BACKGROUND
genetic linkage maps are valuable resources that provide a framework for many genomic analyses.
they are an important tool for many genetic applications, including mapping of quantitative trait loci , comparative mapping, identifying dna markers for fingerprinting, analyses of population genetics and phylogenetics, genome sequence assembly, relating physical and recombination distances along the genome, and map-based cloning of genes.
the wide applications of linkage maps and their importance in genetic research has led to numerous linkage mapping projects being undertaken in plants  <cit> .
carnation , in the caryophyllaceae, is one of the major floricultural crops in japan and around the world.
hundreds of cultivars are grown around the world and many new varieties are bred and registered every year  <cit> .
more than  <dig> dianthus species have been recorded  <cit> .
many dianthus species are distributed throughout europe and asia, and the distribution of the genus extends from arctic north america to mountainous sites in africa  <cit> .
high-quality commercial carnation cultivars are usually obtained by inter- or intraspecific hybridization  <cit> .
most of the carnation cultivars are diploid with a chromosome number of 2n = 2x =  <dig>  <cit> .
the reported nuclear dna content of carnation is  <dig> – <dig>  pg/2c  <cit> .
therefore, carnation has a comparatively small nuclear genome  approximately four times the size of the arabidopsis thaliana genome   <cit> .
according to the plant c-values database , the genome of carnation is very small compared with those of other ornamental flowers such as rosa hybrida , antirrhinum majus , petunia hybrida , chrysanthemum morifolium , and tulipa gesneriana .
to improve the selection efficiency in breeding plants with resistance to carnation bacterial wilt  using d. capitatus ssp.
andrezejowskianus, we constructed the first genetic linkage map for carnation, which comprised  <dig> random amplified polymorphic dna  and nine simple sequence repeat  loci within  <dig> linkage groups   <cit>  .
a qtl analysis identified one major qtl  and two minor qtls  for cbw resistance derived from d. capitatus ssp.
andrezejowskianus.
we also identified a new qtl for cbw resistance , derived from carnation line 85– <dig>  using an ssr-based genetic linkage map .
the “b’ map comprised  <dig> ssr loci, covering  <dig>  cm with an average distance of  <dig>  cm between loci, and  <dig> lgs.
this was the first report of a genetic linkage map based mainly on ssr markers for the genus dianthus.
the ssr markers and the genetic linkage maps developed for carnation in our previous studies would be useful for comparisons between mapping populations, for qtl mapping of important agronomic characteristics, and for phylogenetic analyses.
however, the number of markers was insufficient for these purposes.
also, the small number of markers had resulted in  <dig> lgs being identified on the maps, instead of  <dig> .
we conducted rna-seq analysis and obtained  <dig>  unigenes consisting of  <dig>  contigs and  <dig>  singletons using next-generation sequencing  technology  <cit> .
we identified  <dig>  potential ssrs in  <dig>  of the unigenes.
finally,  <dig>  ssr primer pairs were designed  <cit> .
in this study, we aimed to construct a high-density ssr-based genetic linkage map to serve as a reference for members of dianthus.
we evaluated polymorphisms in the mapping population  using ssr primer pairs designed from data from the rna-seq analysis, the genomic ssr library, and sanger-sequencing expressed sequence tag  analysis  <cit> .
we refined the “a” map by adding the new rapd and ssr loci.
comparative analysis of the refined “a” and “b” maps revealed corresponding lgs between the two linkage maps.
also, we analyzed previously reported genotype and phenotype data to map two qtls  and a flower-type locus onto the refined maps.
RESULTS
genotyping of the 85p population
the name of the mapping population was changed from “b” to “85p” in this study.
screening of  <dig>  ssr primer pairs  derived from the ssr-enriched genomic libraries  <cit>  revealed  <dig> new “gs” ssr markers that were polymorphic in 85p progenies .
therefore, a total of  <dig> “gs” ssr markers, including  <dig> reported previously  <cit> , were polymorphic in the 85p population.
we reanalyzed  <dig>  “ces” primer pairs derived from est analysis  <cit>  using fluorescent fragment analysis instead of electrophoresis on polyacrylamide gels.
this analysis revealed  <dig> new polymorphic “ces” markers .
therefore, a total of  <dig> “ces” ssr markers including  <dig> markers reported previously  <cit>  were polymorphic in the 85p population.
tanase et al.
<cit>  designed  <dig>  ssr primer pairs from  <dig>  potential ssr repeats by rna-seq analysis.
the names of these ssr markers begin with the letters “nes”.
of  <dig> “nes” ssr primers that included di- and tri- nucleotide ssr motifs,  <dig> produced amplicons that were not polymorphic between parents,  <dig> produced no amplicon, weak amplicons, or multi-locus amplified products, and  <dig> produced amplicons that were polymorphic .
additional file  <dig> shows the newly developed ssr markers for the 85p map.
we identified  <dig> ssr markers that showed single-locus polymorphisms in our mapping population, including  <dig> ssr markers mapped in our previous report  <cit> , seven markers  derived from first genomic ssr libraries  <cit> , two markers  developed by smulders et al.
<cit> , and dcest <dig> derived from publicly available ests.
of these,  <dig> were genomic ssr markers and the other  <dig> were est-ssr markers.
refined map construction for 85p population
linkage analysis using a minimum lod score of  <dig>  revealed  <dig> lgs .
this result indicated the existence of several minor lgs consisting of a small number of loci within less than  <dig> cm.
to clarify the relationships among the minor lgs, we transferred all ssr loci in the five minor lgs  to the “np” map updated from the “a” map described in yagi et al.
<cit>  by adding rapd and ssr loci .
thirteen ssr loci out of  <dig> loci on lg 85p_ <dig>  were mapped to lower regions of lg np_ <dig> .
six ssr loci out of seven loci on lg 85p_14- <dig>  and five out of seven loci on lg 85p_14- <dig>  were mapped to lg np_ <dig> .
eleven out of  <dig> loci on lg 85p_15- <dig>  and two out of six loci on lg 85p_15- <dig>  mapped to lg np_ <dig> .
the positions of all ssr loci mapped to both lgs were conserved between lgs.
these results indicated that four of the five minor lgs could be integrated into two lgs.
consequently, the number of lgs corresponded to the basic chromosomal number in carnation .
the lg names and their order on the 85p map follow those used in a previous report  <cit>  .
the total length of the 85p map was  <dig>  cm, with an average distance of  <dig>  cm between loci.
the lgs ranged in size from  <dig>  cm  to  <dig>  cm , with an average length of  <dig>  cm.
the number of loci mapped per lg ranged from  <dig>  to  <dig> , with an average of  <dig> .
the marker density  ranged from  <dig>   to  <dig>  , with an average of  <dig> .
the entire marker density was  <dig> .
two published but previously unmapped loci, ces <dig> and ces <dig>  were mapped onto lg 85p_15- <dig> in this study .
of the  <dig> ssr loci,  <dig> showed segregation patterns that fitted the expected segregation ratio of 1:2: <dig>  while the segregations of  <dig> loci  and  <dig> locus  differed from the expected ratio.
most of the loci showing different segregation ratios were located on lg 85p_  <dig> ; the others were on lgs 85p_  <dig>   <dig>   <dig>  and  <dig>
comparison of lgs between two maps using ssr markers
to reveal the relationships for other lgs between the 85p and np maps, more than five ssr loci in each lg of the 85p map were tested in the refined np map.
this analysis revealed that at least three loci were connected to the homologous lg .
the 85p and np maps had  <dig> loci in common.
the map position of each ssr locus showed good collinearity between homologous lgs.
we identified  <dig> loci in carnation using the two linkage maps.
refined map construction for np population
the np map was significantly improved from our previous report  <cit> .
we constructed the np map using  <dig> lines derived from a cross between ‘carnation nou no.
1’ and ‘pretty favvare’.
linkage analysis using a minimum lod score of  <dig>  revealed  <dig> lgs consisting of  <dig> loci covering  <dig>  cm .
the lg names on the np map were rearranged to be consistent with homologous lgs on the 85p map.
we mapped  <dig> new ssr markers that showed single-locus polymorphisms onto the np map.
of those,  <dig> ssr loci were derived from the genomic sequence;  <dig> were “gs” ssr loci derived from the ssr-enriched genomic library developed by yagi et al.
<cit> ,  <dig> were “cb” or “cf” loci derived from the ssr-enriched genomic library developed by kimura et al.
<cit> , and dcd <dig> on lg np_ <dig> was developed by smulders et al.
<cit> .
the other  <dig> ssr loci were derived from est information; ms-dcdia <dig> on lg np_ <dig> and ms-dinmadsbox on lg np_ <dig> were developed by smulders et al.
<cit> ,  <dig> were “ces” ssr loci derived from sanger-based est analysis  <cit> , four were “dcest” ssr loci derived from public est information, and  <dig> were “nes” ssr loci derived from rna-seq analysis using ngs technology.
these newly mapped ssr loci for the np map are shown in additional file  <dig>
eight new rapd markers  were mapped onto lgs np_ <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> .
we tested  <dig> ethylene biosynthesis and response pathway gene-specific sequence-tagged site  primers designed by de benedetti et al.
<cit> .
among these  <dig> sts markers, one derived from dcers was mapped onto lg np_ <dig> and one derived from eil <dig>  was mapped onto lg np_ <dig>  both of these genes encode putative ethylene receptors.
in total,  <dig> ssr,  <dig> rapd, and two sts loci were mapped onto the np map.
the total length of the map was  <dig>  cm, with an average distance of  <dig>  cm between loci .
linkage groups ranged in size from  <dig>  cm  to  <dig>  cm , with an average length of  <dig>  cm.
the number of loci mapped per lg ranged from  <dig>  to  <dig> , with an average of  <dig> .
the marker density  ranged from  <dig>   to  <dig>  , with an average of  <dig> .
the entire marker density was  <dig> .
of the  <dig> loci, the segregation of  <dig> loci fitted the expected 1: <dig> ratio, while the segregations of  <dig>  and  <dig>  loci deviated from the expected ratio .
most of the deviated loci were located on lg np_  <dig>   <dig>  and  <dig>
mapping of qtls and a flower-type locus
we mapped qtls onto the two maps by analyzing data reported previously  <cit> .
the mapped qtls are shown in figure  <dig> and listed in table  <dig>  a major qtl for cbw resistance derived from line 85– <dig>  was mapped onto lg 85p_ <dig>  there were no other qtls with an lod score above  <dig> , which was the minimum lod score after  <dig> permutation tests  <cit> .
a new marker linked to cbw <dig>  ces <dig>  was identified in this analysis, in addition to the cbw4-linked markers ces <dig> and ces <dig> identified previously  <cit> .
for the np map, qtls for cbw resistance derived from d. capitatus ssp.
andrezejowskianus were mapped in our previous study  <cit> .
cbw <dig> and cbw <dig> were mapped onto lg np_ <dig>  and lg np_ <dig> , respectively.
the nearest marker to cbw <dig> was wg <dig>  the same result obtained previously  <cit> .
there were no other significant qtls with an lod score above  <dig> , which was the minimum lod score after  <dig> permutation tests, hence, cbw <dig>  which had a low lod score of  <dig>  in our previous study  <cit> , was not detected in this study.
flower color is an important trait in ornamentals.
therefore, we identified qtls related to the anthocyanin content in petals  in our previous study  <cit> .
cap <dig>  and cap <dig>  were mapped onto lg np_ <dig> and lg np_ <dig>  respectively.
we also mapped a phenotypic locus for flower-type.
we identified a single flower-type locus in a previous study  <cit> .
the single flower-type locus derived from d. capitatus ssp.
andrezejowskianus was mapped onto lg np_ <dig>  in addition to the four co-segregating rapd markers identified previously, four ssr markers  were identified in this study.
*thresholds were determined by  <dig>  permutation tests using winqtl cartographer software.
discussion
high-density linkage maps are useful for locating genes of interest for marker-assisted selection and to identify qtls.
in this study, we revised previously constructed linkage maps to add new markers and increase their resolution.
in total,  <dig> single-locus ssr markers are mapped,  <dig> of which are new markers.
we have mapped a large number of ssr markers in the linkage map of carnation, which is not a model crop and had few available ssrs.
recently, ngs has been used to develop several types of genetic markers such as restriction site associated dna  and single nucleotide polymorphism  markers, including ssr markers  <cit> .
one full ngs shotgun run can yield thousands of genome-wide ssr markers.
we obtained  <dig> , <dig> high-quality cdna reads by  <dig> pyrosequencing technology and finally designed  <dig>  ssr primer pairs  <cit> .
we mapped  <dig> single-locus ssr markers  of  <dig> tested ssr primer sets obtained from ngs analysis.
the success ratio was similar to that obtained using “ces” markers derived from sanger-sequencing est analysis in this mapping population .
ngs technologies allow the efficient identification of large numbers of ssr loci at a fraction of the cost and effort of traditional ssr isolation approaches  <cit> .
therefore, these technologies will increase the amount of genome information and improve the quality of genomic resources for many plant species, especially ornamental species that tend to have complex genome structures   <cit> .
the ratio of distorted loci was larger in the np map  than in the 85p map .
most of the distorted loci were located on lg 85p_  <dig>  on the 85p map, but the corresponding lg on the np map, np_ <dig>  had no distorted loci .
for the np map, most of the loci located on np_ <dig>   <dig>   <dig> were distorted.
thus, the regions of distortion differed between the two linkage maps.
distorted segregations in linkage maps have been reported for other ornamentals, including rhododendron <cit> , diploid rose  <cit> , asiatic hybrid lily  <cit> , and japanese gentian  <cit> .
the causes of segregation distortion have been suggested to include aneuploidy, chromosomal translocation, competition among gametes, and the inheritance of alleles affecting the viability of the zygote, embryo, or seedling  <cit> .
‘pretty favvare’ is the common mapping parent for both maps, therefore, discordance between normal carnation and ‘carnation nou.
no.1’, which is derived from an interspecific cross, may account for the higher ratio of distorted loci on the np map.
linkage analysis for the 85p map revealed  <dig> lgs.
comparative analysis between lgs using ssr loci in the updated np map revealed that four minor lgs could be integrated into two lgs.
for the np map, the addition of a large number of ssr markers converged the map to  <dig> lgs.
homologous lgs from different populations were identified using the same anchored ssr loci.
these results showed that the present lgs would correspond to the basic chromosome number in carnation .
even though  <dig> ssr loci were added to the 85p map, the total map length was only  <dig>  cm longer than that of the previously constructed map  and many markers were mapped to the same locus .
this might be because of low recombination frequencies in this mapping population, or homozygous chromosomal regions between parental lines.
in a linkage map for pear, the short coverage of lgs revealed that large regions of lgs were homozygous  <cit> .
thus, these findings might suggest a limitation for constructing a saturated linkage map using this population.
we analyzed previously reported genotype and phenotype data to map qtls for cbw and flower color pigmentation, and the flower-type locus, onto the refined maps.
this allowed us to identify some new markers linked to those traits.
as well as rapd markers, co-dominant ssr markers linked to the flower-type locus would be useful, although further analyses are needed to assess their effectiveness as selection markers.
cbw <dig> , was not detected in this study.
this was probably because of the change in analytical method, i.e., from interval mapping using mapmaker software  <cit>  to composite interval mapping  using winqtl cartographer  <cit> .
moreover, the expansion and rearrangement of the map would also affect the ability to detect this locus.
our revised maps described in this paper will allow more precise qtl analysis, including identification of loci associated with various attributes such as flower vase life, an important trait for ornamental plants that has not yet been mapped in the mapping population.
we identified a total of  <dig> loci including  <dig> ssr in carnation using the two linkage maps.
we believe that these linkage maps will serve as reference genetic linkage maps for carnation and that many of the mapped ssr markers will be useful to compare these maps with others constructed using data from different mapping populations.
currently, we are producing another mapping population and will construct a new linkage map.
moreover, whole genome sequencing by the ngs system is underway.
CONCLUSIONS
we improved two carnation linkage maps and mapped a large number of ssr loci.
the genetic linkage map derived from a cross between the cbw-resistant line 85– <dig> and susceptible ‘pretty favvare’ comprising  <dig> ssr loci covering  <dig>  cm, with an average distance of  <dig>  cm between loci.
the genetic linkage map derived from a cross between cbw-resistant ‘carnation nou no.1’ and susceptible ‘pretty favvare’ comprised  <dig> ssr,  <dig> rapd, and two sts loci covering  <dig>  cm, with an average  <dig>  cm distance between loci.
these two refined maps were anchored with  <dig> ssrs loci.
we identified  <dig> loci in carnation using the two linkage maps.
the number of lgs was coincident with the haploid chromosome number of carnation .
our genetic linkage maps, combined with the ssr markers developed in this study, represent reference genetic linkage maps for members of the genus dianthus, including carnation, which will be useful for mapping qtls associated with various traits, and for improving elite carnation cultivars in breeding programs.

 genetic linkage maps are valuable resources that provide a framework for many genomic analyses. they are an important tool for many genetic applications, including mapping of quantitative trait loci, comparative mapping, identifying dna markers for fingerprinting, analyses of population genetics and phylogenetics, genome sequence assembly, relating physical and recombination distances along the genome, and map-based cloning of genes. the wide applications of linkage maps and their importance in genetic research has led to numerous linkage mapping projects being undertaken in plants. carnation, in the caryophyllaceae, is one of the major floricultural crops in japan and around the world. hundreds of cultivars are grown around the world and many new varieties are bred and registered every year. more than dianthus species have been recorded. many dianthus species are distributed throughout europe and asia, and the distribution of the genus extends from arctic north america to mountainous sites in africa. high-quality commercial carnation cultivars are usually obtained by inter- or intraspecific hybridization. most of the carnation cultivars are diploid with a chromosome number of 2n = 2x = . the reported nuclear dna content of carnation is – pg/2c. therefore, carnation has a comparatively small nuclear genome approximately four times the size of the arabidopsis thaliana genome. according to the plant c-values database, the genome of carnation is very small compared with those of other ornamental flowers such as rosa hybrida, antirrhinum majus, petunia hybrida, chrysanthemum morifolium, and tulipa gesneriana. to improve the selection efficiency in breeding plants with resistance to carnation bacterial wilt using d. capitatus ssp. andrezejowskianus, we constructed the first genetic linkage map for carnation, which comprised random amplified polymorphic dna and nine simple sequence repeat loci within linkage groups. a qtl analysis identified one major qtl and two minor qtls for cbw resistance derived from d. capitatus ssp. andrezejowskianus. we also identified a new qtl for cbw resistance, derived from carnation line 85– using an ssr-based genetic linkage map. the “b’ map comprised ssr loci, covering cm with an average distance of cm between loci, and lgs. this was the first report of a genetic linkage map based mainly on ssr markers for the genus dianthus. the ssr markers and the genetic linkage maps developed for carnation in our previous studies would be useful for comparisons between mapping populations, for qtl mapping of important agronomic characteristics, and for phylogenetic analyses. however, the number of markers was insufficient for these purposes. also, the small number of markers had resulted in lgs being identified on the maps, instead of. we conducted rna-seq analysis and obtained unigenes consisting of contigs and singletons using next-generation sequencing technology. we identified potential ssrs in of the unigenes. finally, ssr primer pairs were designed. in this study, we aimed to construct a high-density ssr-based genetic linkage map to serve as a reference for members of dianthus. we evaluated polymorphisms in the mapping population using ssr primer pairs designed from data from the rna-seq analysis, the genomic ssr library, and sanger-sequencing expressed sequence tag analysis. we refined the “a” map by adding the new rapd and ssr loci. comparative analysis of the refined “a” and “b” maps revealed corresponding lgs between the two linkage maps. also, we analyzed previously reported genotype and phenotype data to map two qtls and a flower-type locus onto the refined maps.  genotyping of the 85p population the name of the mapping population was changed from “b” to “85p” in this study. screening of ssr primer pairs derived from the ssr-enriched genomic libraries revealed new “gs” ssr markers that were polymorphic in 85p progenies. therefore, a total of “gs” ssr markers, including reported previously, were polymorphic in the 85p population. we reanalyzed “ces” primer pairs derived from est analysis using fluorescent fragment analysis instead of electrophoresis on polyacrylamide gels. this analysis revealed new polymorphic “ces” markers. therefore, a total of “ces” ssr markers including markers reported previously were polymorphic in the 85p population. tanase et al. designed ssr primer pairs from potential ssr repeats by rna-seq analysis. the names of these ssr markers begin with the letters “nes”. of “nes” ssr primers that included di- and tri- nucleotide ssr motifs, produced amplicons that were not polymorphic between parents, produced no amplicon, weak amplicons, or multi-locus amplified products, and produced amplicons that were polymorphic. additional file shows the newly developed ssr markers for the 85p map. we identified ssr markers that showed single-locus polymorphisms in our mapping population, including ssr markers mapped in our previous report, seven markers derived from first genomic ssr libraries, two markers developed by smulders et al., and dcest derived from publicly available ests. of these, were genomic ssr markers and the other were est-ssr markers. refined map construction for 85p population linkage analysis using a minimum lod score of revealed lgs. this result indicated the existence of several minor lgs consisting of a small number of loci within less than cm. to clarify the relationships among the minor lgs, we transferred all ssr loci in the five minor lgs to the “np” map updated from the “a” map described in yagi et al. by adding rapd and ssr loci. thirteen ssr loci out of loci on lg 85p_ were mapped to lower regions of lg np_. six ssr loci out of seven loci on lg 85p_14- and five out of seven loci on lg 85p_14- were mapped to lg np_. eleven out of loci on lg 85p_15- and two out of six loci on lg 85p_15- mapped to lg np_. the positions of all ssr loci mapped to both lgs were conserved between lgs. these results indicated that four of the five minor lgs could be integrated into two lgs. consequently, the number of lgs corresponded to the basic chromosomal number in carnation. the lg names and their order on the 85p map follow those used in a previous report. the total length of the 85p map was cm, with an average distance of cm between loci. the lgs ranged in size from cm to cm, with an average length of cm. the number of loci mapped per lg ranged from to, with an average of. the marker density ranged from to, with an average of. the entire marker density was. two published but previously unmapped loci, ces and ces were mapped onto lg 85p_15- in this study. of the ssr loci, showed segregation patterns that fitted the expected segregation ratio of 1:2: while the segregations of loci and locus differed from the expected ratio. most of the loci showing different segregation ratios were located on lg 85p_; the others were on lgs 85p_ and comparison of lgs between two maps using ssr markers to reveal the relationships for other lgs between the 85p and np maps, more than five ssr loci in each lg of the 85p map were tested in the refined np map. this analysis revealed that at least three loci were connected to the homologous lg. the 85p and np maps had loci in common. the map position of each ssr locus showed good collinearity between homologous lgs. we identified loci in carnation using the two linkage maps. refined map construction for np population the np map was significantly improved from our previous report. we constructed the np map using lines derived from a cross between ‘carnation nou no. 1’ and ‘pretty favvare’. linkage analysis using a minimum lod score of revealed lgs consisting of loci covering cm. the lg names on the np map were rearranged to be consistent with homologous lgs on the 85p map. we mapped new ssr markers that showed single-locus polymorphisms onto the np map. of those, ssr loci were derived from the genomic sequence; were “gs” ssr loci derived from the ssr-enriched genomic library developed by yagi et al., were “cb” or “cf” loci derived from the ssr-enriched genomic library developed by kimura et al., and dcd on lg np_ was developed by smulders et al.. the other ssr loci were derived from est information; ms-dcdia on lg np_ and ms-dinmadsbox on lg np_ were developed by smulders et al., were “ces” ssr loci derived from sanger-based est analysis, four were “dcest” ssr loci derived from public est information, and were “nes” ssr loci derived from rna-seq analysis using ngs technology. these newly mapped ssr loci for the np map are shown in additional file eight new rapd markers were mapped onto lgs np_. we tested ethylene biosynthesis and response pathway gene-specific sequence-tagged site primers designed by de benedetti et al.. among these sts markers, one derived from dcers was mapped onto lg np_ and one derived from eil was mapped onto lg np_ both of these genes encode putative ethylene receptors. in total, ssr, rapd, and two sts loci were mapped onto the np map. the total length of the map was cm, with an average distance of cm between loci. linkage groups ranged in size from cm to cm, with an average length of cm. the number of loci mapped per lg ranged from to, with an average of. the marker density ranged from to, with an average of. the entire marker density was. of the loci, the segregation of loci fitted the expected 1: ratio, while the segregations of and loci deviated from the expected ratio. most of the deviated loci were located on lg np_ and mapping of qtls and a flower-type locus we mapped qtls onto the two maps by analyzing data reported previously. the mapped qtls are shown in figure  and listed in table  a major qtl for cbw resistance derived from line 85– was mapped onto lg 85p_ there were no other qtls with an lod score above, which was the minimum lod score after permutation tests. a new marker linked to cbw ces was identified in this analysis, in addition to the cbw4-linked markers ces and ces identified previously. for the np map, qtls for cbw resistance derived from d. capitatus ssp. andrezejowskianus were mapped in our previous study. cbw and cbw were mapped onto lg np_ and lg np_, respectively. the nearest marker to cbw was wg the same result obtained previously. there were no other significant qtls with an lod score above, which was the minimum lod score after permutation tests, hence, cbw which had a low lod score of in our previous study, was not detected in this study. flower color is an important trait in ornamentals. therefore, we identified qtls related to the anthocyanin content in petals in our previous study. cap and cap were mapped onto lg np_ and lg np_ respectively. we also mapped a phenotypic locus for flower-type. we identified a single flower-type locus in a previous study. the single flower-type locus derived from d. capitatus ssp. andrezejowskianus was mapped onto lg np_ in addition to the four co-segregating rapd markers identified previously, four ssr markers were identified in this study. *thresholds were determined by permutation tests using winqtl cartographer software. discussion high-density linkage maps are useful for locating genes of interest for marker-assisted selection and to identify qtls. in this study, we revised previously constructed linkage maps to add new markers and increase their resolution. in total, single-locus ssr markers are mapped, of which are new markers. we have mapped a large number of ssr markers in the linkage map of carnation, which is not a model crop and had few available ssrs. recently, ngs has been used to develop several types of genetic markers such as restriction site associated dna and single nucleotide polymorphism markers, including ssr markers. one full ngs shotgun run can yield thousands of genome-wide ssr markers. we obtained, high-quality cdna reads by pyrosequencing technology and finally designed ssr primer pairs. we mapped single-locus ssr markers of tested ssr primer sets obtained from ngs analysis. the success ratio was similar to that obtained using “ces” markers derived from sanger-sequencing est analysis in this mapping population. ngs technologies allow the efficient identification of large numbers of ssr loci at a fraction of the cost and effort of traditional ssr isolation approaches. therefore, these technologies will increase the amount of genome information and improve the quality of genomic resources for many plant species, especially ornamental species that tend to have complex genome structures. the ratio of distorted loci was larger in the np map than in the 85p map. most of the distorted loci were located on lg 85p_ on the 85p map, but the corresponding lg on the np map, np_ had no distorted loci. for the np map, most of the loci located on np_ were distorted. thus, the regions of distortion differed between the two linkage maps. distorted segregations in linkage maps have been reported for other ornamentals, including rhododendron, diploid rose, asiatic hybrid lily, and japanese gentian. the causes of segregation distortion have been suggested to include aneuploidy, chromosomal translocation, competition among gametes, and the inheritance of alleles affecting the viability of the zygote, embryo, or seedling. ‘pretty favvare’ is the common mapping parent for both maps, therefore, discordance between normal carnation and ‘carnation nou. no.1’, which is derived from an interspecific cross, may account for the higher ratio of distorted loci on the np map. linkage analysis for the 85p map revealed lgs. comparative analysis between lgs using ssr loci in the updated np map revealed that four minor lgs could be integrated into two lgs. for the np map, the addition of a large number of ssr markers converged the map to lgs. homologous lgs from different populations were identified using the same anchored ssr loci. these results showed that the present lgs would correspond to the basic chromosome number in carnation. even though ssr loci were added to the 85p map, the total map length was only cm longer than that of the previously constructed map and many markers were mapped to the same locus. this might be because of low recombination frequencies in this mapping population, or homozygous chromosomal regions between parental lines. in a linkage map for pear, the short coverage of lgs revealed that large regions of lgs were homozygous. thus, these findings might suggest a limitation for constructing a saturated linkage map using this population. we analyzed previously reported genotype and phenotype data to map qtls for cbw and flower color pigmentation, and the flower-type locus, onto the refined maps. this allowed us to identify some new markers linked to those traits. as well as rapd markers, co-dominant ssr markers linked to the flower-type locus would be useful, although further analyses are needed to assess their effectiveness as selection markers. cbw, was not detected in this study. this was probably because of the change in analytical method, i.e., from interval mapping using mapmaker software to composite interval mapping using winqtl cartographer. moreover, the expansion and rearrangement of the map would also affect the ability to detect this locus. our revised maps described in this paper will allow more precise qtl analysis, including identification of loci associated with various attributes such as flower vase life, an important trait for ornamental plants that has not yet been mapped in the mapping population. we identified a total of loci including ssr in carnation using the two linkage maps. we believe that these linkage maps will serve as reference genetic linkage maps for carnation and that many of the mapped ssr markers will be useful to compare these maps with others constructed using data from different mapping populations. currently, we are producing another mapping population and will construct a new linkage map. moreover, whole genome sequencing by the ngs system is underway.  we improved two carnation linkage maps and mapped a large number of ssr loci. the genetic linkage map derived from a cross between the cbw-resistant line 85– and susceptible ‘pretty favvare’ comprising ssr loci covering cm, with an average distance of cm between loci. the genetic linkage map derived from a cross between cbw-resistant ‘carnation nou no.1’ and susceptible ‘pretty favvare’ comprised ssr, rapd, and two sts loci covering cm, with an average cm distance between loci. these two refined maps were anchored with ssrs loci. we identified loci in carnation using the two linkage maps. the number of lgs was coincident with the haploid chromosome number of carnation. our genetic linkage maps, combined with the ssr markers developed in this study, represent reference genetic linkage maps for members of the genus dianthus, including carnation, which will be useful for mapping qtls associated with various traits, and for improving elite carnation cultivars in breeding programs. 
genetic linkage maps are important tools for many genetic applications including mapping of quantitative trait loci, identifying dna markers for fingerprinting, and map-based gene cloning. we previously reported a random amplified polymorphic dna -based genetic linkage map derived from dianthus capitatus ssp. andrezejowskianus and a simple sequence repeat -based genetic linkage map constructed using data from intraspecific f populations; however, the number of markers was insufficient, and so the number of linkage groups did not coincide with the number of chromosomes. we improved the ssr-based genetic linkage map using ssr markers derived from a genomic library, expression sequence tags, and rna-seq data. we added new ssrs, eight rapd, and two sequence-tagged site loci to refine the rapd-based genetic linkage map, which comprised lgs consisting of loci covering cm. the improved genetic linkage maps and ssr markers developed in this study will serve as reference genetic linkage maps for members of the genus dianthus, including carnation, and will be useful for mapping qtls associated with various traits, and for improving carnation breeding programs. we also mapped qtls for two traits and a phenotypic locus for flower-type by analyzing previously reported genotype and phenotype data. linkage analysis revealed that ssr loci could be mapped to linkage groups covering cm. we identified loci in carnation using the two linkage maps. therefore, we aimed to produce a high-density genetic map to improve its usefulness for breeding purposes and genetic research. comparison of five minor lgs covering less than cm with lgs in our previous rapd-based genetic map suggested that four lgs could be integrated into two lgs by anchoring common ssr loci.
 genetic linkage maps are important tools for many genetic applications including mapping of quantitative trait loci, identifying dna markers for fingerprinting, and map-based gene cloning. carnation is an important ornamental flower worldwide. we previously reported a random amplified polymorphic dna -based genetic linkage map derived from dianthus capitatus ssp. andrezejowskianus and a simple sequence repeat -based genetic linkage map constructed using data from intraspecific f populations; however, the number of markers was insufficient, and so the number of linkage groups did not coincide with the number of chromosomes. therefore, we aimed to produce a high-density genetic map to improve its usefulness for breeding purposes and genetic research.  we improved the ssr-based genetic linkage map using ssr markers derived from a genomic library, expression sequence tags, and rna-seq data. linkage analysis revealed that ssr loci could be mapped to linkage groups covering cm. comparison of five minor lgs covering less than cm with lgs in our previous rapd-based genetic map suggested that four lgs could be integrated into two lgs by anchoring common ssr loci. consequently, the number of lgs corresponded to the number of chromosomes. we added new ssrs, eight rapd, and two sequence-tagged site loci to refine the rapd-based genetic linkage map, which comprised lgs consisting of loci covering cm. the two maps had ssr loci in common, and most of the positions of markers were conserved between them. we identified loci in carnation using the two linkage maps. we also mapped qtls for two traits and a phenotypic locus for flower-type by analyzing previously reported genotype and phenotype data.  the improved genetic linkage maps and ssr markers developed in this study will serve as reference genetic linkage maps for members of the genus dianthus, including carnation, and will be useful for mapping qtls associated with various traits, and for improving carnation breeding programs. carnationdianthus caryophyllus lestlinkage mapnext-generation sequencing technology rapdstsssr 