BACKGROUND
sugarcane is a member of the poaceae family that contributes 75% of the worlds sugar supply and is being heralded as an important source as an alternative biofuel .
however, knowledge of the genetic architecture of this crop currently lags behind that of other crops of the poaceae family such as wheat, barley and rice.
it is important to uncover the genome structure of a particular crop to guide informed decisions in the identification of important genes/traits that can be utilized during breeding and germplasm introgression.
modern sugarcane cultivars  have complex polyploid, aneuploid genomes that make classical genetic, molecular genetic, and breeding studies difficult to interpret, as information on the structure and organisation of the genome is incomplete .
they are essentially hybrids between the polyploid domesticated species, saccharum officinarum l  and the polyploid wild species, s. spontaneum l .
the cultivated hybrids have undergone a series of backcrosses to s. officinarum as the female parent to recover agronomically adapted genotypes with a high sugar yield.
using in situ hybridisation,  determined that the basic chromosome number of s. officinarum is x =  <dig> and for s. spontaneum is x =  <dig>  with 10-15% of the genome of modern sugarcane cultivars contributed by s. spontaneum, 80% s. officinarum and 11-15% recombinant chromosomes between the two species .
studies have shown that chromosome pairing is mostly as bivalents, although an assortment of meiotic irregularities can occur and contribute to the formation of aneuploid gametes .
sugarcane clones are propagated vegetatively and are stable over many years some cultivars have been grown commercially for over 20 years .
genetic studies of sugarcane have been difficult due to the polyploid and highly heterozygous nature of the genome.
for example, molecular markers such as restriction fragment length polymorphism  and simple sequence repeats  can reveal several markers or alleles at one locus.
they can be single dose , double dose  or multi dose where it is often not possible to know how many copies are at a particular locus.
to date, a number of genetic maps have been constructed using single dose  markers.
single dose markers are used in coupling to construct linkage groups, and multi allelic markers and markers in repulsion are used to sort these linkage groups into homology groups.
the genetic maps generated to date have utilized the following markers; random amplified polymorphic dna  , rflps  and amplified fragment length polymorphism   and simple sequence repeats  .
even the most extensive of these genetic maps still only covers approximately half of the sugarcane genome .
recently, the development of a high-throughput marker system, diversity arrays technology , has enabled the creation of medium-density genetic maps for plants with complex genomes and limited sequence information available .
dart arrays generate whole genome fingerprints by scoring the presence versus absence of dna fragments in genomic representations generated from genomic dna samples through the process of complexity reduction .
dart maps have now been generated for many plants in the grass family, including rice , barley , wheat  and sorghum .
in an effort to increase the marker density for sugarcane, a diversity array was developed using a combination of s. officinarum and s. spontaneum accessions and sugarcane cultivars .
this has resulted in a medium density genetic map for sugarcane with enough sequence information to allow comparative mapping .
comparative genomics is a powerful tool that can provide a starting point for accelerating progress in revealing the genomic structure of crops that are lacking in the necessary genomic tools .
aligning markers or partial sequences to fully sequenced genomes enables researchers to deduce information about gene content and order of a particular crop of interest.
sugarcane is a member of the andropogoneae tribe, within the grass family poaceae, along with other tropical grasses such as sorghum , maize  and rice .
evolutionary studies have speculated that sorghum and maize may have diverged from a common ancestor about  <dig> million years ago  after which maize underwent polyploidation and subsequent reorganisation of the genome.
in contrast, rice and the maize/sorghum lineage may have diverged from a common ancestor around  <dig> million years ago and show much greater levels of chromosome structural rearrangement .
whilst, sugarcane and sorghum may have shared a common ancestor less than  <dig> million years ago and retain very similar gene order .
this close evolutionary relationship has led to the use of sorghum as a model crop for comparative genetic studies to gain a greater understanding of the genome of sugarcane .
the availability of the complete genome sequence of rice, maize and more recently sorghum, and the close evolutionary history of these species, has allowed some powerful comparative genomic studies  to be undertaken to gain an understanding of the genetic architecture of other crops where genomic sequence is lacking.
in this current study we have taken advantage of the development of a new, high-throughput marker system  to increase the marker density combined with the addition of snp and est-ssr markers.
this has led to an increase in genome coverage which will aid in the identification of qtl regions containing agronomically important genes of interest for sugarcane breeders.
furthermore, the availability of the genome sequence of maize, rice and more specifically sorghum has allowed a detailed study of the syntenic relationships between these crops.
the generation of this denser genetic map will ultimately lead to a greater understanding of the genetic architecture of the sugarcane genome which had previously been lacking.
this understanding will now allow the interrogation of important regions and identification of target genes for breeding.
RESULTS
assembly of saccharum linkage groups into homologous groups
the genetic linkage map of q <dig> presented in additional file  <dig> was arranged into eight hgs which represents the lowest saccahrum basic chromosome number .
initially this was carried out using the multiple allelic loci detected by single ssr, rflp or snp markers.
the location of the lgs was then verified using sequence information from all the  <dig> markers with a known sequence that had a primary correspondence to the sorghum genome at a significance level of p < e− <dig>  this allowed the  <dig> lgs to be condensed into the eight hgs .
cultivar q <dig> has a total of  <dig> chromosomes of which 15– <dig> are inherited from s. spontaneum, 82– <dig> are inherited from s. officianrum and 11– <dig> are recombinant between the two species .
alignment to sorghum
the sequence information from the dart, snp, rflp and the est derived ssr markers were used to align the sugarcane lgs to the sorghum genome sequence .
this was achieved by comparing the dna sequence of the  <dig> markers with a known sequence to the genome sequence of sorghum using blastn homology searching.
at the significance threshold of p <  <dig> × 10− <dig> there were  <dig> primary correspondences and a further  <dig> secondary and tertiary correspondences where the sequence aligned to a further one or two sites at p <  <dig> × 10− <dig> but a lower significance level than the primary correspondence .
in combination with the sequence information from  <dig> markers,  <dig> alleles from ssr, snp and rflp loci gave a total of  <dig> links between the sugarcane genetic map and the sorghum genome.
using these  <dig> informative markers the majority of the sugarcane linkage groups could be assigned to a single sorghum chromosome with high confidence  at the macrosyntenic level.
as s. spontaneum has a basic chromosome number of x =  <dig>  the  <dig> hgs of saccharum are aligned to the ten chromosome sequences of sorghum.
a composite map was produced for each hg to increase the number of markers that could be used for alignment between the saccharum hgs and the sorghum genome.
the sugarcane lgs within a hg where possible were condensed into single consensus lgs.
this was only possible where the lgs shared two or more markers in common.
the consensus lg for each hg was compared with the sorghum genome .
in some cases the lgs could not be included as there were not enough markers in common between the lgs within the hg.
for example hg <dig> aligned to sb <dig> and  <dig> using the consensus sequence but when all the lgs are considered parts of lgs also aligned to sb <dig> and sb <dig> .
in total over 92% of the sorghum genome was covered by these composite lgs.
sorghum bicolor
interchromosomal rearrangements between saccharum and sorghum chromosomes
hg1
when all the lgs within this hg are combined 99% of sb <dig> is covered by this hg .
this hg is highly syntenic to sb <dig> with 80% of the markers with sequence hits of e-values ≤ − <dig> aligning to sb <dig>  only on lg <dig> did there appear to be an interchromosomal rearrangement with  <dig> markers that had homology to sb <dig> inserted at the end of the lg , this same lg has an intrachromosomal rearrangement.
hg2
hg <dig> aligned to sb <dig> and sb <dig> with  <dig> lgs containing markers aligned to both chromosomes.
of all the markers with a known sequence, over 90% hit these chromosomes indicating a high level of synteny.
when a composite lg was created from the lgs over 96% of sb <dig> and sb <dig> were covered by this hg .
the lgs that demonstrated synteny with both chromosomes could be explained in the most part by one translocation event although lg1a exhibited a more complex rearrangement that was supported by gene sequence markers .
hg3
hg <dig> was highly syntenic to sb <dig> and the consensus lg covered  <dig> % of this sorghum chromosome .
of the markers with known sequence only lg  <dig> contained markers that had significant homology to another sorghum chromosome .
hg4
hg <dig> was syntenic to sb <dig> and the consensus lg covered  <dig> % of this chromosome .
only lg1b contained any significant markers that had homology to other chromosomes and as they were at the end of the lg could be due to spurious linkage.
hg5
hg <dig> aligned to two sorghum chromosomes sb <dig> and sb <dig>  the coverage was over 90% for both chromosomes.
five of the  <dig> lgs contained significant homology to both chromosomes and lgs  <dig>   <dig>   <dig> and  <dig> all represented a single translocation event at a similar position in the lg .
lg <dig> also contain the lower part of sb <dig> but more markers are needed to verify this.
hg6
hg <dig> aligned to sb <dig> but contained a large number of markers that aligned to different chromosomes.
of the markers that had known sequence 67% mapped to other chromosomes than sb <dig>  including sb <dig>  sb <dig> and sb <dig>  when the composite lg was considered, 98% of sb <dig> was covered, 50% of sb <dig> and sb <dig> and 12% of sb <dig>  .
although the majority of the lgs seemed to contain a backbone of sb <dig>  lg  <dig>   <dig> and  <dig> all contained a region that had homology to sb <dig>  in addition lg <dig> contained a group of markers with homology to sb <dig> which appeared from a single translocation event at the end of sb <dig>
hg7
this hg aligned to sb <dig> with the composite lg covering 97% of the chromosome .
although a number of the markers did align to different chromosomes  there was no real pattern observed.
this could be due to low marker coverage as this hg contained the smallest number of markers of any of the hgs .
hg8
this hg aligned to sb <dig> and sb <dig> and the composite lgs covered approximately 98% of sb <dig> and 40% of sb <dig> .
only lg <dig>   <dig> and  <dig> had markers with homology to both chromosomes.
the majority of the lg  aligned only to sb <dig> which is the largest sorghum chromosome.
sb <dig> is the smallest sorghum chromosome and only  <dig> lgs aligned to this chromosome.
lg  <dig> and  <dig> both had translocations at the end of sb <dig> but lg <dig> contained at least two rearrangements.
less than half of sb <dig> was present in this hg, which could be due to low polymorphism levels of this chromosome or it is possible that the rest of the chromosome is present in other hgs.
rearrangements between saccharum and sorghum chromosomes
four major rearrangements were identified from syntenic analysis between the sugarcane genetic map and the sorghum genome .
two of these account for the reduction in basic chromosome number from x =  <dig> in sorghum and s. officinarum to x =  <dig> in s. spontaneum.
the complicated nature of the hybrid sugarcane genome means that only a few of the lgs within a hg have the translocation, for example in hg <dig>   <dig> of the lgs display the translocation, the other lgs align only to one of the two chromosomes .
this recombination results in the two sorghum chromosomes combining into one sugarcane hg.
this translocation event reduces the basic chromosome number of sugarcane .
the other translocation event is in hg <dig> which aligns to sb <dig> and sb <dig> and again reduces the chromosome number to its final basic chromosome number of x =  <dig> .
the other major rearrangements had no effect on basic chromosome number but involved rearrangements between chromosomes.
hg <dig> aligns in the main to sb <dig> but one lg contains a significant translocation from sb <dig> covering approximately 50% of sb <dig>  the last rearrangement is in hg <dig> which aligns to sb <dig> and sb <dig> ; this appears to be a simple translocation between sb <dig> and half of sb <dig> and is present in  <dig> lgs within this hg .
analysis of the dart sequences
in total,  <dig> dart clones were sequenced and a proportion of these were also used to generate the genetic map.
these sequences produced a total sequence length of  <dig> ,170 bp with an average length of 465 bp.
these dart sequences were assembled into a total of  <dig> sequences, comprised of  <dig> contigs and  <dig> singletons.
of these  <dig> unique sequences,  <dig> hit against the saccharum est dataset,  <dig> to the s. bicolor est dataset,  <dig> to the z. mays est dataset and  <dig> to the o. sativa est dataset.
a venn diagram representing the assembled dart sequences that aligned to each of the relevant datasets is displayed in figure  <dig>  table  <dig> contains the number of ests in each dataset from the different genomes and the total number of assembled dart sequences that aligned to each dataset.
saccharum
sorghum bicolor
zea mays
oryza sativa
the dart markers aligned to all ten sorghum chromosomes with the largest number having homology to sb <dig> .
of the  <dig> dart markers  <dig> were mapped into the q <dig> linkage map.
all hgs contained dart markers,  <dig> were from unique sequences which resulted in 18% redundancy of the mapped markers.
of the mapped dart markers 34% had hits to genes .
atotal number of individual dart markers.
btotal number of unique markers.
genome sequence comparison
a results table of the dart sequence comparison against the genomes of s. bicolor, z. mays, and o. sativa is provided in the additional file  <dig>  a total of  <dig> assembled dart sequences aligned to an annotated gene in the s. bicolor genome, while a similar number  did not align to the genome at all.
a small proportion of the dart sequences aligned to repeats .
the categories of repeats against which the dart sequences aligned on the s. bicolor genome are displayed in figure  <dig>
.
bicolor genome including genes, repeats, the boundary between a repeat and genes and other which are regions that are not annotated.
s. bicolor
genome against which assembled dart sequences aligned.
in total,  <dig> assembled dart sequences which hit against the s. bicolor genome sequence also hit against the z. mays genome sequence, of which  <dig>  aligned to regions syntenic between these two species .
in contrast, only  <dig> assembled dart sequences hitting against the s. bicolor genome also hit against the o. sativa genome sequence, of which only  <dig>  aligned in regions syntenic between these two species.
the ratios of syntenic to non-syntenic region alignments for each category of annotation are displayed in figures  <dig> and  <dig>  in the case of both z. mays and o. sativa, dart sequences aligning to annotated genes most frequently aligned to syntenic regions .
s. bicolor
and
z. mays
syntenic to non-syntenic regions.
s. bicolor
and
o. sativa
syntenic to non-syntenic regions.
discussion
comparison of the sugarcane genetic map to the sorghum genome
to date only low resolution maps have been used to compare the sugarcane genetic map to the sorghum genome .
these maps were constructed with low-copy rflp markers that were selected for their ability to provide a signal in cross-hybridisation thereby limiting the ability to assess orthologous and paralogous relationships in gene families, since comparative mapping by rflp often identifies paralogous rather than orthologous sequences .
the large number of markers with known sequence in this study made it possible for the first time to align a significant part of the sugarcane genetic map to the sorghum genome sequence .
although with  <dig> sequences this analysis is at the macro level and only the major structural changes are identified, at the micro level there could be many more rearrangements but more markers and sequence information is needed to verify these.
to use as many markers as possible in the comparison to sorghum a composite linkage map was generated where possible for each hg.
this was then aligned to the sorghum genome sequence .
six hgs aligned to one sorghum chromosome and four hgs aligned in the main to two sorghum chromosomes.
for all chromosomes there were regions that appeared to contain markers from other chromosomes  but to be considered for further analysis a number of linked markers from the same chromosomes showing homology to gene sequences had to be identified.
this left four regions that showed strong likelihood of being due to rearrangement.
a major translocation was identified in hg <dig>  where lg <dig> aligned to sb <dig> and sb <dig>  this result agrees with previous attempts to align sugarcane to sorghum  although the greater number of markers in this map means a higher level of resolution of the alignment.
two sugarcane populations were studied by  and they determined that no more than four or five major interchromosomal translocations had occurred since the saccharum-sorghum divergence.
they also identified a translocation between sb <dig> and sb <dig> in both s. spontaneum parents and the hybrid parents of their populations.
they identified additional possible translocations between sorghum chromosomes  <dig> and  <dig> in the hybrid parent and  <dig> and  <dig> and  <dig> and  <dig> in the s. spontaneum parent, these were not verified in this study.
they did not identify the sb5/sb <dig> translocation that was identified in this analysis and by .
these two major translocations resulted in both cases in the condensation of two chromosomes into one.
this process is known in many lineages and is demonstrated within the grass family where all grasses have evolved from an ancestral genome of  <dig> chromosomes .
a further  <dig> hgs contained translocated chromosomes although these did not result in reduced chromosome number.
hg <dig> aligned in the main to sb <dig> but  <dig> of the  <dig> lgs also aligned to sb <dig> indicating a translocation between sb <dig> and sb <dig>  an interchromosomal rearrangement between lgs that aligned to sb <dig> and sb <dig> was also identified by  in the hybrid parent of the cross studied.
the additional interchromosomal rearrangement in hg <dig> lg <dig> which aligns to sb <dig> and sb <dig> has not previously been identified .
translocations
a more detailed comparison of the four major interchromosomal rearrangements  revealed that in some cases the rearrangement of the grass ancestral genome that has occurred in maize is the same as in sugarcane .
the chromosomal rearrangement seen in hg <dig> on lg <dig>  which aligns to sb <dig> and part of sb <dig>  corresponds to maize chromosome  <dig> .
maize diverged from sorghum approximately  <dig> million years ago  followed by allopolyploidisation approximately  <dig> mya .
the present maize chromosomes are a result of numerous rearrangements which reduced the chromosome number from  <dig> to  <dig> .
recent work by  has shown that the whole genome duplication  which occurred in the ancestor of both maize and sorghum  <dig> mya has resulted in large duplicated blocks in their genomes.
sb <dig> of sorghum shares a duplicated block with sb <dig> these two chromosomes have recombined to form maize m <dig>  the same rearrangement has occurred in sugarcane as seen in lg <dig> in hg <dig> .
the translocation identified in hg <dig> which aligns to sb <dig> and sb <dig> is also seen in the top of maize m <dig> .
parts of hg <dig> also align to m <dig> and m <dig> which are the duplicated ancestral regions .
the rearrangement in hg <dig> which aligns to sb <dig> and sb <dig> again is present in maize and aligns to m <dig> .
hg <dig> aligns to sorghum sb <dig> and sb <dig> which also aligns to maize m <dig> and m <dig>  interestingly the ancestral fusion event in sb <dig> is retained in both maize and sugarcane although a further rearrangement of this chromosome has occurred uniquely in sugarcane .
saccharum spontaneum
sugarcane being a polyploid, is more tolerant of gene deletions and rearrangements than a diploid.
it would appear that the rearrangements in hg <dig> and hg <dig> are major chromosome rearrangements to account for the smaller number of basic chromosomes in s. spontaneum.
a sparse rflp map was generated by  of a s. spontaneum clone ses <dig> using  <dig> single dose rflp markers.
from comparative mapping of the rflp clones to sorghum and maize, hg <dig> of this map contains clones umc <dig> and umc <dig> which align to sb <dig> and sg <dig> and umc <dig> which align to sb <dig> and are on the same lg.
this is comparable to the q <dig> hg <dig> which aligns to sb <dig> and sb <dig>  similarly rflp markers in hg <dig> of ses <dig> align to sb <dig> and sb <dig> as does the q <dig> hg <dig>  the translocations in hg <dig> and hg <dig> appear to be inherited from the s. spontaneum part of the genome of q <dig> but with the current data it is not possible to distinguish s. spontaneum chromosome from s. officinarum chromosomes.
however, as s. officinarum is x =  <dig>  the assumption is that at least the two rearrangements in hg <dig> and hg <dig> are inherited from the s. spontaneum part of the genome.
the other rearrangements in hg <dig> and hg <dig> could be inherited from either or both species.
comparison within the poaceae
recently a detailed genetic map was constructed for another member of the andropogoneae: poaceae, miscanthus sinensis.
this species has a diploid inheritance patterns  and when aligned to sorghum contains two sub-genomes, each syntenic to the entire sorghum genome with one major structural rearrangement which resulted in the condensation of two chromosomes into one and a reduction in chromosome number to x =  <dig> .
in this case the translocated lg aligned to sb <dig> and sb <dig> a translocation not seen in saccharum but again seen in maize on m <dig>  interestingly  found that the m. sinensis lgs that were syntenic to sb <dig> had the lowest percentage of markers that mapped to sorghum, a similar finding to switchgrass.
the data suggests that linkages syntenic to sb <dig> have encountered more evolutionary changes than other chromosomes after the species diverged from each other.
similarly hg <dig> in sacharrum which is syntenic to sb <dig> has undergone more rearrangement than other hgs.
recent work by  comparing sequenced genomes of grasses has demonstrated that the chromosome number variation/reduction from the n =  <dig> common paleo-ancestor to the n =  <dig> of sorghum could be due to the insertion of a chromosome in the centromeric region of another chromosome although these reductions could also come about through pericentric inversions followed by reciprocal translocations .
the comparison of the q <dig> saccharum genetic map to the sorghum and maize genomes demonstrates saccharum contains the same two ancestral fusion events.
moreover, out of the  <dig> major translocations identified,  <dig> of them have also occurred in maize , one of which resulted in a reduction in chromosome number.
s. officinarum is an octoploid and s. spontaneum ranges from 2n = 5x =  <dig> to 16x =  <dig>  in chromosome number.
it would seem probable that after the whole genome duplication that also occurred in maize the saccharum ancestor underwent some of the same rearrangements as maize then diverged and under went further wgd events.
more sequence analysis is needed to determine how these events occurred but in evolutionary terms they would have been recent .
whole genome duplication and the rapid and massive structural changes which have occurred in the poaceae and to a greater extent in the saccharum might explain the ability of these polyploids to adapt and survive environmental conditions that are not tolerated by their diploid ancestors.
within the saccharum there are no known diploids as all species are polyploid.
s. spontaneum has the widest distribution from japan and indonesia through the indian subcontinent to the mediterranean and africa  and the largest range of ploidy levels .
it has been reported that polyploidy provides increased vigour  and provided an advantage for colonizing a wider range of environments .
dart sequence comparison to est and genome sequences
it is to be expected that the highest number of darts would align to ests from saccharum as the genome of origin.
interestingly, a higher number of darts align to the maize est dataset  than to the sorghum  est dataset, even though sorghum is generally thought to be a much closer relative to sugarcane  .
this may be attributable to the higher volume of repetitive dna in the maize genome , some of which may be expressed and represented in the est database but missing from the sorghum est database which is much smaller than the maize database.
as would be expected, the least number of dart sequences aligned to the rice est dataset, which is the most distant relative of the species surveyed.
a proportionally low number of dart sequences aligned to annotated repeats  in the sorghum genome compared to the number of darts aligned to annotated genes , given that 55% of the sorghum genome is known to be repetitive and the repetitive content of sugarcane has been demonstrated to be similar to that of sorghum .
this suggests that the enrichment for unique regions applied in the production of the dart markers has largely succeeded.
interestingly,  <dig>  of the dart marker sequences can be observed in figure  <dig> to display no significant alignment to the sorghum genome, suggesting that these originate from divergent regions of sugarcane.
CONCLUSIONS
both the analysis of the sequence data and the comparative mapping has indicated that sugarcane is closely related to both maize and sorghum.
three out of the four translocations identified from the sugarcane genetic map are also present in the maize genome .
these events occurred in maize after a wgd which also occurred in the ancestors of sugarcane indicating its possible sugarcane diverged after the initial rearrangements occurred.
this study has highlighted the need for generation of a denser genetic map and ultimately the sugarcane genome sequence to allow the determination of the evolution of these three closely related species, sorghum, maize and sugarcane.

 sugarcane is a member of the poaceae family that contributes 75% of the worlds sugar supply and is being heralded as an important source as an alternative biofuel. however, knowledge of the genetic architecture of this crop currently lags behind that of other crops of the poaceae family such as wheat, barley and rice. it is important to uncover the genome structure of a particular crop to guide informed decisions in the identification of important genes/traits that can be utilized during breeding and germplasm introgression. modern sugarcane cultivars have complex polyploid, aneuploid genomes that make classical genetic, molecular genetic, and breeding studies difficult to interpret, as information on the structure and organisation of the genome is incomplete. they are essentially hybrids between the polyploid domesticated species, saccharum officinarum l and the polyploid wild species, s. spontaneum l. the cultivated hybrids have undergone a series of backcrosses to s. officinarum as the female parent to recover agronomically adapted genotypes with a high sugar yield. using in situ hybridisation, determined that the basic chromosome number of s. officinarum is x =  and for s. spontaneum is x =  with 10-15% of the genome of modern sugarcane cultivars contributed by s. spontaneum, 80% s. officinarum and 11-15% recombinant chromosomes between the two species. studies have shown that chromosome pairing is mostly as bivalents, although an assortment of meiotic irregularities can occur and contribute to the formation of aneuploid gametes. sugarcane clones are propagated vegetatively and are stable over many years some cultivars have been grown commercially for over 20 years. genetic studies of sugarcane have been difficult due to the polyploid and highly heterozygous nature of the genome. for example, molecular markers such as restriction fragment length polymorphism and simple sequence repeats can reveal several markers or alleles at one locus. they can be single dose, double dose or multi dose where it is often not possible to know how many copies are at a particular locus. to date, a number of genetic maps have been constructed using single dose markers. single dose markers are used in coupling to construct linkage groups, and multi allelic markers and markers in repulsion are used to sort these linkage groups into homology groups. the genetic maps generated to date have utilized the following markers; random amplified polymorphic dna, rflps and amplified fragment length polymorphism and simple sequence repeats. even the most extensive of these genetic maps still only covers approximately half of the sugarcane genome. recently, the development of a high-throughput marker system, diversity arrays technology, has enabled the creation of medium-density genetic maps for plants with complex genomes and limited sequence information available. dart arrays generate whole genome fingerprints by scoring the presence versus absence of dna fragments in genomic representations generated from genomic dna samples through the process of complexity reduction. dart maps have now been generated for many plants in the grass family, including rice, barley, wheat and sorghum. in an effort to increase the marker density for sugarcane, a diversity array was developed using a combination of s. officinarum and s. spontaneum accessions and sugarcane cultivars. this has resulted in a medium density genetic map for sugarcane with enough sequence information to allow comparative mapping. comparative genomics is a powerful tool that can provide a starting point for accelerating progress in revealing the genomic structure of crops that are lacking in the necessary genomic tools. aligning markers or partial sequences to fully sequenced genomes enables researchers to deduce information about gene content and order of a particular crop of interest. sugarcane is a member of the andropogoneae tribe, within the grass family poaceae, along with other tropical grasses such as sorghum, maize and rice. evolutionary studies have speculated that sorghum and maize may have diverged from a common ancestor about million years ago after which maize underwent polyploidation and subsequent reorganisation of the genome. in contrast, rice and the maize/sorghum lineage may have diverged from a common ancestor around million years ago and show much greater levels of chromosome structural rearrangement. whilst, sugarcane and sorghum may have shared a common ancestor less than million years ago and retain very similar gene order. this close evolutionary relationship has led to the use of sorghum as a model crop for comparative genetic studies to gain a greater understanding of the genome of sugarcane. the availability of the complete genome sequence of rice, maize and more recently sorghum, and the close evolutionary history of these species, has allowed some powerful comparative genomic studies to be undertaken to gain an understanding of the genetic architecture of other crops where genomic sequence is lacking. in this current study we have taken advantage of the development of a new, high-throughput marker system to increase the marker density combined with the addition of snp and est-ssr markers. this has led to an increase in genome coverage which will aid in the identification of qtl regions containing agronomically important genes of interest for sugarcane breeders. furthermore, the availability of the genome sequence of maize, rice and more specifically sorghum has allowed a detailed study of the syntenic relationships between these crops. the generation of this denser genetic map will ultimately lead to a greater understanding of the genetic architecture of the sugarcane genome which had previously been lacking. this understanding will now allow the interrogation of important regions and identification of target genes for breeding.  assembly of saccharum linkage groups into homologous groups the genetic linkage map of q presented in additional file was arranged into eight hgs which represents the lowest saccahrum basic chromosome number. initially this was carried out using the multiple allelic loci detected by single ssr, rflp or snp markers. the location of the lgs was then verified using sequence information from all the markers with a known sequence that had a primary correspondence to the sorghum genome at a significance level of p < e− this allowed the lgs to be condensed into the eight hgs. cultivar q has a total of chromosomes of which 15– are inherited from s. spontaneum, 82– are inherited from s. officianrum and 11– are recombinant between the two species. alignment to sorghum the sequence information from the dart, snp, rflp and the est derived ssr markers were used to align the sugarcane lgs to the sorghum genome sequence. this was achieved by comparing the dna sequence of the markers with a known sequence to the genome sequence of sorghum using blastn homology searching. at the significance threshold of p <  × 10− there were primary correspondences and a further secondary and tertiary correspondences where the sequence aligned to a further one or two sites at p <  × 10− but a lower significance level than the primary correspondence. in combination with the sequence information from markers, alleles from ssr, snp and rflp loci gave a total of links between the sugarcane genetic map and the sorghum genome. using these informative markers the majority of the sugarcane linkage groups could be assigned to a single sorghum chromosome with high confidence at the macrosyntenic level. as s. spontaneum has a basic chromosome number of x =  the hgs of saccharum are aligned to the ten chromosome sequences of sorghum. a composite map was produced for each hg to increase the number of markers that could be used for alignment between the saccharum hgs and the sorghum genome. the sugarcane lgs within a hg where possible were condensed into single consensus lgs. this was only possible where the lgs shared two or more markers in common. the consensus lg for each hg was compared with the sorghum genome. in some cases the lgs could not be included as there were not enough markers in common between the lgs within the hg. for example hg aligned to sb and using the consensus sequence but when all the lgs are considered parts of lgs also aligned to sb and sb. in total over 92% of the sorghum genome was covered by these composite lgs. sorghum bicolor interchromosomal rearrangements between saccharum and sorghum chromosomes hg1 when all the lgs within this hg are combined 99% of sb is covered by this hg. this hg is highly syntenic to sb with 80% of the markers with sequence hits of e-values ≤ − aligning to sb only on lg did there appear to be an interchromosomal rearrangement with markers that had homology to sb inserted at the end of the lg, this same lg has an intrachromosomal rearrangement. hg2 hg aligned to sb and sb with lgs containing markers aligned to both chromosomes. of all the markers with a known sequence, over 90% hit these chromosomes indicating a high level of synteny. when a composite lg was created from the lgs over 96% of sb and sb were covered by this hg. the lgs that demonstrated synteny with both chromosomes could be explained in the most part by one translocation event although lg1a exhibited a more complex rearrangement that was supported by gene sequence markers. hg3 hg was highly syntenic to sb and the consensus lg covered % of this sorghum chromosome. of the markers with known sequence only lg contained markers that had significant homology to another sorghum chromosome. hg4 hg was syntenic to sb and the consensus lg covered % of this chromosome. only lg1b contained any significant markers that had homology to other chromosomes and as they were at the end of the lg could be due to spurious linkage. hg5 hg aligned to two sorghum chromosomes sb and sb the coverage was over 90% for both chromosomes. five of the lgs contained significant homology to both chromosomes and lgs and all represented a single translocation event at a similar position in the lg. lg also contain the lower part of sb but more markers are needed to verify this. hg6 hg aligned to sb but contained a large number of markers that aligned to different chromosomes. of the markers that had known sequence 67% mapped to other chromosomes than sb including sb sb and sb when the composite lg was considered, 98% of sb was covered, 50% of sb and sb and 12% of sb. although the majority of the lgs seemed to contain a backbone of sb lg and all contained a region that had homology to sb in addition lg contained a group of markers with homology to sb which appeared from a single translocation event at the end of sb hg7 this hg aligned to sb with the composite lg covering 97% of the chromosome. although a number of the markers did align to different chromosomes there was no real pattern observed. this could be due to low marker coverage as this hg contained the smallest number of markers of any of the hgs. hg8 this hg aligned to sb and sb and the composite lgs covered approximately 98% of sb and 40% of sb. only lg and had markers with homology to both chromosomes. the majority of the lg aligned only to sb which is the largest sorghum chromosome. sb is the smallest sorghum chromosome and only lgs aligned to this chromosome. lg and both had translocations at the end of sb but lg contained at least two rearrangements. less than half of sb was present in this hg, which could be due to low polymorphism levels of this chromosome or it is possible that the rest of the chromosome is present in other hgs. rearrangements between saccharum and sorghum chromosomes four major rearrangements were identified from syntenic analysis between the sugarcane genetic map and the sorghum genome. two of these account for the reduction in basic chromosome number from x =  in sorghum and s. officinarum to x =  in s. spontaneum. the complicated nature of the hybrid sugarcane genome means that only a few of the lgs within a hg have the translocation, for example in hg of the lgs display the translocation, the other lgs align only to one of the two chromosomes. this recombination results in the two sorghum chromosomes combining into one sugarcane hg. this translocation event reduces the basic chromosome number of sugarcane. the other translocation event is in hg which aligns to sb and sb and again reduces the chromosome number to its final basic chromosome number of x = . the other major rearrangements had no effect on basic chromosome number but involved rearrangements between chromosomes. hg aligns in the main to sb but one lg contains a significant translocation from sb covering approximately 50% of sb the last rearrangement is in hg which aligns to sb and sb; this appears to be a simple translocation between sb and half of sb and is present in lgs within this hg. analysis of the dart sequences in total, dart clones were sequenced and a proportion of these were also used to generate the genetic map. these sequences produced a total sequence length of,170 bp with an average length of 465 bp. these dart sequences were assembled into a total of sequences, comprised of contigs and singletons. of these unique sequences, hit against the saccharum est dataset, to the s. bicolor est dataset, to the z. mays est dataset and to the o. sativa est dataset. a venn diagram representing the assembled dart sequences that aligned to each of the relevant datasets is displayed in figure  table  contains the number of ests in each dataset from the different genomes and the total number of assembled dart sequences that aligned to each dataset. saccharum sorghum bicolor zea mays oryza sativa the dart markers aligned to all ten sorghum chromosomes with the largest number having homology to sb. of the dart markers were mapped into the q linkage map. all hgs contained dart markers, were from unique sequences which resulted in 18% redundancy of the mapped markers. of the mapped dart markers 34% had hits to genes. atotal number of individual dart markers. btotal number of unique markers. genome sequence comparison a results table of the dart sequence comparison against the genomes of s. bicolor, z. mays, and o. sativa is provided in the additional file a total of assembled dart sequences aligned to an annotated gene in the s. bicolor genome, while a similar number did not align to the genome at all. a small proportion of the dart sequences aligned to repeats. the categories of repeats against which the dart sequences aligned on the s. bicolor genome are displayed in figure . bicolor genome including genes, repeats, the boundary between a repeat and genes and other which are regions that are not annotated. s. bicolor genome against which assembled dart sequences aligned. in total, assembled dart sequences which hit against the s. bicolor genome sequence also hit against the z. mays genome sequence, of which aligned to regions syntenic between these two species. in contrast, only assembled dart sequences hitting against the s. bicolor genome also hit against the o. sativa genome sequence, of which only aligned in regions syntenic between these two species. the ratios of syntenic to non-syntenic region alignments for each category of annotation are displayed in figures  and in the case of both z. mays and o. sativa, dart sequences aligning to annotated genes most frequently aligned to syntenic regions. s. bicolor and z. mays syntenic to non-syntenic regions. s. bicolor and o. sativa syntenic to non-syntenic regions. discussion comparison of the sugarcane genetic map to the sorghum genome to date only low resolution maps have been used to compare the sugarcane genetic map to the sorghum genome. these maps were constructed with low-copy rflp markers that were selected for their ability to provide a signal in cross-hybridisation thereby limiting the ability to assess orthologous and paralogous relationships in gene families, since comparative mapping by rflp often identifies paralogous rather than orthologous sequences. the large number of markers with known sequence in this study made it possible for the first time to align a significant part of the sugarcane genetic map to the sorghum genome sequence. although with sequences this analysis is at the macro level and only the major structural changes are identified, at the micro level there could be many more rearrangements but more markers and sequence information is needed to verify these. to use as many markers as possible in the comparison to sorghum a composite linkage map was generated where possible for each hg. this was then aligned to the sorghum genome sequence. six hgs aligned to one sorghum chromosome and four hgs aligned in the main to two sorghum chromosomes. for all chromosomes there were regions that appeared to contain markers from other chromosomes but to be considered for further analysis a number of linked markers from the same chromosomes showing homology to gene sequences had to be identified. this left four regions that showed strong likelihood of being due to rearrangement. a major translocation was identified in hg where lg aligned to sb and sb this result agrees with previous attempts to align sugarcane to sorghum although the greater number of markers in this map means a higher level of resolution of the alignment. two sugarcane populations were studied by and they determined that no more than four or five major interchromosomal translocations had occurred since the saccharum-sorghum divergence. they also identified a translocation between sb and sb in both s. spontaneum parents and the hybrid parents of their populations. they identified additional possible translocations between sorghum chromosomes and in the hybrid parent and and and and in the s. spontaneum parent, these were not verified in this study. they did not identify the sb5/sb translocation that was identified in this analysis and by. these two major translocations resulted in both cases in the condensation of two chromosomes into one. this process is known in many lineages and is demonstrated within the grass family where all grasses have evolved from an ancestral genome of chromosomes. a further hgs contained translocated chromosomes although these did not result in reduced chromosome number. hg aligned in the main to sb but of the lgs also aligned to sb indicating a translocation between sb and sb an interchromosomal rearrangement between lgs that aligned to sb and sb was also identified by in the hybrid parent of the cross studied. the additional interchromosomal rearrangement in hg lg which aligns to sb and sb has not previously been identified. translocations a more detailed comparison of the four major interchromosomal rearrangements revealed that in some cases the rearrangement of the grass ancestral genome that has occurred in maize is the same as in sugarcane. the chromosomal rearrangement seen in hg on lg which aligns to sb and part of sb corresponds to maize chromosome. maize diverged from sorghum approximately million years ago followed by allopolyploidisation approximately mya. the present maize chromosomes are a result of numerous rearrangements which reduced the chromosome number from to. recent work by has shown that the whole genome duplication which occurred in the ancestor of both maize and sorghum mya has resulted in large duplicated blocks in their genomes. sb of sorghum shares a duplicated block with sb these two chromosomes have recombined to form maize m the same rearrangement has occurred in sugarcane as seen in lg in hg. the translocation identified in hg which aligns to sb and sb is also seen in the top of maize m. parts of hg also align to m and m which are the duplicated ancestral regions. the rearrangement in hg which aligns to sb and sb again is present in maize and aligns to m. hg aligns to sorghum sb and sb which also aligns to maize m and m interestingly the ancestral fusion event in sb is retained in both maize and sugarcane although a further rearrangement of this chromosome has occurred uniquely in sugarcane. saccharum spontaneum sugarcane being a polyploid, is more tolerant of gene deletions and rearrangements than a diploid. it would appear that the rearrangements in hg and hg are major chromosome rearrangements to account for the smaller number of basic chromosomes in s. spontaneum. a sparse rflp map was generated by of a s. spontaneum clone ses using single dose rflp markers. from comparative mapping of the rflp clones to sorghum and maize, hg of this map contains clones umc and umc which align to sb and sg and umc which align to sb and are on the same lg. this is comparable to the q hg which aligns to sb and sb similarly rflp markers in hg of ses align to sb and sb as does the q hg the translocations in hg and hg appear to be inherited from the s. spontaneum part of the genome of q but with the current data it is not possible to distinguish s. spontaneum chromosome from s. officinarum chromosomes. however, as s. officinarum is x =  the assumption is that at least the two rearrangements in hg and hg are inherited from the s. spontaneum part of the genome. the other rearrangements in hg and hg could be inherited from either or both species. comparison within the poaceae recently a detailed genetic map was constructed for another member of the andropogoneae: poaceae, miscanthus sinensis. this species has a diploid inheritance patterns and when aligned to sorghum contains two sub-genomes, each syntenic to the entire sorghum genome with one major structural rearrangement which resulted in the condensation of two chromosomes into one and a reduction in chromosome number to x = . in this case the translocated lg aligned to sb and sb a translocation not seen in saccharum but again seen in maize on m interestingly found that the m. sinensis lgs that were syntenic to sb had the lowest percentage of markers that mapped to sorghum, a similar finding to switchgrass. the data suggests that linkages syntenic to sb have encountered more evolutionary changes than other chromosomes after the species diverged from each other. similarly hg in sacharrum which is syntenic to sb has undergone more rearrangement than other hgs. recent work by comparing sequenced genomes of grasses has demonstrated that the chromosome number variation/reduction from the n =  common paleo-ancestor to the n =  of sorghum could be due to the insertion of a chromosome in the centromeric region of another chromosome although these reductions could also come about through pericentric inversions followed by reciprocal translocations. the comparison of the q saccharum genetic map to the sorghum and maize genomes demonstrates saccharum contains the same two ancestral fusion events. moreover, out of the major translocations identified, of them have also occurred in maize, one of which resulted in a reduction in chromosome number. s. officinarum is an octoploid and s. spontaneum ranges from 2n = 5x =  to 16x =  in chromosome number. it would seem probable that after the whole genome duplication that also occurred in maize the saccharum ancestor underwent some of the same rearrangements as maize then diverged and under went further wgd events. more sequence analysis is needed to determine how these events occurred but in evolutionary terms they would have been recent. whole genome duplication and the rapid and massive structural changes which have occurred in the poaceae and to a greater extent in the saccharum might explain the ability of these polyploids to adapt and survive environmental conditions that are not tolerated by their diploid ancestors. within the saccharum there are no known diploids as all species are polyploid. s. spontaneum has the widest distribution from japan and indonesia through the indian subcontinent to the mediterranean and africa and the largest range of ploidy levels. it has been reported that polyploidy provides increased vigour and provided an advantage for colonizing a wider range of environments. dart sequence comparison to est and genome sequences it is to be expected that the highest number of darts would align to ests from saccharum as the genome of origin. interestingly, a higher number of darts align to the maize est dataset than to the sorghum est dataset, even though sorghum is generally thought to be a much closer relative to sugarcane. this may be attributable to the higher volume of repetitive dna in the maize genome, some of which may be expressed and represented in the est database but missing from the sorghum est database which is much smaller than the maize database. as would be expected, the least number of dart sequences aligned to the rice est dataset, which is the most distant relative of the species surveyed. a proportionally low number of dart sequences aligned to annotated repeats in the sorghum genome compared to the number of darts aligned to annotated genes, given that 55% of the sorghum genome is known to be repetitive and the repetitive content of sugarcane has been demonstrated to be similar to that of sorghum. this suggests that the enrichment for unique regions applied in the production of the dart markers has largely succeeded. interestingly, of the dart marker sequences can be observed in figure  to display no significant alignment to the sorghum genome, suggesting that these originate from divergent regions of sugarcane.  both the analysis of the sequence data and the comparative mapping has indicated that sugarcane is closely related to both maize and sorghum. three out of the four translocations identified from the sugarcane genetic map are also present in the maize genome. these events occurred in maize after a wgd which also occurred in the ancestors of sugarcane indicating its possible sugarcane diverged after the initial rearrangements occurred. this study has highlighted the need for generation of a denser genetic map and ultimately the sugarcane genome sequence to allow the determination of the evolution of these three closely related species, sorghum, maize and sugarcane. 
we have utilized the recently developed sugarcane dart array to increase the marker density within the genetic map. we have used the genetic map of a sugarcane cultivar to generate a consensus genetic map to increase genome coverage for comparison to the sorghum genome. four major chromosome rearrangements were identified between the other four sugarcane hgs and sorghum, two of which were condensations of chromosomes reducing the basic chromosome number of sugarcane from x =  to x =  this macro level of synteny was transferred to other members within the poaceae family such as maize to uncover the important evolutionary relationships that exist between sugarcane and these species. furthermore of the four major chromosome rearrangements identified in this study, three were common to maize providing some evidence that chromosome reduction from a common paleo-ancestor of both maize and sugarcane was driven by the same translocation events seen in both species. comparative mapping of sugarcane to the sorghum genome has revealed new information on the genome structure of sugarcane which will help guide identification of important genes for use in sugarcane breeding. the sequence of these dart markers plus snp and est-ssr markers was then used to form a bridge to the sorghum genomic sequence by blast alignment to start to unravel the complex genomic architecture of sugarcane. these hgs were syntenic to four sorghum chromosomes with from 98% to 100% of these chromosomes covered by these linked markers. the understanding of sugarcane genetics has lagged behind that of other members of the poaceae family such as wheat, rice, barley and sorghum mainly due to the complexity, size and polyploidization of the genome. on a macrosyntenic level a good collinearity was observed between sugarcane and sorghum for of the homology groups.
 the understanding of sugarcane genetics has lagged behind that of other members of the poaceae family such as wheat, rice, barley and sorghum mainly due to the complexity, size and polyploidization of the genome. we have used the genetic map of a sugarcane cultivar to generate a consensus genetic map to increase genome coverage for comparison to the sorghum genome. we have utilized the recently developed sugarcane dart array to increase the marker density within the genetic map. the sequence of these dart markers plus snp and est-ssr markers was then used to form a bridge to the sorghum genomic sequence by blast alignment to start to unravel the complex genomic architecture of sugarcane.  comparative mapping revealed that certain sugarcane chromosomes show greater levels of synteny to sorghum than others. on a macrosyntenic level a good collinearity was observed between sugarcane and sorghum for of the homology groups. these hgs were syntenic to four sorghum chromosomes with from 98% to 100% of these chromosomes covered by these linked markers. four major chromosome rearrangements were identified between the other four sugarcane hgs and sorghum, two of which were condensations of chromosomes reducing the basic chromosome number of sugarcane from x =  to x =  this macro level of synteny was transferred to other members within the poaceae family such as maize to uncover the important evolutionary relationships that exist between sugarcane and these species.  comparative mapping of sugarcane to the sorghum genome has revealed new information on the genome structure of sugarcane which will help guide identification of important genes for use in sugarcane breeding. furthermore of the four major chromosome rearrangements identified in this study, three were common to maize providing some evidence that chromosome reduction from a common paleo-ancestor of both maize and sugarcane was driven by the same translocation events seen in both species. saccharumpoaceaesyntenyorthologytranslocationssnp markers 