BACKGROUND
simple sequence repeats  or microsatellites are short ) tandem repeat nucleotides in dna sequences.
they are ubiquitous in genomes, being found in both eukaryotes and prokaryotes and in any region   <cit> .
because of their high mutation rates via insertion or deletion of one or a few repeat units, ssrs have been developed into one of the most popular sources of codominant markers with high information content during the past several years  <cit> .
they are widely employed in many research areas, including linkage mapping, population genetics, phylogenetics, and comparative genomics  <cit> .
additionally, recent studies reveal that microsatellites may serve an important role in regulating gene expression, protein function, and genome evolution  <cit> .
the conventional strategies of developing ssr markers are screening genomic dna libraries or constructing ssr-enriched libraries  <cit> .
both are usually time-consuming and labor-intensive.
however, the availability of genome sequences allows us to mine for ssrs at the genomic level in silico, and analysis of these ssrs has provided insight into their distributions, putative functions, and evolution  <cit> .
cottons  comprise five tetraploid  and over  <dig> diploid  species  <cit> .
the diploid species are grouped into eight subgenomes, designated a–g and k  <cit> .
the a-genome  is almost twice the size of the d-genome   <cit> .
tetraploid cotton species are thought to have formed through hybridization and subsequent polyploidization between an a-genome and a d-genome species  <cit> .
analyses of genetic maps revealed that diploid a- and d-genomes have a high level of synteny with at- and dt-genomes in tetraploid cotton  <cit> .
the tetraploid g. hirsutum, also known as upland cotton, produces over 90% of the world’s cotton  <cit> .
however, because of the duplicated dna segments and homologous chromosomes in its tetraploid genome, many markers map to at least two positions  <cit> , most on homologous chromosomes, hindering the precise mapping of qualitative character genes or quantitative trait loci  to particular chromosomes.
furthermore, given the narrow genetic base, intraspecific genetic diversity in cottons is relatively low , both among and within g. hirsutum cultivars  <cit> , hindering the development of high-resolution genetic maps and marker-assisted selection  breeding in cottons.
however, chromosome-specific markers with high polymorphism distributed throughout the genome would facilitate chromosome identification in genetic maps of tetraploid cottons, which are instrumental for applications like positional gene cloning, especially in discriminating homologous chromosomes during fine mapping of single traits and qtls.
traditional methods including meiotic fluorescence in situ hybridization  and translocation lines can be used to precisely locate dna markers to chromosomes, but most markers are too small to be used directly as probes in fish.
bacterial artificial chromosome  clones were often used as chromosome-specific markers  <cit> , but the process is time-consuming and labor-intensive, and only a few bacs have been identified as chromosome-specific  <cit> .
large-scale development of chromosome-specific ssrs can be realized only when each chromosome sequence of the cotton genome is obtained.
recently, from the whole-genome sequence of g. raimondii  <cit> , zou et al.
<cit>  identified  <dig>  microsatellites, and  <dig>  primer pairs were designed, but they are insufficient for mining chromosome-specific ssrs when only one progenitor genome sequence is present.
the availability of a very large set of chromosome-specific ssrs distributed throughout the genome would benefit the cotton research and breeding community.
the rapid development of high-throughput dna sequencing technologies have allowed increasing numbers of genomes to be sequenced, especially multiple genomes within a genus, expediting the development of chromosome-specific markers and detailed comparative mapping.
in recent years, the genomes of g. raimondii  <cit>  and g. arboreum  <cit>   have been sequenced; the progenitors of these two diploid species were putative contributors of the a- and d-subgenomes to tetraploid cotton.
these diploid genomes share a high level of synteny or co-linearity with the subgenomes in tetraploid cotton  <cit> .
in spite of the importance of chromosome-specific microsatellite markers to many applications, their systematic and genome-wide characterization in cotton genomes has not yet be conducted.
in the present study, we characterized the distribution and density of microsatellites in g. raimondii and g. arboreum chromosomes and identified chromosome-specific ssrs by comparative analysis.
in addition, a set of highly-polymorphic ssrs were selected to construct a genetic linkage map of two homologous chromosomes  in tetraploid cotton, proving that chromosome-specific ssrs could distinguish homologous chromosomes during genetic mapping.
RESULTS
we analyzed the distributions of ssrs with ≥  <dig> repeat units and a minimum total length of  <dig> bp in each of the  <dig> chromosomes sequences of g. arboreum  and g. raimondii  to understand their general features in cotton genomes.
ssrs on each chromosome were then compared those on the other  <dig> chromosomes  to find chromosome-specific ssrs.
standardized motifs used in sciroko  <cit>  were used to represent all variants on both strands of the dna sequence  for consistency in estimating repeat frequencies.
unless otherwise specified, ssr content was expressed as ‘number of ssrs per million base pairs’ or as relative frequencies  within a particular dataset.
using the chromosome-specific ssrs, we constructed two linkage groups for two homologous chromosomes  in the tetraploid cotton genome to test whether these chromosome-specific ssrs could distinguish homologous chromosomes.
distribution of ssr types in genomes and on individual chromosomes
the ssr content of the g. arboreum and g. raimondii genomic sequences are summarized in table  <dig>  a total of  <dig>  ssrs were detected from  <dig> g. arboreum chromosome sequences, giving an overall density across the chromosomes of  <dig>  ssrs/mb .
<dig>  ssrs were detected in the  <dig> g. raimondii chromosome sequences, giving an overall density of  <dig>  ssrs/mb .
although the g. arboreum genome is about twice the size of the g. raimondii genome the ssr content did not exhibit a two-fold difference; g. raimondii had a much higher ssr density  than g. arboreum .
to enable convenient and selective use of the newly developed ssrs, a blast search to determine redundancy was performed against publically available ssrs in the cottongen database ;  <dig>  ssrs were recognized as duplicates .table  <dig>
distribution of ssrs with ≥  <dig> repeats and a minimum length of  <dig> bp in genomic sequences of
gossypium arboreum
and
gossypium raimondii
sequence type
g. arboreum*
g. raimondii*
count
rel.
freq.
mean repeat number
density
count
rel.
freq.
mean repeat number
density
*only ssrs anchored in chromosomes were considered.
pentanucleotide repeats were the most common ssr type in both g. arboreum and g. raimondii, representing  <dig> % and  <dig>  of all ssrs, respectively, followed by dinucleotide repeats .
no major differences were observed between g. arboreum and g. raimondii, aside from the higher density of ssrs in g. raimondii.
the distribution of ssr types in the two cotton genomes differed from those of cucumber, poplar and grapevine, in which tetranucleotide repeats were the most frequent type, and also from those of rice and arabidopsis, in which trinucleotide repeats were most frequent  <cit> .figure  <dig>
relative frequency  of ssr types in the genomes of
gossypium arboreum
and
gossypium raimondii
.
ssrs are organized by the number of repeats.
the graph was based on a total of  <dig>  and  <dig>  ssrs detected in the genomes of g. arboreum and g. raimondii, respectively.
the ssr contents of each chromosome sequence in g. arboreum and g. raimondii are summarized in table  <dig>  in general, no significant differences were detected among chromosomes within a genome in the distribution of ssrs, except that chr <dig> in g. arboreum had a relative low density.
for each chromosome, the distribution of motifs was consistent with their genome features, pentanucleotides were the most common ssr type followed by dinucleotides in every chromosome, and no motifs were enriched on particular chromosomes.
the variation in ssr density among chromosomes was larger in g. arboreum  than in g. raimondii .table  <dig>
distribution of total and chromosome-specific ssrs among chromosome sequences of
gossypium arboreum
and
gossypium raimondii
chromosome
g. arboreum
g. raimondii
total ssr no.
density
chromosome-specific no.
rel.
freq.
total ssr no.
density
chromosome-specific no.
rel.
freq.
distribution of ssr motifs
detailed analyses of individual repeat motifs were carried out for each type of ssr found in g. arboreum and g. raimondii .
in general, g. raimondii had a higher density of all repeat types except a motifs, which were similarly and significantly overrepresented in both g. arboreum  and g. raimondii genomes .
among dinucleotide repeats, the at motif was dramatically overrepresented in both genomes , representing  <dig> % of the dinucleotide motifs in g. arboreum and  <dig> % in g. raimondii.
at repeats were also the most abundant motif overall in cotton genomes , accounting for  <dig> % and  <dig> % of the total ssrs, respectively.
cg repeats were the least frequent dinucleotides; only five were found in g. arboreum and  <dig> in g. raimondii.figure  <dig>
distribution of di- and trinucleotide repeats in the genomes of
gossypium arboreum
and
gossypium raimondii
.
frequency values are expressed as the number of repeats per million base pairs of sequence.
detailed information on frequencies of individual di- and trinucleotide repeat motifs is provided in additional file  <dig>
among the trinucleotides, repeats of aat were most common in both the g. arboreum  and g. raimondii  genomes .
acg was the rarest trinucleotide motif, unlike in most dicot plants in which ccg is least common   <cit> .
among tetranucleotide repeats, the at-rich motifs aaat, aaag, aatt, and atac were, in that order, the most abundant in both genomes, together representing  <dig> % of all tetramer repeats in g. arboreum and  <dig> % in g. raimondii.
gc-rich repeats, like cccg, acgg and accg, were rarest, with relative frequencies of about  <dig> % .
the most abundant motif aaat had nearly twice the density in g. raimondii than in g. arboreum.
as with the dinucleotides and other kinds of motif, the density in g. raimondii was slightly higher than in g. arboreum.
among pentanucleotide repeats, the at-rich motifs aaaat, aaaag, and aaatt were most frequent in both genomes, together accounting for  <dig> % of all pentanucleotides in g. arboreum and  <dig> % in g. raimondii .
aaaat was the most abundant, outnumbering the next most frequent repeats, aaaag or aaatt, by about three fold.
surprisingly, although gc-rich repeats like ccgcg, ccccc and accgt were the rarest, cccgg motif was relatively abundant in both genomes.
among hexanucleotides, at-rich motifs also predominated in both genomes; aaaaat was the most common hexanucleotide repeat in both genomes, but the next most frequent was aatcag in g. arboreum and aaaaag in g. raimondii.
in general, at-rich motifs were most common, especially a-rich ones .
g. raimondii had higher density of all repeat types than g. arboreum except the a motif , while g. arboreum had more repeats due to larger genome.
for the same ssr type, the genomes tended to accumulate motifs containing more a in tandem repeat.
for example, aaatt was more abundant than aatat in the pentanucleotides, and aaaatt was more abundant than aaatat in the hexanucleotides.
development of chromosome-specific ssrs
we first examined ssr repetition within each chromosome to detect whether a small number of ssr types dominated, given the high repeat content in the cotton genome.
each ssr sequence, including the repeat and both flanking sequences, were compared with all other ssr sequences on the same chromosome.
the average proportion of unissrs was  <dig> % on g. arboreum chromosomes and  <dig> % on g. raimondii chromosomes.
thus, only 20–30% ssrs were repetitive within chromosomes, which is conducive to obtaining chromosome-specific ssrs.
for single repetitive ssrs, g. arboreum had an average of  <dig>  repetitions and g. raimondii and average of  <dig> .
the average maximum number of repetitions was  <dig> in g. arboreum and only  <dig> in g. raimondii, so the former had a higher degree of repetition than the latter .
ssr sequences from each chromosome were then compared with those on the other  <dig> chromosomes ; similar ssrs were then excluded to identify chromosome-specific ssrs for each chromosome.
the genome of g. arboreum had more chromosome-specific ssrs than did g. raimondii.
the average proportion of chromosome-specific ssrs was higher in g. raimondii  than in g. arboreum .
as the ssrs were designed using diploid cotton genome sequences, it is important to check the percentage of ssrs that could be successfully amplified in tetraploid cottons.
of  <dig> successfully designed ssrs,  <dig>  could be amplified in tm- <dig>  hai  <dig>  and liaomian  <dig>  these chromosome-specific ssrs are therefore considered suitable for amplification in tetraploid cotton.
chromosome-specific ssrs could distinguish homologous chromosomes in tetraploid cotton
to determine whether the chromosome-specific ssrs could distinguish homologous chromosomes in other tetraploid cotton species, two homologous linkage groups were constructed using  <dig> ssrs from chr <dig> of g. arboreum and  <dig> ssrs from chr <dig> of g. raimondii.
a total of  <dig> and  <dig> primer pairs were designed from g. arboreum and g. raimondii, respectively.
a bc <dig> population of  <dig> individuals was constructed by crossing tm- <dig> and t <dig> .
<dig>   <dig>  and  <dig> polymorphic markers between the parents were obtained from the  <dig> ssrs designed from chr <dig> of g. arboreum,  <dig> from chr <dig> of g. raimondii, and  <dig> from published genetic maps, respectively.
all  <dig> ssrs were included in the linkage analysis.
two linkage groups, containing  <dig> markers, were constructed:  <dig> markers in linkage group  i, and  <dig> in lg ii .
the other  <dig> loci were not part of any linkage group.
<dig> of the  <dig> polymorphic ssrs from g. arboreum mapped to lg i, and  <dig> of the  <dig> ssrs designed from g. raimondii mapped to lg ii.
no ssr designed from g. arboreum mapped to lg ii, and no ssr designed from g. raimondii mapped to lg i.figure  <dig>
genetic linkage groups of chr <dig> and chr <dig> in tetraploid cotton.
linkage groups were developed from  <dig> individuals of a bc <dig> population derived from tm- <dig> and t <dig> using chromosome-specific ssrs.
ac <dig> represents chromosome-specific ssrs designed from chr <dig> of gossypium arboreum .
dc <dig> represents chromosome-specific ssrs designed from chr <dig> of gossypium raimondii .
markers muss <dig> and nau <dig> mapped only to chr <dig>  and nau <dig> mapped only to chr <dig> in the published genetic maps.
the other published markers, including mghes <dig>  bnl <dig>  bnl <dig>  bnl <dig>  jespr <dig>  mapped to one or more other chromosomes, in addition to chr <dig> or chr <dig> in the published genetic maps.
three markers designed from published resources mapped to lg i; among them, nau <dig> and muss <dig> only mapped to chr <dig> in tetraploid cotton in the published maps  <cit> .
similarly, five markers designed from published work mapped to lg ii, including nau <dig>  which only mapped to chr <dig> in tetraploid cotton  <cit> .
thus, lg i was designated as being on chr <dig>  and lg ii on chr <dig>  other markers mapped to more than one chromosome in different genetic maps, usually both chr <dig> and chr16; as an extreme, jespr <dig> was mapped on chr <dig>  chr <dig>  chr <dig>  and chr <dig> in various maps  <cit> .
clearly, chromosome-specific ssrs designed from diploid cotton genomes could be mapped accurately to corresponding chromosomes in tetraploid cotton.
high polymorphism of designed chromosome-specific ssrs between upland and sealand cotton inbred lines
high polymorphism is the most important characteristic for molecular markers, even those designed to be chromosome-specific.
although there were several thousand chromosome-specific ssrs it was important to identify the most polymorphic ones for map construction and gene mapping.
ssr polymorphism is correlated with repeat length, and dinucleotide at-rich repeats are more polymorphic than other kinds of repeats  <cit> .
therefore, the ssrs designed in this study were selected from longer repeats of dinucleotide at-rich chromosome-specific ssrs.
however, the polymorphism rate was almost identical between at-repeat ssrs and public ssr markers.
this is likely due to the near-isogenic relationship of the two parents , which limit the polymorphism of all kinds of markers to a very low level.
to test whether the at-repeat ssrs were more polymorphic than normal ssrs, a further upland cotton variety  and a sealand cotton variety  were employed for genotyping against tm- <dig>  fifty-two percent of the at-repeat ssrs showed polymorphism between tm- <dig> and hai  <dig>  while 40% of the public ssrs showed polymorphism.
a similar situation was observed between the two upland cottons : 10% of at-repeat ssrs showed polymorphism, versus 6% of public ssrs.
therefore, the at-repeat ssrs selected in this study were deemed more polymorphic than normal ssrs.
discussion
frequency differences between g. arboreum and g. raimondii
genome-wide analysis of ssrs provides valuable information for genetic, genomic, and evolutionary studies.
in this study, we analyzed the distributions and frequencies of ssrs with motifs of 1– <dig> bp and a minimum of three repeat units in two diploid cotton species, whose progenitors were putative contributors of the diploid a- and d-subgenomes to tetraploid cotton.
paradoxically, although the genome of g. arboreum was about twice the size of that of g. raimondii, the total ssr number did not differ by two fold; the ssr frequency was much higher in g. raimondii than in g. arboreum .
previous studies have shown that gypsy-like retrotransposon elements have undergone a massive proliferation in larger genomes, like the a-genome, accounting for a major portion of genome-size change  <cit> .
our recent study on the genome of g. arboreum showed that this species did not double its genome size by whole genome duplication but by repetitive dna amplification, mainly via long-terminal-repeat  retrotransposons .
this result indicated that most microsatellites did not reside in ltrs in the ancestral genome of cotton but in regions of low-copy dna.
because the genome of g. arboreum increased rapidly in size by ltr amplification, the replication slippage events that are thought to produce microsatellites  <cit>  may have occurred relatively more slowly, resulting in the apparent paradox.
given the lack of a whole genome duplication events in g. arboreum, the higher frequency of ssrs in g. raimondii was consistent with the previous report that a higher density of microsatellites is associated with species in which the genome expanded less recently  <cit> .
in general, the ssr motifs were strongly biased towards at-rich sequences.
for example, the number of at repeats was  <dig>  fold higher than the second most abundant dinucleotide motif ,  <dig>  fold higher than the third most abundant dinucleotide motif , and  <dig>  fold hogher than cg motif.
at-rich motifs showed striking differences in frequency between g. arboreum and g. raimondii .
for example, the number of at motifs was  <dig> in g. raimondii and  <dig> in g. arboreum, but ag and ac motifs did not show significant differences, e.g., ag had  <dig> and  <dig> copies, respectively.
similarly, the number of aat trinucleotide motifs was  <dig> in g. raimondii and  <dig> in g. arboreum.
however, aag and atc, the second and third most abundant trinucleotides, did not differ significantly in number, e.g., atc had  <dig> and  <dig> copies, respectively.
some motifs were even less frequent in g. arboreum, such as the fourth-most abundant trinucleotide aac .
thus, at-rich motifs were the most abundant in both genomes and they accounted for a major part of the excess of ssrs in g. arboreum.
chromosome-specific ssrs and homologous chromosomes
many ssr markers have been developed from genomic or est sequences in cotton  <cit>  and used successfully to construct genetic maps and for gene mapping.
however, many of them map to at least two positions in tetraploids, mainly because tetraploid cotton  was derived from two diploid species  <cit> , and there are  <dig> pairs of homologous chromosomes in tetraploid cotton.
distinguishing which chromosome of a pair a given linkage group belongs to is difficult.
our study tested the ability of traditional markers to distinguish homologous chromosomes.
we selected  <dig> and  <dig> markers from chr <dig> and chr <dig>  respectively.
blast analysis showed that  <dig> of  <dig> chr <dig> markers could be detected on chr <dig> of g. arboreum, and  <dig> of  <dig> markers could be detected on chr <dig> of g. raimondii;  <dig> of those detectable markers were shared.
eighty of the  <dig> chr <dig> markers could be detected on chr <dig> of g. raimondii, and  <dig> of  <dig> markers could be detected in g. arboreum, with  <dig> markers shared.
thus, most traditional markers were located on both homologous chromosomes in tetraploid cotton, although some only mapped to one chromosome.
to make associations between chromosomes and their genetic linkage groups, wang et al.
<cit>  developed a set of chromosome-specific bac clones to identify all  <dig> chromosomes.
however, fish is time-consuming and labor-intensive, and bac clones used as cytogenetic markers could not be easily used as molecular markers in genetic maps.
this study benefitted from the genome sequence of two diploid cottons, and a set of chromosome-specific ssrs was developed to identify  <dig> chromosomes by comparing ssr flanking sequences.
diploid cottons were used to represent the subgenomes in tetraploid cotton based on the high co-linearity between diploid and tetraploid cottons  <cit> .
we also constructed two linkage groups for two homologous chromosomes in which putative chromosome-specific ssrs were exactly mapped to their homologous chromosomes.
<dig> of  <dig> polymorphic ssrs designed from g. arboreum mapped to chr <dig>  and  <dig> of  <dig> ssrs designed from g. raimondii mapped to chr <dig>  a-subgenomes in tetraploid cotton are reported to have longer genetic distances than d-subgenomes  <cit> ; this may be why more ssrs mapped to chr <dig> than to chr <dig>  nine markers designed in this study did not map to corresponding chromosomes, this is likely a result of errors occurring due to mismatching of chromosomes between diploid and tetraploid cottons.
the use of high-density linkage groups of homologous chromosomes, constructed using chromosome-specific ssrs, would allow more ssrs to be mapped.
the chromosome-specific ssrs were developed using diploid cotton sequences, and although the co-linearity between diploid and tetraploid cottons is obvious  <cit>  many ssrs may have changed chromosome location during polyploidization and subsequent genome evolution.
furthermore, the occurrence of misassembles in the cotton genome, especially the a genome , may have had an impact on our findings.
therefore, a single chromosome-specific ssr cannot represent the entire chromosome.
we suggest that a linkage group containing at least three chromosome-specific ssrs will be indicative of a corresponding chromosome.
high polymorphism of selected ssrs between upland and sealand cottons
high polymorphism is an important character for markers used in high-density genetic map construction and fine mapping of target genes.
there are two possible mechanisms for microsatellite evolution: slippage of the dna polymerase and unequal crossing-over.
the first mechanism, which generate gains or losses of one or a few repeat unit, accounts for most microsatellite mutations  <cit> .
polymerase slippage rates are highest in dinucleotides, followed by tri- and tetranucleotides, as illustrated in human, mouse, fruit fly, and yeast  <cit> .
there are significant positive relationships between repeat length and mutation rate in human  <cit> , fruit fly  <cit> , and yeast  <cit>  microsatellites.
in this study, all chromosome-specific ssrs of each motif were ranked by size.
the longest  <dig> ssrs from chr <dig> of g. arboreum and from chr <dig> of g. raimondii were selected from the dinucleotide at-rich ssrs.
primer pairs were successfully designed to recover high polymorphism.
however, two upland cotton varieties, tm- <dig> and t <dig>  had only 4% polymorphism.
given that ssrs are not highly polymorphic  among or within g. hirsutum cultivars  <cit> , this polymorphism rate was reasonable.
the near-isogenic relationship of tm- <dig> and t <dig> might also limit the polymorphisms recovered by these selected ssrs; t <dig> was derived from tm- <dig> by several generation of backcrossing  <cit> .
several studies have suggested that there is a high frequency of ssr polymorphisms between g. hirsutum and g. barbadense  <cit> .
han et al.
<cit>  detected  <dig> % polymorphism for est–ssrs between these two species, and yu et al.
<cit>  observed  <dig> % polymorphism.
guo et al.
<cit>  and yu et al.
<cit>  detected  <dig> % and  <dig> % polymorphism, respectively, between g. hirsutum and g. barbadense using ssr markers derived from genomic dna.
similarly, yu et al.
<cit>  showed  <dig> % polymorphism between cri  <dig>  and hai <dig> .
the discrepancy in polymorphism rates may be due to different genetic distances between cotton varieties, different kinds of ssrs, or the number of ssrs used.
in this study, there was a 52% polymorphism rate between g. hirsutum and g. barbadense, and a 10% polymorphic rate within g. hirsutum for the selected chromosome-specific ssrs.
however, a 40% polymorphism rate was observed between g. hirsutum and g. barbadense, and a 6% rate within g. hirsutum when using previously published markers.
it seems that the selected ssrs are more polymorphic than normal markers.
therefore, candidate ssrs with the highest polymorphism from this set can be chosen for map construction or gene mapping.
use of chromosome-specific ssr resources for cotton research
chromosome-specific ssrs are efficient tools for chromosome identification, gene location, genetic mapping, qtl tracking, and marker-assisted breeding of cotton.
their greatest advantage is their chromosome specificity derived from the flanking sequences unique to individual chromosomes.
this character is very useful in anchoring linkage groups to chromosomes during genetic mapping and in mapping of qualitative-trait genes and in qtl tracking.
in general, the number of linkage groups in genetic maps is always more than the number of chromosomes in the genomes, and these linkage groups are often assigned to chromosomes based on chromosome-anchored markers by comparison to previous maps  <cit> .
many linkage groups, especially small groups with few markers, are not assigned to chromosomes, because no marker anchors the group to any particular chromosome.
however, genetic maps constructed using chromosome-specific ssrs could solve this problem, because even the smallest linkage groups contain chromosome-specific markers that could be used to assign them to chromosomes.
furthermore, the chromosome-specific ssrs can also be used for gene mapping.
the chromosome-specific ssrs were not clustered in specific regions but distributed evenly across all the chromosomes, so they can be widely used in fingerprinting, genetic diversity analyses, and other applications.
to make the chromosome-specific ssrs more user friendly, they were sorted by motif type and repeat length .
researchers can choose the most polymorphic candidate ssrs according to position and personal preference.
the chromosome-specific ssrs also have shortcomings.
they were developed from diploid species based on high co-linearity between diploid and tetraploid cottons assuming no chromosome rearrangements.
some may not chromosome-specific or may not exist on the expected chromosomes in tetraploid cottons.
however, multiple chromosome-specific ssrs in the same linkage group could solve this problem.
another issue is that the chromosome-specific ssrs are not robust for comparative evolutionary analyses among chromosomes because similar ssrs may not be found on other chromosomes, but they are efficient tools for comparing different genomes because almost no noise could be detected across chromosomes.
finally, recent progress in genome sequencing in many species has rapidly advanced map-based cloning and marker-assisted breeding  <cit> .
however, because of the scarcity of highly-polymorphic, chromosome-specific molecular markers in cotton, no gene has been cloned through a map-based strategy.
the chromosomal loci of many economically-important genes are yet unknown to  <cit> , so marker-assisted breeding in cotton is still underdeveloped relative to some other crops.
in the present study, chromosome-specific ssrs for each chromosome were developed that could distinguish homologous chromosomes in tetraploid cotton.
the candidate ssrs had high polymorphism, and their exact positions were listed.
this very large set of chromosome-specific ssrs, distributed evenly throughout the chromosomes, provides an invaluable resource for cotton genome researches, and it will facilitate the construction of high-resolution maps for positional gene-cloning and marker-assisted breeding.
CONCLUSIONS
the current work characterized microsatellites in cotton and developed chromosome-specific ssrs for map construction and gene mapping.
a total of  <dig>  and  <dig>  ssrs were detected on the  <dig> chromosomes of g. arboreum and g. raimondii, respectively.
pentanucleotides were the most common ssr type in both g. arboreum and g. raimondii.
no major differences were observed between the two species except a higher density of ssrs in g. raimondii.
chromosome-specific ssrs were obtained by comparing ssr sequences from each chromosome with those from the other  <dig> chromosomes.
there was an average of  <dig>  ssrs per chromosome.
we proved that the chromosome-specific ssrs could distinguish homologous chromosomes in genetic linkage maps of tetraploid cotton.
no crossing of markers between the two chromosomes was observed.
in addition, for convenience, all of the ssrs were sorted by motif type and repeat length for each chromosome.
these ssrs will facilitate a number of genetic and genomic studies in cotton, including anchoring linkage groups to genetic maps, positional gene-cloning, fingerprinting, and genetic diversity and comparative evolutionary analyses among gossypium species.

 simple sequence repeats or microsatellites are short ) tandem repeat nucleotides in dna sequences. they are ubiquitous in genomes, being found in both eukaryotes and prokaryotes and in any region. because of their high mutation rates via insertion or deletion of one or a few repeat units, ssrs have been developed into one of the most popular sources of codominant markers with high information content during the past several years. they are widely employed in many research areas, including linkage mapping, population genetics, phylogenetics, and comparative genomics. additionally, recent studies reveal that microsatellites may serve an important role in regulating gene expression, protein function, and genome evolution. the conventional strategies of developing ssr markers are screening genomic dna libraries or constructing ssr-enriched libraries. both are usually time-consuming and labor-intensive. however, the availability of genome sequences allows us to mine for ssrs at the genomic level in silico, and analysis of these ssrs has provided insight into their distributions, putative functions, and evolution. cottons comprise five tetraploid and over diploid species. the diploid species are grouped into eight subgenomes, designated a–g and k. the a-genome is almost twice the size of the d-genome. tetraploid cotton species are thought to have formed through hybridization and subsequent polyploidization between an a-genome and a d-genome species. analyses of genetic maps revealed that diploid a- and d-genomes have a high level of synteny with at- and dt-genomes in tetraploid cotton. the tetraploid g. hirsutum, also known as upland cotton, produces over 90% of the world’s cotton. however, because of the duplicated dna segments and homologous chromosomes in its tetraploid genome, many markers map to at least two positions, most on homologous chromosomes, hindering the precise mapping of qualitative character genes or quantitative trait loci to particular chromosomes. furthermore, given the narrow genetic base, intraspecific genetic diversity in cottons is relatively low, both among and within g. hirsutum cultivars, hindering the development of high-resolution genetic maps and marker-assisted selection breeding in cottons. however, chromosome-specific markers with high polymorphism distributed throughout the genome would facilitate chromosome identification in genetic maps of tetraploid cottons, which are instrumental for applications like positional gene cloning, especially in discriminating homologous chromosomes during fine mapping of single traits and qtls. traditional methods including meiotic fluorescence in situ hybridization and translocation lines can be used to precisely locate dna markers to chromosomes, but most markers are too small to be used directly as probes in fish. bacterial artificial chromosome clones were often used as chromosome-specific markers, but the process is time-consuming and labor-intensive, and only a few bacs have been identified as chromosome-specific. large-scale development of chromosome-specific ssrs can be realized only when each chromosome sequence of the cotton genome is obtained. recently, from the whole-genome sequence of g. raimondii, zou et al. identified microsatellites, and primer pairs were designed, but they are insufficient for mining chromosome-specific ssrs when only one progenitor genome sequence is present. the availability of a very large set of chromosome-specific ssrs distributed throughout the genome would benefit the cotton research and breeding community. the rapid development of high-throughput dna sequencing technologies have allowed increasing numbers of genomes to be sequenced, especially multiple genomes within a genus, expediting the development of chromosome-specific markers and detailed comparative mapping. in recent years, the genomes of g. raimondii and g. arboreum have been sequenced; the progenitors of these two diploid species were putative contributors of the a- and d-subgenomes to tetraploid cotton. these diploid genomes share a high level of synteny or co-linearity with the subgenomes in tetraploid cotton. in spite of the importance of chromosome-specific microsatellite markers to many applications, their systematic and genome-wide characterization in cotton genomes has not yet be conducted. in the present study, we characterized the distribution and density of microsatellites in g. raimondii and g. arboreum chromosomes and identified chromosome-specific ssrs by comparative analysis. in addition, a set of highly-polymorphic ssrs were selected to construct a genetic linkage map of two homologous chromosomes in tetraploid cotton, proving that chromosome-specific ssrs could distinguish homologous chromosomes during genetic mapping.  we analyzed the distributions of ssrs with ≥  repeat units and a minimum total length of bp in each of the chromosomes sequences of g. arboreum and g. raimondii to understand their general features in cotton genomes. ssrs on each chromosome were then compared those on the other chromosomes to find chromosome-specific ssrs. standardized motifs used in sciroko were used to represent all variants on both strands of the dna sequence for consistency in estimating repeat frequencies. unless otherwise specified, ssr content was expressed as ‘number of ssrs per million base pairs’ or as relative frequencies within a particular dataset. using the chromosome-specific ssrs, we constructed two linkage groups for two homologous chromosomes in the tetraploid cotton genome to test whether these chromosome-specific ssrs could distinguish homologous chromosomes. distribution of ssr types in genomes and on individual chromosomes the ssr content of the g. arboreum and g. raimondii genomic sequences are summarized in table  a total of ssrs were detected from g. arboreum chromosome sequences, giving an overall density across the chromosomes of ssrs/mb. ssrs were detected in the g. raimondii chromosome sequences, giving an overall density of ssrs/mb. although the g. arboreum genome is about twice the size of the g. raimondii genome the ssr content did not exhibit a two-fold difference; g. raimondii had a much higher ssr density than g. arboreum. to enable convenient and selective use of the newly developed ssrs, a blast search to determine redundancy was performed against publically available ssrs in the cottongen database; ssrs were recognized as duplicates.table distribution of ssrs with ≥  repeats and a minimum length of bp in genomic sequences of gossypium arboreum and gossypium raimondii sequence type g. arboreum* g. raimondii* count rel. freq. mean repeat number density count rel. freq. mean repeat number density *only ssrs anchored in chromosomes were considered. pentanucleotide repeats were the most common ssr type in both g. arboreum and g. raimondii, representing % and of all ssrs, respectively, followed by dinucleotide repeats. no major differences were observed between g. arboreum and g. raimondii, aside from the higher density of ssrs in g. raimondii. the distribution of ssr types in the two cotton genomes differed from those of cucumber, poplar and grapevine, in which tetranucleotide repeats were the most frequent type, and also from those of rice and arabidopsis, in which trinucleotide repeats were most frequent.figure relative frequency of ssr types in the genomes of gossypium arboreum and gossypium raimondii. ssrs are organized by the number of repeats. the graph was based on a total of and ssrs detected in the genomes of g. arboreum and g. raimondii, respectively. the ssr contents of each chromosome sequence in g. arboreum and g. raimondii are summarized in table  in general, no significant differences were detected among chromosomes within a genome in the distribution of ssrs, except that chr in g. arboreum had a relative low density. for each chromosome, the distribution of motifs was consistent with their genome features, pentanucleotides were the most common ssr type followed by dinucleotides in every chromosome, and no motifs were enriched on particular chromosomes. the variation in ssr density among chromosomes was larger in g. arboreum than in g. raimondii.table distribution of total and chromosome-specific ssrs among chromosome sequences of gossypium arboreum and gossypium raimondii chromosome g. arboreum g. raimondii total ssr no. density chromosome-specific no. rel. freq. total ssr no. density chromosome-specific no. rel. freq. distribution of ssr motifs detailed analyses of individual repeat motifs were carried out for each type of ssr found in g. arboreum and g. raimondii. in general, g. raimondii had a higher density of all repeat types except a motifs, which were similarly and significantly overrepresented in both g. arboreum and g. raimondii genomes. among dinucleotide repeats, the at motif was dramatically overrepresented in both genomes, representing % of the dinucleotide motifs in g. arboreum and % in g. raimondii. at repeats were also the most abundant motif overall in cotton genomes, accounting for % and % of the total ssrs, respectively. cg repeats were the least frequent dinucleotides; only five were found in g. arboreum and in g. raimondii.figure distribution of di- and trinucleotide repeats in the genomes of gossypium arboreum and gossypium raimondii. frequency values are expressed as the number of repeats per million base pairs of sequence. detailed information on frequencies of individual di- and trinucleotide repeat motifs is provided in additional file among the trinucleotides, repeats of aat were most common in both the g. arboreum and g. raimondii genomes. acg was the rarest trinucleotide motif, unlike in most dicot plants in which ccg is least common. among tetranucleotide repeats, the at-rich motifs aaat, aaag, aatt, and atac were, in that order, the most abundant in both genomes, together representing % of all tetramer repeats in g. arboreum and % in g. raimondii. gc-rich repeats, like cccg, acgg and accg, were rarest, with relative frequencies of about %. the most abundant motif aaat had nearly twice the density in g. raimondii than in g. arboreum. as with the dinucleotides and other kinds of motif, the density in g. raimondii was slightly higher than in g. arboreum. among pentanucleotide repeats, the at-rich motifs aaaat, aaaag, and aaatt were most frequent in both genomes, together accounting for % of all pentanucleotides in g. arboreum and % in g. raimondii. aaaat was the most abundant, outnumbering the next most frequent repeats, aaaag or aaatt, by about three fold. surprisingly, although gc-rich repeats like ccgcg, ccccc and accgt were the rarest, cccgg motif was relatively abundant in both genomes. among hexanucleotides, at-rich motifs also predominated in both genomes; aaaaat was the most common hexanucleotide repeat in both genomes, but the next most frequent was aatcag in g. arboreum and aaaaag in g. raimondii. in general, at-rich motifs were most common, especially a-rich ones. g. raimondii had higher density of all repeat types than g. arboreum except the a motif, while g. arboreum had more repeats due to larger genome. for the same ssr type, the genomes tended to accumulate motifs containing more a in tandem repeat. for example, aaatt was more abundant than aatat in the pentanucleotides, and aaaatt was more abundant than aaatat in the hexanucleotides. development of chromosome-specific ssrs we first examined ssr repetition within each chromosome to detect whether a small number of ssr types dominated, given the high repeat content in the cotton genome. each ssr sequence, including the repeat and both flanking sequences, were compared with all other ssr sequences on the same chromosome. the average proportion of unissrs was % on g. arboreum chromosomes and % on g. raimondii chromosomes. thus, only 20–30% ssrs were repetitive within chromosomes, which is conducive to obtaining chromosome-specific ssrs. for single repetitive ssrs, g. arboreum had an average of repetitions and g. raimondii and average of. the average maximum number of repetitions was in g. arboreum and only in g. raimondii, so the former had a higher degree of repetition than the latter. ssr sequences from each chromosome were then compared with those on the other chromosomes; similar ssrs were then excluded to identify chromosome-specific ssrs for each chromosome. the genome of g. arboreum had more chromosome-specific ssrs than did g. raimondii. the average proportion of chromosome-specific ssrs was higher in g. raimondii than in g. arboreum. as the ssrs were designed using diploid cotton genome sequences, it is important to check the percentage of ssrs that could be successfully amplified in tetraploid cottons. of successfully designed ssrs, could be amplified in tm- hai and liaomian these chromosome-specific ssrs are therefore considered suitable for amplification in tetraploid cotton. chromosome-specific ssrs could distinguish homologous chromosomes in tetraploid cotton to determine whether the chromosome-specific ssrs could distinguish homologous chromosomes in other tetraploid cotton species, two homologous linkage groups were constructed using ssrs from chr of g. arboreum and ssrs from chr of g. raimondii. a total of and primer pairs were designed from g. arboreum and g. raimondii, respectively. a bc population of individuals was constructed by crossing tm- and t. and polymorphic markers between the parents were obtained from the ssrs designed from chr of g. arboreum, from chr of g. raimondii, and from published genetic maps, respectively. all ssrs were included in the linkage analysis. two linkage groups, containing markers, were constructed: markers in linkage group i, and in lg ii. the other loci were not part of any linkage group. of the polymorphic ssrs from g. arboreum mapped to lg i, and of the ssrs designed from g. raimondii mapped to lg ii. no ssr designed from g. arboreum mapped to lg ii, and no ssr designed from g. raimondii mapped to lg i.figure genetic linkage groups of chr and chr in tetraploid cotton. linkage groups were developed from individuals of a bc population derived from tm- and t using chromosome-specific ssrs. ac represents chromosome-specific ssrs designed from chr of gossypium arboreum. dc represents chromosome-specific ssrs designed from chr of gossypium raimondii. markers muss and nau mapped only to chr and nau mapped only to chr in the published genetic maps. the other published markers, including mghes bnl bnl bnl jespr mapped to one or more other chromosomes, in addition to chr or chr in the published genetic maps. three markers designed from published resources mapped to lg i; among them, nau and muss only mapped to chr in tetraploid cotton in the published maps. similarly, five markers designed from published work mapped to lg ii, including nau which only mapped to chr in tetraploid cotton. thus, lg i was designated as being on chr and lg ii on chr other markers mapped to more than one chromosome in different genetic maps, usually both chr and chr16; as an extreme, jespr was mapped on chr chr chr and chr in various maps. clearly, chromosome-specific ssrs designed from diploid cotton genomes could be mapped accurately to corresponding chromosomes in tetraploid cotton. high polymorphism of designed chromosome-specific ssrs between upland and sealand cotton inbred lines high polymorphism is the most important characteristic for molecular markers, even those designed to be chromosome-specific. although there were several thousand chromosome-specific ssrs it was important to identify the most polymorphic ones for map construction and gene mapping. ssr polymorphism is correlated with repeat length, and dinucleotide at-rich repeats are more polymorphic than other kinds of repeats. therefore, the ssrs designed in this study were selected from longer repeats of dinucleotide at-rich chromosome-specific ssrs. however, the polymorphism rate was almost identical between at-repeat ssrs and public ssr markers. this is likely due to the near-isogenic relationship of the two parents, which limit the polymorphism of all kinds of markers to a very low level. to test whether the at-repeat ssrs were more polymorphic than normal ssrs, a further upland cotton variety and a sealand cotton variety were employed for genotyping against tm- fifty-two percent of the at-repeat ssrs showed polymorphism between tm- and hai while 40% of the public ssrs showed polymorphism. a similar situation was observed between the two upland cottons: 10% of at-repeat ssrs showed polymorphism, versus 6% of public ssrs. therefore, the at-repeat ssrs selected in this study were deemed more polymorphic than normal ssrs. discussion frequency differences between g. arboreum and g. raimondii genome-wide analysis of ssrs provides valuable information for genetic, genomic, and evolutionary studies. in this study, we analyzed the distributions and frequencies of ssrs with motifs of 1– bp and a minimum of three repeat units in two diploid cotton species, whose progenitors were putative contributors of the diploid a- and d-subgenomes to tetraploid cotton. paradoxically, although the genome of g. arboreum was about twice the size of that of g. raimondii, the total ssr number did not differ by two fold; the ssr frequency was much higher in g. raimondii than in g. arboreum. previous studies have shown that gypsy-like retrotransposon elements have undergone a massive proliferation in larger genomes, like the a-genome, accounting for a major portion of genome-size change. our recent study on the genome of g. arboreum showed that this species did not double its genome size by whole genome duplication but by repetitive dna amplification, mainly via long-terminal-repeat retrotransposons. this result indicated that most microsatellites did not reside in ltrs in the ancestral genome of cotton but in regions of low-copy dna. because the genome of g. arboreum increased rapidly in size by ltr amplification, the replication slippage events that are thought to produce microsatellites may have occurred relatively more slowly, resulting in the apparent paradox. given the lack of a whole genome duplication events in g. arboreum, the higher frequency of ssrs in g. raimondii was consistent with the previous report that a higher density of microsatellites is associated with species in which the genome expanded less recently. in general, the ssr motifs were strongly biased towards at-rich sequences. for example, the number of at repeats was fold higher than the second most abundant dinucleotide motif, fold higher than the third most abundant dinucleotide motif, and fold hogher than cg motif. at-rich motifs showed striking differences in frequency between g. arboreum and g. raimondii. for example, the number of at motifs was in g. raimondii and in g. arboreum, but ag and ac motifs did not show significant differences, e.g., ag had and copies, respectively. similarly, the number of aat trinucleotide motifs was in g. raimondii and in g. arboreum. however, aag and atc, the second and third most abundant trinucleotides, did not differ significantly in number, e.g., atc had and copies, respectively. some motifs were even less frequent in g. arboreum, such as the fourth-most abundant trinucleotide aac. thus, at-rich motifs were the most abundant in both genomes and they accounted for a major part of the excess of ssrs in g. arboreum. chromosome-specific ssrs and homologous chromosomes many ssr markers have been developed from genomic or est sequences in cotton and used successfully to construct genetic maps and for gene mapping. however, many of them map to at least two positions in tetraploids, mainly because tetraploid cotton was derived from two diploid species, and there are pairs of homologous chromosomes in tetraploid cotton. distinguishing which chromosome of a pair a given linkage group belongs to is difficult. our study tested the ability of traditional markers to distinguish homologous chromosomes. we selected and markers from chr and chr respectively. blast analysis showed that of chr markers could be detected on chr of g. arboreum, and of markers could be detected on chr of g. raimondii; of those detectable markers were shared. eighty of the chr markers could be detected on chr of g. raimondii, and of markers could be detected in g. arboreum, with markers shared. thus, most traditional markers were located on both homologous chromosomes in tetraploid cotton, although some only mapped to one chromosome. to make associations between chromosomes and their genetic linkage groups, wang et al. developed a set of chromosome-specific bac clones to identify all chromosomes. however, fish is time-consuming and labor-intensive, and bac clones used as cytogenetic markers could not be easily used as molecular markers in genetic maps. this study benefitted from the genome sequence of two diploid cottons, and a set of chromosome-specific ssrs was developed to identify chromosomes by comparing ssr flanking sequences. diploid cottons were used to represent the subgenomes in tetraploid cotton based on the high co-linearity between diploid and tetraploid cottons. we also constructed two linkage groups for two homologous chromosomes in which putative chromosome-specific ssrs were exactly mapped to their homologous chromosomes. of polymorphic ssrs designed from g. arboreum mapped to chr and of ssrs designed from g. raimondii mapped to chr a-subgenomes in tetraploid cotton are reported to have longer genetic distances than d-subgenomes; this may be why more ssrs mapped to chr than to chr nine markers designed in this study did not map to corresponding chromosomes, this is likely a result of errors occurring due to mismatching of chromosomes between diploid and tetraploid cottons. the use of high-density linkage groups of homologous chromosomes, constructed using chromosome-specific ssrs, would allow more ssrs to be mapped. the chromosome-specific ssrs were developed using diploid cotton sequences, and although the co-linearity between diploid and tetraploid cottons is obvious many ssrs may have changed chromosome location during polyploidization and subsequent genome evolution. furthermore, the occurrence of misassembles in the cotton genome, especially the a genome, may have had an impact on our findings. therefore, a single chromosome-specific ssr cannot represent the entire chromosome. we suggest that a linkage group containing at least three chromosome-specific ssrs will be indicative of a corresponding chromosome. high polymorphism of selected ssrs between upland and sealand cottons high polymorphism is an important character for markers used in high-density genetic map construction and fine mapping of target genes. there are two possible mechanisms for microsatellite evolution: slippage of the dna polymerase and unequal crossing-over. the first mechanism, which generate gains or losses of one or a few repeat unit, accounts for most microsatellite mutations. polymerase slippage rates are highest in dinucleotides, followed by tri- and tetranucleotides, as illustrated in human, mouse, fruit fly, and yeast. there are significant positive relationships between repeat length and mutation rate in human, fruit fly, and yeast microsatellites. in this study, all chromosome-specific ssrs of each motif were ranked by size. the longest ssrs from chr of g. arboreum and from chr of g. raimondii were selected from the dinucleotide at-rich ssrs. primer pairs were successfully designed to recover high polymorphism. however, two upland cotton varieties, tm- and t had only 4% polymorphism. given that ssrs are not highly polymorphic among or within g. hirsutum cultivars, this polymorphism rate was reasonable. the near-isogenic relationship of tm- and t might also limit the polymorphisms recovered by these selected ssrs; t was derived from tm- by several generation of backcrossing. several studies have suggested that there is a high frequency of ssr polymorphisms between g. hirsutum and g. barbadense. han et al. detected % polymorphism for est–ssrs between these two species, and yu et al. observed % polymorphism. guo et al. and yu et al. detected % and % polymorphism, respectively, between g. hirsutum and g. barbadense using ssr markers derived from genomic dna. similarly, yu et al. showed % polymorphism between cri and hai. the discrepancy in polymorphism rates may be due to different genetic distances between cotton varieties, different kinds of ssrs, or the number of ssrs used. in this study, there was a 52% polymorphism rate between g. hirsutum and g. barbadense, and a 10% polymorphic rate within g. hirsutum for the selected chromosome-specific ssrs. however, a 40% polymorphism rate was observed between g. hirsutum and g. barbadense, and a 6% rate within g. hirsutum when using previously published markers. it seems that the selected ssrs are more polymorphic than normal markers. therefore, candidate ssrs with the highest polymorphism from this set can be chosen for map construction or gene mapping. use of chromosome-specific ssr resources for cotton research chromosome-specific ssrs are efficient tools for chromosome identification, gene location, genetic mapping, qtl tracking, and marker-assisted breeding of cotton. their greatest advantage is their chromosome specificity derived from the flanking sequences unique to individual chromosomes. this character is very useful in anchoring linkage groups to chromosomes during genetic mapping and in mapping of qualitative-trait genes and in qtl tracking. in general, the number of linkage groups in genetic maps is always more than the number of chromosomes in the genomes, and these linkage groups are often assigned to chromosomes based on chromosome-anchored markers by comparison to previous maps. many linkage groups, especially small groups with few markers, are not assigned to chromosomes, because no marker anchors the group to any particular chromosome. however, genetic maps constructed using chromosome-specific ssrs could solve this problem, because even the smallest linkage groups contain chromosome-specific markers that could be used to assign them to chromosomes. furthermore, the chromosome-specific ssrs can also be used for gene mapping. the chromosome-specific ssrs were not clustered in specific regions but distributed evenly across all the chromosomes, so they can be widely used in fingerprinting, genetic diversity analyses, and other applications. to make the chromosome-specific ssrs more user friendly, they were sorted by motif type and repeat length. researchers can choose the most polymorphic candidate ssrs according to position and personal preference. the chromosome-specific ssrs also have shortcomings. they were developed from diploid species based on high co-linearity between diploid and tetraploid cottons assuming no chromosome rearrangements. some may not chromosome-specific or may not exist on the expected chromosomes in tetraploid cottons. however, multiple chromosome-specific ssrs in the same linkage group could solve this problem. another issue is that the chromosome-specific ssrs are not robust for comparative evolutionary analyses among chromosomes because similar ssrs may not be found on other chromosomes, but they are efficient tools for comparing different genomes because almost no noise could be detected across chromosomes. finally, recent progress in genome sequencing in many species has rapidly advanced map-based cloning and marker-assisted breeding. however, because of the scarcity of highly-polymorphic, chromosome-specific molecular markers in cotton, no gene has been cloned through a map-based strategy. the chromosomal loci of many economically-important genes are yet unknown to, so marker-assisted breeding in cotton is still underdeveloped relative to some other crops. in the present study, chromosome-specific ssrs for each chromosome were developed that could distinguish homologous chromosomes in tetraploid cotton. the candidate ssrs had high polymorphism, and their exact positions were listed. this very large set of chromosome-specific ssrs, distributed evenly throughout the chromosomes, provides an invaluable resource for cotton genome researches, and it will facilitate the construction of high-resolution maps for positional gene-cloning and marker-assisted breeding.  the current work characterized microsatellites in cotton and developed chromosome-specific ssrs for map construction and gene mapping. a total of and ssrs were detected on the chromosomes of g. arboreum and g. raimondii, respectively. pentanucleotides were the most common ssr type in both g. arboreum and g. raimondii. no major differences were observed between the two species except a higher density of ssrs in g. raimondii. chromosome-specific ssrs were obtained by comparing ssr sequences from each chromosome with those from the other chromosomes. there was an average of ssrs per chromosome. we proved that the chromosome-specific ssrs could distinguish homologous chromosomes in genetic linkage maps of tetraploid cotton. no crossing of markers between the two chromosomes was observed. in addition, for convenience, all of the ssrs were sorted by motif type and repeat length for each chromosome. these ssrs will facilitate a number of genetic and genomic studies in cotton, including anchoring linkage groups to genetic maps, positional gene-cloning, fingerprinting, and genetic diversity and comparative evolutionary analyses among gossypium species. 
recently, the genomes of two diploid species were sequenced whose progenitors were putative contributors of the at- and dt-subgenomes to tetraploid cotton. the ssrs reported here will facilitate a number of genetic and genomic studies in cotton, including construction of high-density genetic maps, positional gene cloning, fingerprinting, and genetic diversity and comparative evolutionary analyses among gossypium species. to confirm their chromosome specificity, these ssrs were used to distinguish two homologous chromosomes in tetraploid cotton through linkage group construction. consequently, many markers in cotton were mapped to multiple positions during linkage genetic map construction, posing a challenge to anchoring linkage groups and mapping economically-important genes to particular chromosomes. chromosome-specific ssrs are efficient tools for chromosome identification by anchoring linkage groups to particular chromosomes during genetic mapping and are especially useful in mapping of qualitative-trait genes or quantitative trait loci with just a few markers. in this study, simple sequence repeats on each chromosome in the two diploid genomes were characterized. because longer dinucleotide at-rich repeats were the most polymorphic in previous reports, the ssrs on each chromosome were sorted by motif type and repeat length for convenient selection. chromosome-specific ssrs were developed by comparative analysis and proved to distinguish chromosomes. chromosome-specific ssrs were obtained by comparing ssr flanking sequences from each chromosome with those from the other chromosomes. the chromosome-specific ssrs and previously-reported chromosome markers were grouped together, and no marker mapped to another homologous chromosome, proving that the chromosome-specific ssrs were unique and could distinguish homologous chromosomes in tetraploid cotton. the primer sequences of all chromosome-specific ssrs were also made publicly available. these sequences provide a powerful tool for developing chromosome-specific markers given the high level of synteny among tetraploid and diploid cotton genomes. chromosome-specific markers could solve this problem. tetraploid cotton contains two sets of homologous chromosomes, the at- and dt-subgenomes.
 tetraploid cotton contains two sets of homologous chromosomes, the at- and dt-subgenomes. consequently, many markers in cotton were mapped to multiple positions during linkage genetic map construction, posing a challenge to anchoring linkage groups and mapping economically-important genes to particular chromosomes. chromosome-specific markers could solve this problem. recently, the genomes of two diploid species were sequenced whose progenitors were putative contributors of the at- and dt-subgenomes to tetraploid cotton. these sequences provide a powerful tool for developing chromosome-specific markers given the high level of synteny among tetraploid and diploid cotton genomes. in this study, simple sequence repeats on each chromosome in the two diploid genomes were characterized. chromosome-specific ssrs were developed by comparative analysis and proved to distinguish chromosomes.  a total of and ssrs were detected on the chromosomes of gossypium arboreum l. and gossypium raimondii ulbrich, respectively. chromosome-specific ssrs were obtained by comparing ssr flanking sequences from each chromosome with those from the other chromosomes. the average was per chromosome. to confirm their chromosome specificity, these ssrs were used to distinguish two homologous chromosomes in tetraploid cotton through linkage group construction. the chromosome-specific ssrs and previously-reported chromosome markers were grouped together, and no marker mapped to another homologous chromosome, proving that the chromosome-specific ssrs were unique and could distinguish homologous chromosomes in tetraploid cotton. because longer dinucleotide at-rich repeats were the most polymorphic in previous reports, the ssrs on each chromosome were sorted by motif type and repeat length for convenient selection. the primer sequences of all chromosome-specific ssrs were also made publicly available.  chromosome-specific ssrs are efficient tools for chromosome identification by anchoring linkage groups to particular chromosomes during genetic mapping and are especially useful in mapping of qualitative-trait genes or quantitative trait loci with just a few markers. the ssrs reported here will facilitate a number of genetic and genomic studies in cotton, including construction of high-density genetic maps, positional gene cloning, fingerprinting, and genetic diversity and comparative evolutionary analyses among gossypium species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords chromosome-specificssrtetraploid cottongenome-wideissue-copyright-statement© the author 2015 