BACKGROUND
gentianaceae , comprises  <dig> genera and more than  <dig>  species that are distributed worldwide.
the genus gentiana is the largest genus in the gentianaceae and includes over  <dig> species  <cit> .
among them, two major floricultural species, gentiana triflora and gentiana scabra, the so-called japanese gentians, have been cultivated in japan since the 1960s.
in addition to japanese gentians, the important floricultural plant eustoma , otherwise known as lisianthus, and a pharmaceutical plant, swertia , also belong to the family gentianaceae.
japanese gentians are among the most popular floricultural plants in japan, and have unique vivid blue flowers  <cit> .
the main japanese gentian is g. triflora, which usually blooms from july to september in japan.
g. scabra blooms later, from september to november, and has some valuable traits, such as an open corolla and resistance to gentian brown leaf spot caused by mycochaetophora gentianae <cit> .
the chromosome numbers of g. triflora and g. scabra are 2n= <dig>  whereas those of european pharmaceutical gentians, including gentiana lutea and gentiana punctata, are 2n= <dig>  it is difficult to produce interspecific hybrids among members of the genus gentiana because of the different basal chromosome numbers.
the nuclear dna content in g. triflora is  <dig>  to  <dig>  pg/2c, and that in g. scabra is  <dig>  to  <dig>  pg/2c  <cit> .
thus, these two japanese gentians have similar genome sizes , which are larger than those of some other plants, e.g.
arabidopsis thaliana   <cit> , medicago truncatula   <cit> , rice   <cit> , and tomato   <cit> .
there has been extensive molecular and physiological research on flower colors, flowering times, and cold tolerance of winter buds of japanese gentians  <cit> .
based on these molecular studies, several genetic markers have been developed.
for example, we developed genetic markers that discriminate among blue, pink, and white flower color  <cit> .
to protect breeders’ rights, markers based on fragment length polymorphisms  were developed to identify japanese gentian cultivars  <cit> .
the haplotypes of w14/ <dig> alleles, which encode an esterase involved in cold tolerance of winter buds, provided data that clarified phylogenetic relationships in the genus gentiana, and allowed analysis of the pedigree and breeding history of cultivars derived from those gentiana spp.
<cit> .
genetic linkage maps are useful for studies on genomic structure and evolution, and for identification of monogenic traits or mendelian components of quantitative trait loci .
therefore, they are very useful for breeding programs and are the basis for future positional gene cloning  <cit> .
they are also very useful for marker-assisted breeding and introgression of beneficial traits into floricultural plants.
genetic linkage maps have been constructed for several floricultural plants, including rose   <cit> , carnation   <cit> , asiatic hybrid lily   <cit> , and periwinkle   <cit> .
however, no genetic linkage maps have been developed for the genus gentiana or for any member of the family gentianaceae, including economically important species.
the majority of floricultural plants are allogamous, but most linkage maps for crop plants have been obtained from segregating populations derived from crosses between inbred lines.
therefore, genetic linkage maps for allogamous and vegetatively reproducing species have been constructed using a double pseudo testcross strategy  <cit> .
conversely, the genetic linkage map for ryegrass  was constructed using a backcross population involving a double haploid  tester  <cit> .
japanese gentians are also highly heterozygous plants that suffer from inbreeding depression.
to obtain pure lines, the androgenic double haploid dh line, aki6ps, was produced recently in g. triflora using anther culture  <cit> .
here, we report the construction of a genetic linkage map for japanese gentian, g. scabra, using a bc <dig> population backcrossed with g. triflora dh line aki6ps and  <dig> genetic markers.
this is the first reported genetic linkage map for japanese gentians and the first for any species belonging to the family gentianaceae.
our results are important for understanding the genetic basis of traits and to apply molecular markers linked to mapped loci for marker-assisted selection in gentian breeding programs.
RESULTS
simple sequence repeat markers
the enriched ssr libraries from the dh line aki6ps  yielded  <dig>  clones using roche  <dig> pyrosequencing.
of these,  <dig>  clones  contained ssr motifs.
only  <dig> clones  were suitable for designing primer sets for ssr markers; the rest comprised reads that were too short  to design primers from the proximal sequences of the ssr motif.
therefore, in addition to next generation sequencing,  <dig>  clones from the enriched ssr libraries were subjected to sanger dna sequencing analysis.
as a result,  <dig>  clones  had ssr motifs within the sequence reads, and  <dig> clones  could be used to design primer sets for ssr markers.
these results indicated that the conventional strategy using sanger sequencing was more efficient than next generation sequencing for developing ssr markers, without considering cost, labor, and time.
we screened  <dig>  ssr markers using both parents and one f <dig> individual.
most ssr markers showed no or unstable amplification, or produced amplified fragments with unexpected lengths.
eighty ssr markers detected significant polymorphisms as co-dominant markers.
in addition,  <dig> ssr markers were dominant markers, amplifying pcr fragments from sp6a <dig> and f <dig> but not from aki6ps .
ultimately,  <dig> ssr markers were available to construct the genetic linkage map.
sequence data for the markers have been deposited in genbank/embl/ddbj nucleotide sequence databases under the accession numbers .
retrotransposon microsatellite amplified polymorphism markers
to increase the number of available genetic markers for japanese gentian, we developed some retrotransposon microsatellite amplified polymorphism  using a retrotransposon.
a retrotransposable element gstrim <dig>  has been reported in g. scabra <cit> .
therefore, to isolate novel ltrs of retrotransposons from japanese gentians, we used ipbs technology as described by kalendar et al.
<cit>  .
genomic dna was amplified using eight ipbs primers , and then subjected to sequencing analysis.
three putative ltr sequences,  <dig> bp ,  <dig> bp  and  <dig> bp , were found in the comparative sequence analyses among  <dig> independent clones.
although eight primers could be designed from the three putative ltr sequences, a few amplified fragments were obtained using each ltr primer during inter-retrotransposon amplified polymorphism  analysis .
therefore, each ltr primer was explored in combination with issr primers to create remap markers .
issr primer sets provided from the university of british columbia  were pre-screened using aki6ps genomic dna.
seventeen primers amplified more than eight stable fragments .
seven primer sets combining ltr and issr, as shown in table  <dig>  produced reproducible amplified fragments.
these primer combinations provided stable and reproducible results at an annealing temperature of 60°c.
remap markers were more efficient and reproducible than irap and issr markers in japanese gentians.
amplified fragment length polymorphism markers
our preliminary experiments showed that conventional amplified fragment length polymorphism  amplification using combinations of selective primers with three selective nucleotides resulted in too many faint and overlapping fragments in gentian .
to solve this problem, we added a fourth selective nucleotide to the mse i primer, as described by remington et al.
<cit>  .
aflp and rapd markers are dominantly inherited; therefore, we identified only g. scabra sp6a1-specific fragments among many amplified fragments because aki6ps-specific fragments were not segregated in the bc <dig> population backcrossed with g. triflora aki6ps.
fourteen primer sets, which produced more than  <dig> sp6a1-specific fragments per primer combination, were selected to generate the genetic linkage map .
long random amplification polymorphic dna markers
we first screened for reproducible and stable random amplification polymorphic dna  markers using  <dig> primers, as described by debener and mattiesch  <cit>  and by yamagishi et al.
<cit> .
among them, twelve 15-mer primers  amplified bands reproducibly, and were selected for genetic analysis of japanese gentians.
an average of  <dig> fragments  with two to seven sp6a1-specific fragments were amplified per primer.
functional markers
in japanese gentians, there has been extensive physiological and molecular research on flower pigmentation, flowering time, and cold tolerance of winter buds, and the nucleotide sequences of some important genes have already been deposited in public databases.
in addition, our previous study reported significant insertions/deletions  in introns and untranslated regions, representing molecular polymorphisms that could be used to discriminate among cultivars and species of japanese gentians  <cit> .
therefore, we first identified the intron and proximal sequences of chalcone synthase  and the flowering locus t <dig>  and ft <dig> genes from both g. triflora aki6ps and g. scabra sp6a <dig>  comparative sequencing analysis identified significant in/del polymorphisms with the intron regions of chs and ft <dig>  and marker primer sets were designed in the proximity of the in/dels of each gene .
for chalcone isomerase , flavanone 3-hydroxylase , flavonoid 3′,5′-hydroxylase , anthocyanidin synthase , myb <dig>  basic helix loop helix  <dig> , terminal flowering  <dig>  and w14/ <dig> genes, we used the primer sets reported in previous studies  <cit> .
no in/dels were detected in the genomic sequences of ft <dig> and tfl <dig> genes, but some snps were detected using restriction enzymes.
therefore, ft <dig> and tfl <dig> markers were distinguished between aki6ps and sp6a <dig> as cleaved amplified polymorphic sequence  markers, when their amplified fragments were digested by pst i and sau3a i, respectively.
segregation of stem pigmentation trait
the g. triflora breeding line aki6ps has a green stem color, while stems of the g. scabra breeding line sp6a <dig> and f <dig> are red because of the accumulation of anthocyanins.
among bc <dig> progeny,  <dig> and  <dig> individuals showed green- and red-stem color phenotypes, respectively.
segregation of stem color fitted a 1: <dig> ratio based on a chi-square test at p =  <dig> , suggesting monogenetic inheritance of this trait.
genetic map construction
we used a population of  <dig> bc <dig> from g. scabra sp6a <dig> backcrossed with g. triflora aki6ps dh line .
in total,  <dig> markers, comprising  <dig> ssr markers,  <dig> aflp markers,  <dig> rapd markers,  <dig> remap markers and  <dig> functional markers, and one phenotypic marker were grouped with a lod score of  <dig> .
as a result,  <dig> linkage groups containing at least two markers could be assembled with  <dig> markers .
thirty-five markers were unmapped and ungrouped in this linkage map, and more rapd  and remap  markers were excluded from the linkage groups, compared with ssr , aflp  and functional markers .
of the  <dig> functional markers, ft <dig> was closely linked to ft <dig>  an ortholog of ft <dig>  stem color locus as a phenotypic trait was closely linked to the m03e1- <dig> aflp marker in lg <dig>  in total,  <dig> linkage groups were identified, covering  <dig>  cm with an average distance of  <dig>  cm between each pair of markers.
the size of the linkage groups ranged from  <dig>  to  <dig>  cm.
twenty-seven,  <dig>  and  <dig> markers showed distorted segregation at the 5%, 1%, and  <dig> % levels, respectively, based on the chi-square test.
five linkage groups  were assembled from markers with distorted segregation at the  <dig> % level in the genetic map of japanese gentian.
discussion
we used  <dig> flx titanium pyrosequencing of an ssr-enriched library to develop ssr markers from japanese gentians.
recently, next generation sequencing has been used successfully to develop several genetic markers, including ssrs  <cit> .
in our study, out of approximately  <dig>  sequences from the enriched ssr library, only  <dig> % could be used to design primers suitable for ssr markers.
in contrast,  <dig> % of the sequences derived from conventional sanger dna sequencing were suitable for designing primers for ssr markers.
this is because the reads obtained from pyrosequencing in this study are shorter and less accurate than those obtained from sanger sequencing.
similarly, in the honey bee  and zebrafish , only  <dig> % to  <dig> % sequences obtained by  <dig> flx titanium pyrosequencing could be used to design primers for ssr markers  <cit> .
in spite of its flaws, next generation sequencing is cheaper and less labor-intensive than conventional sanger dna sequencing.
as next generation sequencing technology develops further, for example, the long-read protocols based on the gs flx+ system, this technology will become more efficient for development of ssr markers.
retrotransposons can be used as molecular markers because their integration creates new junctions between genomic dna and their conserved ends  <cit> .
irap and remap are simple methods that do not require restriction enzyme digestion or ligation to generate marker bands  <cit> .
however, the limiting factor for developing genetic markers based on ltr retrotransposons for new plant species is the availability or otherwise of retrotransposon sequences.
in japanese gentian, three dispersed transposable elements, gstrim <dig>  tgt <dig> , and gtmite <dig>  have been identified  <cit> ; however, there are low copy numbers of tgt <dig> and gtmite <dig> in the gentian genome  <cit> .
we used ipbs amplification  <cit> , which is based on the universal presence of primer binding sites  of ltr retrotransposons, to identify new ltr sequences from japanese gentian.
three putative ltr sequences were predicted from overlapping regions among independent sequences obtained by ipbs amplification.
these sequences did not correspond to the terminal direct repeat  of gstrim <dig> and ltr of tgt <dig>  we designed primers from these newly identified ltr sequences .
however, no or few fragments were amplified by the putative ltr primers from any japanese gentian lines, probably because the ltr retrotransposons were too far apart for efficient amplification.
therefore, these ltr primers were not suitable as irap markers.
conversely, remap primers yielded more reproducible and stable fragments in japanese gentians than irap and issr, and were used to generate the genetic linkage map.
rapd and aflp technologies are conventional molecular markers.
their advantages are that they do not require sequence information and have a relatively low start-up cost  <cit> .
however, certain procedures must be modified to use rapd and aflp markers for japanese gentians, probably because the large genome size and heterogeneity.
rapds using10-mer primers and aflp using three selective nucleotides resulted in too many faint and overlapping fragments , whereas long-rapds using 15-mer primers amplified reproducible and stable fragments at higher annealing temperatures.
the use of 15-mer primers also led to more effective amplification of polymorphic fragments than 10-mer primers in grape  <cit> , rose  <cit> , and asiatic hybrid lily  <cit> .
the aflp markers produced reproducible fragments using mse i-selective primers with four selective nucleotides in japanese gentians.
in conifer, which has a very large genome , the use of selective primers with a fourth selective nucleotide also improved amplification of reproducible fragments  <cit> .
therefore, markers developed by modified rapd and aflp were used to generate the gentian genetic linkage map .
a genetic linkage map with  <dig> markers was constructed using an interspecific bc <dig> population.
nineteen linkage groups were obtained for the bc <dig> population at an lod value of  <dig> , and encompassed  <dig>  cm .
analysis of published saturated maps for tomato   <cit> , melon   <cit> , and lettuce   <cit>  using the joinmap program showed that an average map distance of  <dig> to  <dig> cm can be expected per chromosome regardless of its physical length.
we estimate the map length of japanese gentians  to be  <dig>  to  <dig>  cm.
the present genetic linkage map probably covers less than half of the genome; therefore, more genetic markers are required to saturate the linkage map of japanese gentians.
in the bc <dig> progeny of japanese gentians,  <dig> % of markers showed segregation distortion.
these skewed segregations were also reported in other ornamental plants species, such as the diploid rose  <cit>  and asiatic hybrid lily  <cit> .
the causes of segregation distortion are not well understood.
segregation distortion has been reported in rhododendron <cit> , asiatic lily  <cit>  and carnation  <cit> , and it was assumed to represent hybrid sterility genes and gametophytic selection genes in interspecific crossing.
the bc <dig> population used in this study was derived from interspecific crossing between g. triflora and g. scabra, and the reciprocal cross showed hybrid weakness.
therefore, hybrid sterility and gametophytic selection genes might be responsible for the segregation distortion in japanese gentian populations.
functional markers are those derived from polymorphic sites within genes responsible for phenotype traits  <cit> .
ten functional markers for genes involved in important traits, such as flower pigmentation , flowering time  and cold tolerance of winter buds , were mapped to the  <dig> linkage groups of japanese gentian .
our previous research revealed that white-flowered gentians resulted from the functional deficiency of ans, a structural enzyme, or myb <dig>  an anthocyanin biosynthetic transcription factor.
genetic analysis showed that the ans locus was not linked to the myb <dig> locus  <cit> .
consistent with these observations, ans and myb <dig> were assembled into different linkage groups: lg <dig> and lg <dig>  respectively .
interestingly, two orthologs of the flowering hormone florigen, ft <dig> and ft <dig>  were closely linked .
these findings will be useful for determining the molecular functions of these genes and to study the genetic regulation of flowering time in gentian, although further research is necessary.
the mapping of functional markers corresponding to important agricultural traits will be accelerated by further molecular and physiological studies on japanese gentians.
our genetic linkage map will be useful for mapping qtls associated with various traits, and for improving japanese gentian breeding programs.
the map is also applicable to other members of the gentianaceae family, including several other economically important species.
CONCLUSIONS
this study presents the first genetic linkage map for japanese gentian.
the map was constructed using four different types of molecular markers.
it was produced from genotypes of  <dig> bc <dig> progeny derived from a dh line and included  <dig> markers .
the map revealed  <dig> linkage groups that covered  <dig>  cm, with an average intermarker distance of  <dig>  cm.
this map is a starting point for mapping single or quantitative trait loci affecting agronomically important phenotypes, and will be useful for research on gentian genetics and breeding.

 gentianaceae, comprises genera and more than species that are distributed worldwide. the genus gentiana is the largest genus in the gentianaceae and includes over species. among them, two major floricultural species, gentiana triflora and gentiana scabra, the so-called japanese gentians, have been cultivated in japan since the 1960s. in addition to japanese gentians, the important floricultural plant eustoma, otherwise known as lisianthus, and a pharmaceutical plant, swertia, also belong to the family gentianaceae. japanese gentians are among the most popular floricultural plants in japan, and have unique vivid blue flowers. the main japanese gentian is g. triflora, which usually blooms from july to september in japan. g. scabra blooms later, from september to november, and has some valuable traits, such as an open corolla and resistance to gentian brown leaf spot caused by mycochaetophora gentianae. the chromosome numbers of g. triflora and g. scabra are 2n= whereas those of european pharmaceutical gentians, including gentiana lutea and gentiana punctata, are 2n= it is difficult to produce interspecific hybrids among members of the genus gentiana because of the different basal chromosome numbers. the nuclear dna content in g. triflora is to pg/2c, and that in g. scabra is to pg/2c. thus, these two japanese gentians have similar genome sizes, which are larger than those of some other plants, e.g. arabidopsis thaliana, medicago truncatula, rice, and tomato. there has been extensive molecular and physiological research on flower colors, flowering times, and cold tolerance of winter buds of japanese gentians. based on these molecular studies, several genetic markers have been developed. for example, we developed genetic markers that discriminate among blue, pink, and white flower color. to protect breeders’ rights, markers based on fragment length polymorphisms were developed to identify japanese gentian cultivars. the haplotypes of w14/ alleles, which encode an esterase involved in cold tolerance of winter buds, provided data that clarified phylogenetic relationships in the genus gentiana, and allowed analysis of the pedigree and breeding history of cultivars derived from those gentiana spp.. genetic linkage maps are useful for studies on genomic structure and evolution, and for identification of monogenic traits or mendelian components of quantitative trait loci. therefore, they are very useful for breeding programs and are the basis for future positional gene cloning. they are also very useful for marker-assisted breeding and introgression of beneficial traits into floricultural plants. genetic linkage maps have been constructed for several floricultural plants, including rose, carnation, asiatic hybrid lily, and periwinkle. however, no genetic linkage maps have been developed for the genus gentiana or for any member of the family gentianaceae, including economically important species. the majority of floricultural plants are allogamous, but most linkage maps for crop plants have been obtained from segregating populations derived from crosses between inbred lines. therefore, genetic linkage maps for allogamous and vegetatively reproducing species have been constructed using a double pseudo testcross strategy. conversely, the genetic linkage map for ryegrass was constructed using a backcross population involving a double haploid tester. japanese gentians are also highly heterozygous plants that suffer from inbreeding depression. to obtain pure lines, the androgenic double haploid dh line, aki6ps, was produced recently in g. triflora using anther culture. here, we report the construction of a genetic linkage map for japanese gentian, g. scabra, using a bc population backcrossed with g. triflora dh line aki6ps and genetic markers. this is the first reported genetic linkage map for japanese gentians and the first for any species belonging to the family gentianaceae. our results are important for understanding the genetic basis of traits and to apply molecular markers linked to mapped loci for marker-assisted selection in gentian breeding programs.  simple sequence repeat markers the enriched ssr libraries from the dh line aki6ps yielded clones using roche pyrosequencing. of these, clones contained ssr motifs. only clones were suitable for designing primer sets for ssr markers; the rest comprised reads that were too short to design primers from the proximal sequences of the ssr motif. therefore, in addition to next generation sequencing, clones from the enriched ssr libraries were subjected to sanger dna sequencing analysis. as a result, clones had ssr motifs within the sequence reads, and clones could be used to design primer sets for ssr markers. these results indicated that the conventional strategy using sanger sequencing was more efficient than next generation sequencing for developing ssr markers, without considering cost, labor, and time. we screened ssr markers using both parents and one f individual. most ssr markers showed no or unstable amplification, or produced amplified fragments with unexpected lengths. eighty ssr markers detected significant polymorphisms as co-dominant markers. in addition, ssr markers were dominant markers, amplifying pcr fragments from sp6a and f but not from aki6ps. ultimately, ssr markers were available to construct the genetic linkage map. sequence data for the markers have been deposited in genbank/embl/ddbj nucleotide sequence databases under the accession numbers. retrotransposon microsatellite amplified polymorphism markers to increase the number of available genetic markers for japanese gentian, we developed some retrotransposon microsatellite amplified polymorphism using a retrotransposon. a retrotransposable element gstrim has been reported in g. scabra. therefore, to isolate novel ltrs of retrotransposons from japanese gentians, we used ipbs technology as described by kalendar et al.. genomic dna was amplified using eight ipbs primers, and then subjected to sequencing analysis. three putative ltr sequences, bp, bp and bp, were found in the comparative sequence analyses among independent clones. although eight primers could be designed from the three putative ltr sequences, a few amplified fragments were obtained using each ltr primer during inter-retrotransposon amplified polymorphism analysis. therefore, each ltr primer was explored in combination with issr primers to create remap markers. issr primer sets provided from the university of british columbia were pre-screened using aki6ps genomic dna. seventeen primers amplified more than eight stable fragments. seven primer sets combining ltr and issr, as shown in table  produced reproducible amplified fragments. these primer combinations provided stable and reproducible results at an annealing temperature of 60°c. remap markers were more efficient and reproducible than irap and issr markers in japanese gentians. amplified fragment length polymorphism markers our preliminary experiments showed that conventional amplified fragment length polymorphism amplification using combinations of selective primers with three selective nucleotides resulted in too many faint and overlapping fragments in gentian. to solve this problem, we added a fourth selective nucleotide to the mse i primer, as described by remington et al.. aflp and rapd markers are dominantly inherited; therefore, we identified only g. scabra sp6a1-specific fragments among many amplified fragments because aki6ps-specific fragments were not segregated in the bc population backcrossed with g. triflora aki6ps. fourteen primer sets, which produced more than sp6a1-specific fragments per primer combination, were selected to generate the genetic linkage map. long random amplification polymorphic dna markers we first screened for reproducible and stable random amplification polymorphic dna markers using primers, as described by debener and mattiesch and by yamagishi et al.. among them, twelve 15-mer primers amplified bands reproducibly, and were selected for genetic analysis of japanese gentians. an average of fragments with two to seven sp6a1-specific fragments were amplified per primer. functional markers in japanese gentians, there has been extensive physiological and molecular research on flower pigmentation, flowering time, and cold tolerance of winter buds, and the nucleotide sequences of some important genes have already been deposited in public databases. in addition, our previous study reported significant insertions/deletions in introns and untranslated regions, representing molecular polymorphisms that could be used to discriminate among cultivars and species of japanese gentians. therefore, we first identified the intron and proximal sequences of chalcone synthase and the flowering locus t and ft genes from both g. triflora aki6ps and g. scabra sp6a comparative sequencing analysis identified significant in/del polymorphisms with the intron regions of chs and ft and marker primer sets were designed in the proximity of the in/dels of each gene. for chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3′,5′-hydroxylase, anthocyanidin synthase, myb basic helix loop helix, terminal flowering and w14/ genes, we used the primer sets reported in previous studies. no in/dels were detected in the genomic sequences of ft and tfl genes, but some snps were detected using restriction enzymes. therefore, ft and tfl markers were distinguished between aki6ps and sp6a as cleaved amplified polymorphic sequence markers, when their amplified fragments were digested by pst i and sau3a i, respectively. segregation of stem pigmentation trait the g. triflora breeding line aki6ps has a green stem color, while stems of the g. scabra breeding line sp6a and f are red because of the accumulation of anthocyanins. among bc progeny, and individuals showed green- and red-stem color phenotypes, respectively. segregation of stem color fitted a 1: ratio based on a chi-square test at p =, suggesting monogenetic inheritance of this trait. genetic map construction we used a population of bc from g. scabra sp6a backcrossed with g. triflora aki6ps dh line. in total, markers, comprising ssr markers, aflp markers, rapd markers, remap markers and functional markers, and one phenotypic marker were grouped with a lod score of. as a result, linkage groups containing at least two markers could be assembled with markers. thirty-five markers were unmapped and ungrouped in this linkage map, and more rapd and remap markers were excluded from the linkage groups, compared with ssr, aflp and functional markers. of the functional markers, ft was closely linked to ft an ortholog of ft stem color locus as a phenotypic trait was closely linked to the m03e1- aflp marker in lg in total, linkage groups were identified, covering cm with an average distance of cm between each pair of markers. the size of the linkage groups ranged from to cm. twenty-seven, and markers showed distorted segregation at the 5%, 1%, and % levels, respectively, based on the chi-square test. five linkage groups were assembled from markers with distorted segregation at the % level in the genetic map of japanese gentian. discussion we used flx titanium pyrosequencing of an ssr-enriched library to develop ssr markers from japanese gentians. recently, next generation sequencing has been used successfully to develop several genetic markers, including ssrs. in our study, out of approximately sequences from the enriched ssr library, only % could be used to design primers suitable for ssr markers. in contrast, % of the sequences derived from conventional sanger dna sequencing were suitable for designing primers for ssr markers. this is because the reads obtained from pyrosequencing in this study are shorter and less accurate than those obtained from sanger sequencing. similarly, in the honey bee and zebrafish, only % to % sequences obtained by flx titanium pyrosequencing could be used to design primers for ssr markers. in spite of its flaws, next generation sequencing is cheaper and less labor-intensive than conventional sanger dna sequencing. as next generation sequencing technology develops further, for example, the long-read protocols based on the gs flx+ system, this technology will become more efficient for development of ssr markers. retrotransposons can be used as molecular markers because their integration creates new junctions between genomic dna and their conserved ends. irap and remap are simple methods that do not require restriction enzyme digestion or ligation to generate marker bands. however, the limiting factor for developing genetic markers based on ltr retrotransposons for new plant species is the availability or otherwise of retrotransposon sequences. in japanese gentian, three dispersed transposable elements, gstrim tgt, and gtmite have been identified; however, there are low copy numbers of tgt and gtmite in the gentian genome. we used ipbs amplification, which is based on the universal presence of primer binding sites of ltr retrotransposons, to identify new ltr sequences from japanese gentian. three putative ltr sequences were predicted from overlapping regions among independent sequences obtained by ipbs amplification. these sequences did not correspond to the terminal direct repeat of gstrim and ltr of tgt we designed primers from these newly identified ltr sequences. however, no or few fragments were amplified by the putative ltr primers from any japanese gentian lines, probably because the ltr retrotransposons were too far apart for efficient amplification. therefore, these ltr primers were not suitable as irap markers. conversely, remap primers yielded more reproducible and stable fragments in japanese gentians than irap and issr, and were used to generate the genetic linkage map. rapd and aflp technologies are conventional molecular markers. their advantages are that they do not require sequence information and have a relatively low start-up cost. however, certain procedures must be modified to use rapd and aflp markers for japanese gentians, probably because the large genome size and heterogeneity. rapds using10-mer primers and aflp using three selective nucleotides resulted in too many faint and overlapping fragments, whereas long-rapds using 15-mer primers amplified reproducible and stable fragments at higher annealing temperatures. the use of 15-mer primers also led to more effective amplification of polymorphic fragments than 10-mer primers in grape, rose, and asiatic hybrid lily. the aflp markers produced reproducible fragments using mse i-selective primers with four selective nucleotides in japanese gentians. in conifer, which has a very large genome, the use of selective primers with a fourth selective nucleotide also improved amplification of reproducible fragments. therefore, markers developed by modified rapd and aflp were used to generate the gentian genetic linkage map. a genetic linkage map with markers was constructed using an interspecific bc population. nineteen linkage groups were obtained for the bc population at an lod value of, and encompassed cm. analysis of published saturated maps for tomato, melon, and lettuce using the joinmap program showed that an average map distance of to cm can be expected per chromosome regardless of its physical length. we estimate the map length of japanese gentians to be to cm. the present genetic linkage map probably covers less than half of the genome; therefore, more genetic markers are required to saturate the linkage map of japanese gentians. in the bc progeny of japanese gentians, % of markers showed segregation distortion. these skewed segregations were also reported in other ornamental plants species, such as the diploid rose and asiatic hybrid lily. the causes of segregation distortion are not well understood. segregation distortion has been reported in rhododendron, asiatic lily and carnation, and it was assumed to represent hybrid sterility genes and gametophytic selection genes in interspecific crossing. the bc population used in this study was derived from interspecific crossing between g. triflora and g. scabra, and the reciprocal cross showed hybrid weakness. therefore, hybrid sterility and gametophytic selection genes might be responsible for the segregation distortion in japanese gentian populations. functional markers are those derived from polymorphic sites within genes responsible for phenotype traits. ten functional markers for genes involved in important traits, such as flower pigmentation, flowering time and cold tolerance of winter buds, were mapped to the linkage groups of japanese gentian. our previous research revealed that white-flowered gentians resulted from the functional deficiency of ans, a structural enzyme, or myb an anthocyanin biosynthetic transcription factor. genetic analysis showed that the ans locus was not linked to the myb locus. consistent with these observations, ans and myb were assembled into different linkage groups: lg and lg respectively. interestingly, two orthologs of the flowering hormone florigen, ft and ft were closely linked. these findings will be useful for determining the molecular functions of these genes and to study the genetic regulation of flowering time in gentian, although further research is necessary. the mapping of functional markers corresponding to important agricultural traits will be accelerated by further molecular and physiological studies on japanese gentians. our genetic linkage map will be useful for mapping qtls associated with various traits, and for improving japanese gentian breeding programs. the map is also applicable to other members of the gentianaceae family, including several other economically important species.  this study presents the first genetic linkage map for japanese gentian. the map was constructed using four different types of molecular markers. it was produced from genotypes of bc progeny derived from a dh line and included markers. the map revealed linkage groups that covered cm, with an average intermarker distance of cm. this map is a starting point for mapping single or quantitative trait loci affecting agronomically important phenotypes, and will be useful for research on gentian genetics and breeding. 
in this study, we developed a genetic linkage map to improve breeding programs of japanese gentians. this is the first reported genetic linkage map for japanese gentians and for any species belonging to the family gentianaceae. one phenotypic trait and functional markers related to genes controlling flower color, flowering time and cold tolerance were assigned to the linkage map, confirming its utility. as demonstrated by mapping of functional markers and the stem color trait, our results will help to explain the genetic basis of agronomic important traits, and will be useful for marker-assisted selection in gentian breeding programs. japanese gentians are amongst the most popular floricultural plants in japan. however, genomic resources for japanese gentians have not yet been developed, mainly because of the heterozygous genome structure conserved by outcrossing, the long juvenile period, and limited knowledge about the inheritance of important traits. using bc progeny from g. scabra backcrossed with a g. triflora double haploid line, linkage groups were constructed with a total of markers. our map will also be an important resource for further genetic analyses such as mapping of quantitative trait loci and map-based cloning of genes in this species. we also developed retrotransposon microsatellite amplified polymorphism markers combining retrotransposon and inter-simple sequence repeat markers. in addition to ssr and remap markers, modified amplified fragment length polymorphism and random amplification polymorphic dna markers were developed. to increase the number of molecular markers, we identified three putative long terminal repeat sequences using the recently developed inter-primer binding site method.
 japanese gentians are amongst the most popular floricultural plants in japan. however, genomic resources for japanese gentians have not yet been developed, mainly because of the heterozygous genome structure conserved by outcrossing, the long juvenile period, and limited knowledge about the inheritance of important traits. in this study, we developed a genetic linkage map to improve breeding programs of japanese gentians.  enriched simple sequence repeat libraries from a g. triflora double haploid line yielded almost clones using pyrosequencing technology, % of which could be used to design ssr markers. to increase the number of molecular markers, we identified three putative long terminal repeat sequences using the recently developed inter-primer binding site method. we also developed retrotransposon microsatellite amplified polymorphism markers combining retrotransposon and inter-simple sequence repeat markers. in addition to ssr and remap markers, modified amplified fragment length polymorphism and random amplification polymorphic dna markers were developed. using bc progeny from g. scabra backcrossed with a g. triflora double haploid line, linkage groups were constructed with a total of markers. one phenotypic trait and functional markers related to genes controlling flower color, flowering time and cold tolerance were assigned to the linkage map, confirming its utility.  this is the first reported genetic linkage map for japanese gentians and for any species belonging to the family gentianaceae. as demonstrated by mapping of functional markers and the stem color trait, our results will help to explain the genetic basis of agronomic important traits, and will be useful for marker-assisted selection in gentian breeding programs. our map will also be an important resource for further genetic analyses such as mapping of quantitative trait loci and map-based cloning of genes in this species. aflpfunctional markergenetic linkage mapipbsjapanese gentianmarkerrapdremapssr 