromosomes; that is, fewer syntenic genes were mapped to this genomic region. such an interruption of synteny in the proximal region of wheat homoeologous group chromosomes relative to rice chromosome os has been previously described by the mapping of orthologous genes. it has also been documented between chromosome 6d of ae. tauschii and os based on high-resolution physical mapping. using the genomic sequences of the mtp bac clones in this region, we estimated the number of genes in these bins that were homologous to the rice genes by a blast analysis. from mtp clones in the three bins, we identified gene sequences homologous to the rice genes. we found and gene sequences that were homologous to os and os respectively, and 25– gene sequences were derived from other chromosomes in rice. we investigated the colinearity of the sequences that were homologous to os based on their positions on os but we failed to detect a certain level of colinearity. the estimated proportion of nonsyntenic genes in this region was very high compared with the proportion in the entire chromosome 6b based on our survey sequence data, as noted previously. furthermore, a recent genome sequencing study reported that the proportion of nonsyntenic genes in the proximal region of chromosome 3b was 28 %, and even in distal regions, the proportions were and 53 %. these results suggest that the interruption was unusual; it was not due to the simple integration of chromosomal segments from a rice chromosome other than os into this region but rather to the abundant and random accumulation of nonsyntenic genes that might disrupt the synteny between wheat chromosome 6b and rice chromosome although we do not currently have any data to explain the mechanisms underlying the disruption in colinearity in this region, it might be due to structural differences in the centromeric region, including the previously described centromere shifting. large interruptions of approximately 30–40 mb of the genomic region of bradi and sb were also found. the interruption between 6b and bradi revealed characteristics of the chromosomal structures of bradi which was postulated to correspond to three rice chromosomes, and its pericentromeric 10–45 mb region was syntenic with os and os. therefore, the interruption between 6b and bradi resulted from the insertion of other chromosomal segments in bradi the cause of the interruption between 6b and sb remains unclear, although the small number of annotated gene in this 30-mb genomic region corresponding to the proximal region of sb is estimated to be one of the causes. in summary, we could infer the structural features of chromosome 6b with high resolution based on the the physical localizations of genic markers obtained by rh mapping. the results provide information to understand the molecular and biological mechanisms underlying the genomic divergence and chromosomal evolution of wheat.  here, we provide a bac-based physical map of wheat chromosome 6b with an estimated size of  mb that covers 91 % of the chromosome, which allowed us to select mtp bac clones for the map-based genomic sequencing of chromosome 6b. the development of anchor markers and the construction of a high-resolution rh map permitted the successful localization of of the assembled contigs to their chromosomal regions, representing 87 % of the entire chromosome. the establishment and analysis of the integrated physical map also led to the discovery of several important features of chromosome 6b, including the fine chromosomal localization and organization of nor-b and the centromere, the gene distribution patterns and the evolutionary history of chromosomal rearrangements among grass species. furthermore, the use of marker information from syntenic genes led to the identification of chromosomal regions that were highly conserved or frequently rearranged, which is useful for our understanding of the complexity of genome evolution in wheat. 
the order and direction of contigs, corresponding to 87 % of the total length of 6b, were determined. because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the international wheat genome sequencing consortium chose a strategy that involves constructing bacterial artificial chromosome -based physical maps of individual chromosomes and performing bac-by-bac sequencing. more importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6b. here, we report the construction of a physical map of chromosome 6b with the goal of revealing the structural features of the third largest chromosome in wheat. we assembled informative bac contigs representing 91 % of the entire physical length of wheat chromosome 6b. the contigs were integrated into a radiation hybrid map of chromosome 6b, with one linkage group consisting of loci with markers. analysis of the virtual gene order along 6b using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6b. we constructed a reliable physical map of chromosome 6b, enabling us to analyze its genomic structure and evolutionary progression. centromerechromosomal rearrangementchromosome 6bdna markergene ordernucleolus organizer regionbac physical maprh mapsyntenywheatissue-copyright-statement© the author 2015 we also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the rh map and the assembled bac clone sequences.
 a complete genome sequence is an essential tool for the genetic improvement of wheat. because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the international wheat genome sequencing consortium chose a strategy that involves constructing bacterial artificial chromosome -based physical maps of individual chromosomes and performing bac-by-bac sequencing. here, we report the construction of a physical map of chromosome 6b with the goal of revealing the structural features of the third largest chromosome in wheat.  we assembled informative bac contigs representing 91 % of the entire physical length of wheat chromosome 6b. the contigs were integrated into a radiation hybrid map of chromosome 6b, with one linkage group consisting of loci with markers. the order and direction of contigs, corresponding to 87 % of the total length of 6b, were determined. we also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the rh map and the assembled bac clone sequences. analysis of the virtual gene order along 6b using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6b.  we constructed a reliable physical map of chromosome 6b, enabling us to analyze its genomic structure and evolutionary progression. more importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6b. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords centromerechromosomal rearrangementchromosome 6bdna markergene ordernucleolus organizer regionbac physical maprh mapsyntenywheatissue-copyright-statement© the author 2015 