BACKGROUND
high-density linkage maps facilitate not only the understanding of genome structure and comparative genomic studies, but also quantitative trait loci  mapping and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection .
mas is an effective method for accelerating the screening of target genes using tightly-linked molecular markers.
effective mas for qtls or specific genes has been reported in crops such as barley  <cit> , rice  <cit>  and tomato  <cit> .
however, it is difficult to employ mas in conifers, primarily because their genomes are very big and complex and are largely uncharacterized.
indeed, very few dna markers linked to major genes have been reported in conifers; the only ones of note are some dominant markers linked to a major dominant resistance gene in pinus lambertiana  <cit> , p. monticola  <cit> , p. taeda  <cit> , and p. thunbergii  <cit> .
sugi  is an allogamous, diploid, wind-pollinated conifer species with a haploid chromosome number  of  <dig> .
using flow cytometry, the dna content of its haploid cells was estimated to be  <dig>  pg/c  <cit> , which corresponds to a haploid genome size of  <dig>  gb as calculated using the following expression: genome size  =  <dig>  ×  <dig> × dna content   <cit> .
c. japonica is frequently used for commercial afforestation in japan, and about 45% of all the man-made forests of japan are composed of this species.
however, since the 1970s, the incidence of c. japonica pollinosis in japan has risen in line with the increasing number of man-made c. japonica forests  <cit> .
today, c. japonica pollinosis is a serious social problem in japan, affecting almost 20% of the population.
in  <dig>  a genetically male-sterile c. japonica tree whose sterility is determined by a major recessive gene  was found in toyama prefecture  <cit> ; this gene is expected play an important role in breeding for reduced pollen dispersal.
since the discovery of this male-sterile individual, considerable effort has been expended on characterizing male sterility in c. japonica, identifying male-sterile and plus-trees, creating artificial crosses between male-sterile and plus-trees, and propagating male-sterile trees  <cit> .
male-sterile trees are currently identified by direct inspection of the male strobili using a magnifying glass or a microscope.
conversely, plus-trees that are heterozygous for ms <dig> have been identified by examining segregation data for the progeny arising from artificial crosses; such trees provide important breeding material for seed production while avoiding the problems associated with inbreeding depression.
however, these methods are very time-, labour-, and space-intensive; as such, it would be extremely useful to develop a mas-based method for selecting trees carrying the male-fertile gene.
a composite linkage map for c. japonica was constructed using data for two pedigrees, yi and ko  <cit> .
a total of  <dig> markers were assigned to  <dig> large linkage groups and some small or non-integrated linkage groups; the total observed map length was  <dig>  cm, and the average marker interval was  <dig>  cm.
in general, the most efficient way to study the linkage of a given target gene in a mapping population for which no linkage map is available is to start with a high-density linkage map.
in a recent study on spruces, it was found that the golden gate single nucleotide polymorphism  assay system developed by illumina greatly facilitates the genetic mapping of species whose genomes have not been extensively studied  <cit> .
to design an snp genotyping array, it is necessary to identify a large number of snps.
in the case of c. japonica, a database of  <dig>  expressed sequence tags  has been constructed from cdna libraries obtained from seedlings, inner bark, female strobili, male strobili, pollen, leaves, vegetative buds and heartwood; this database is freely-available on the internet   <cit> .
a number of cdna-based sequence-tagged site  markers have been also identified using these ests  <cit> .
a library of c. japonica snps suitable for use in a golden gate snp array has been identified  <cit> , and complements the existing c. japonica est databases, which will also be useful in identifying candidate genes associated with male gametophyte development and male sterility on the basis of sequence similarity and microarray expression analysis.
this paper reports a study in which a high-density linkage map for c. japonica was constructed and the mapping population was subjected to the goldengate genotyping assay.
using information from the high-density linkage map, we mapped the ms <dig> gene and constructed a partial linkage map around the ms <dig> locus.
this allowed us to identify snp markers that are tightly linked to the ms <dig> gene for use in mas.
the paper also includes a discussion of the importance of high-density maps and mas markers in tree breeding.
methods
plant materials and dna extraction
a high density linkage map for the yi pedigree of c. japonica was constructed using  <dig> half-sib progenies from crosses between two f <dig> plants  × yi <dig> )   <cit> .
in addition, to avoid the effects of inbreeding depression, a partial linkage map around the ms <dig> locus  was constructed for  <dig> progenies of the to-s pedigree, which was derived from a cross between a to <dig> f <dig> plant and the elite tree suzu- <dig> .
the to <dig> f <dig> plant was a cross between 'toyama <dig> ' and 'ohara- <dig> ' and is thus a male-fertile tree that is heterozygous for the ms <dig> gene .
suzu- <dig> is also male-fertile and heterozygous for the ms <dig> gene   <cit> .
of the  <dig> to-s progeny,  <dig> were male-fertile and  <dig> were male-sterile.
the actual segregation ratio thus deviated significantly from the expected ratio of 3: <dig> ; the reason for this is as yet unknown.
needle tissue was collected from the parents and progeny of both the yi and to-s pedigrees.
genomic dna was extracted from individual needles using a modification of the ctab method  <cit> .
for use in the illumina goldengate assay, the extracted dna was purified using a genomic dna purification kit  and its concentration was standardized .
genetic markers
for the yi pedigree, six kinds of genetic markers were used to construct the linkage map: cleaved amplified polymorphic sequences  markers, restriction fragment length polymorphism  markers, microsatellite  markers, est-derived microsatellite  markers, amplicon length polymorphism  markers and single nucleotide polymorphisms  merkers.
the segregation data for  <dig> capss,  <dig> rflps,  <dig> ssrs,  <dig> alp and  <dig> snp markers were obtained in the previous study  <cit> .
in this study, we added  <dig> est-ssr markers and  <dig> snp markers including  <dig> gsnps,  <dig> hrmsnps,  <dig> sssnps and  <dig> measnps to the linkage map for the yi pedigree .
detailed information on these markers can be found at sugi genome database website .
snp genotyping for randomly-developed markers
a large set of snp markers was genotyped using the goldengate assay; collectively, this set is referred to as "gsnps".
for the yi pedigree, multiplexed genotyping of the gsnp markers was carried out using the 1536-plex goldengate array, in accordance with the manufacturer's protocol.
a detailed description of the procedures employed and results obtained in the course of discovering these snps can be found elsewhere  <cit> .
a total of  <dig> - <dig>  μg of genomic dna per sample  was used in the goldengate assay.
to screen the gsnp markers linked to the ms <dig> gene,  <dig> male-sterile progenies and the parents of to-s pedigree were also genotyped using this assay, since the gsnp markers found to be monomorphic in the yi pedigree were excluded from linkage mapping in the to-s pedigree.
the goldengate assay employs highly multiplexed allele-specific extension methods and universal pcr amplification reactions.
the pcr products, which were fluorescently labeled by the incorporation of 5'-labeled primers p <dig>  and p <dig> , were hybridized to capture probes on the beads in the array.
the ratio of the fluorescent signals from  <dig> allele-specific ligation products was used to determine the sample's genotype.
signal intensity data processing, clustering and genotype calling were performed using the genotyping module in the beadstudio software .
genotyping was conducted exclusively on the basis of snps with an illumina gentrain score in excess of  <dig> ; the gentrain score provides a measure of the reliability of snp detection based on the distribution of genotypic classes.
for each analyzed snp, individual genotypes with an illumina gencall score below  <dig>  were excluded; the gencall score provides a measure of the reliability of an individual snp call relative to the distribution of genotypic classes.
high resolution melting  analysis was also used to obtain snp genotyping data for linkage map construction; the snps identified in this way are henceforth referred to as "hrmsnps".
the development procedure and analyzed condition was reported elsewhere  <cit> .
the linkage map also incorporated est-ssr markers; details on the use of this data have been reported previously  <cit> .
screening and genotyping of candidate genes
sequence information for genes related to male gametophyte development and sterility were collected by reviewing the literature on other plant species such as arabidopsis  <cit> , brassica  <cit> , oryza  <cit> , nicotiana  <cit>  and petunia  <cit> .
the sequences from these publications were then compared to the available c. japonica est sequences using tblastn   <cit> .
in addition, genes related to male-sterility were screened by comprehensive expression analysis using a microarray containing around  <dig>  probes derived from  <dig>  tentative consensus sequences obtained from ests.
genes that exhibited at least a four-fold difference in expression between male-sterile and male-fertile strobili during the end of september and mid-october were selected for further analysis .
these time points were chosen because they represent the periods immediately before and immediately after male gametogenesis was observed in male-sterile strobili.
to identify snp markers, pcr primers for the selected candidate genes were designed using the primer <dig> software.
pcr amplifications were carried out using a model  <dig> geneamp pcr system  in reaction mixtures with a total volume of  <dig> μl containing  <dig> mmol/l tris-hcl ,  <dig> mmol/l kcl,  <dig> mmol/l mgcl <dig>   <dig>  mmol/l of each deoxynucleoside triphosphate,  <dig>  μmol/l of each primer,  <dig> ng template dna, and  <dig>  units of taq polymerase .
the following thermal profile was used:  <dig> min denaturation at 94°c, followed by  <dig> cycles of  <dig> s denaturation at 94°c,  <dig> s annealing at 55-64°c, and  <dig> s extension at 72°c, with a final extension step of 72°c for  <dig> min.
amplification products were separated by electrophoresis in 2%  agarose gels run in  <dig> × tae buffer.
the gels were then stained with ethidium bromide and visualized under uv light.
each pcr fragment was sequenced using the bigdye terminator kit  and abi prism  <dig> dna sequencer  to identify snps in the parents of the yi pedigree .
primers for both the forward and the backward direction were used.
snp markers associated with genes having significant similarities to genes from other species that are known to be important in male gametophyte development and/or male-sterility were collectively referred to as sssnps, while snp markers associated with genes exhibiting differential expression between male-fertile and male-infertile individuals were collectively referred to as measnps.
the sssnp and measnp markers were genotyped by sequencing.
for the mapped measnp markers, proteins with high similarities to their original ests were identified from the ncbi refseq database using blastx   <cit>
construction of the linkage map for the yi pedigree
linkage analyses were conducted for all hrmsnp   <cit> , est-ssr  <cit> , gsnp  <cit> , sssnp  and measnp  markers that exhibited polymorphism in the parents of the yi pedigree.
the segregation data for caps, rflp, ssr, snp, and alp markers in the previous study  <cit>  were used together with the genotype data obtained in this work to construct a linkage map.
chi-squared tests were performed for each locus to assess its deviation from the expected mendelian segregation ratio.
loci exhibiting extreme segregation distortion  were excluded from further linkage analysis.
all linkage analyses were performed using the joinmap v <dig>  software with the parameter cp   <cit> .
during the construction of the maps, markers were assigned to tentative linkage groups using logarithm of odds ratio  thresholds of  <dig>  to  <dig> , with increments of  <dig> ; an lod threshold of  <dig>  was ultimately used when defining groups of markers.
map distances were calculated using the kosambi mapping function  <cit> .
for the other parameters such as recombination frequency threshold and a ripple value, default settings were used.
images of the linkage groups were drawn using the mapchart v <dig>  software  <cit> .
estimation of genome length and map coverage
the observed genome length  for the linkage map was calculated as the sum of the sizes of the linkage groups.
the expected genome length, ge, was estimated using method  <dig> of chakravarti et al.
<cit> , in which the total length of the linkage groups is multiplied by the factor /, where m is the number of markers in the linkage groups.
the observed map coverage, co, is the ratio of the observed and the estimated genome lengths, i.e.
go/ge.
the expected genome coverage, ce, was calculated using the equation of bishop et al.
<cit> :
ce=1−
here, r is the haploid number of chromosomes, n is the number of positioned loci, x is the maximum observed map distance between two adjacent assigned markers in cm at or above a minimum lod threshold value of  <dig> , and ge is the estimated genome length.
analysis of marker distribution
if the markers were randomly distributed and the genome is divided in n intervals, the number of markers per interval would follow a poisson distribution with a mean of μ. to determine whether the markers were randomly distributed, all linkage groups were divided into  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> cm intervals.
the number of intervals that contained markers were counted and the average number of markers per interval  was calculated.
if the average number of random occurrences per interval is μ, then the probability that x markers will fall within a given interval is
p=e-uxx!
we compared the actual distribution of markers to that expected for a poisson distribution using the chi-squared test as described by kang et al.
<cit> .
localization of ms <dig> gene
microsatellite markers were used to identify the linkage group on which the target gene is located because of their high polymorphism and straightforward analysis.
thus, to identify the linkage group containing the ms <dig> gene, a total of  <dig> microsatellite markers on the linkage map  that were polymorphic in the parents of the to-s pedigree were genotyped for  <dig> of the male-sterile progeny of the to-s pedigree.
if markers were linked to ms <dig>  they should be significantly deviated from segregation rates expected from parental genotypes.
pcr amplifications were carried out using the model  <dig> geneamp pcr system .
a reaction mixture with a total volume of  <dig> μl was used, consisting of  <dig> × multiplex pcr master mix , fluorescently-labeled forward primers , reverse primers , and  <dig> ng of genomic dna.
the following thermal profile was used:  <dig> min at 94°c, then  <dig> cycles of  <dig> sec at 94°c,  <dig> sec at 55-62°c,  <dig> sec at 72°c, followed by  <dig> min at 72°c; the results were analyzed using a  <dig> genetic analyzer .
the independence of the segregation of the ssr markers and the ms <dig> gene was investigated using chi-square tests to identify markers linked to the ms <dig> gene.
the construction of a partial linkage map around the ms <dig> locus
linkage analysis using est-ssr markers indicated that the ms <dig> gene was located in the 9th linkage group, which is hereafter referred to as "lg <dig> " the markers in lg <dig> that exhibited polymorphism in the parents of the to-s pedigree were therefore used to construct a partial linkage map around the ms <dig> locus.
in addition, gsnp markers that exhibited polymorphism in the parents of the to-s pedigree and whose segregation with the ms <dig> gene deviated significantly from that expected in the absence of linkage  in  <dig> male-sterile progenies were also used in the construction of the partial linkage map.
the markers of lg- <dig> in the to-s pedigree except two microsatellite markers and four caps markers were genotyped on a biomark  <dig>  dynamic array  using kaspar assays.
primer pairs suitable for the kaspar assays were designed on the basis of the sequences of the relevant markers ; see additional file  <dig>  a total of  <dig>  ng of genomic dna per sample  was used for specific target amplification ; the kaspar reactions were performed using the sta products after dilution by a factor of  <dig>  the primers were designed and the assays were performed as specified by the manufacturer.
the data obtained were analyzed using the fluidigm snp genotyping analysis software to obtain genotype calls.
linkage analyses for the to-s pedigree were performed using the same conditions as were used for the yi pedigree.
loci whose segregation patterns deviated significantly from mendelian ratios were not excluded from the further linkage analysis in the to-s pedigree because distortion of loci linked to the ms <dig> gene was expected in this case.
RESULTS
identifying snp markers associated with candidate genes related to male gametophyte development and male sterility
on the basis of the sequence similarity results,  <dig> primer pairs were designed for various candidate genes; of these,  <dig> generated pcr products that could be separated and identified after electrophoresis on a 2% agarose gel.
of these obtained stss,  <dig> were polymorphic in the parents of the yi pedigree .
in the microarray expression analysis, significant differences in expression were observed between trees with male-fertile and male-sterile strobili using probes derived from  <dig> different tentative consensus sequences.
primer pairs were designed on the basis of these sequences and the  <dig> measnp markers that exhibited polymorphism in the parents of the yi pedigree .
genotyping markers
a total of  <dig> gsnp markers from the  <dig> snp array  were successfully genotyped, of which  <dig> exhibited clear segregation within the yi pedigree.
in addition to these gsnp markers,  <dig> capss,  <dig> rflps,  <dig> microsatellites,  <dig> est-ssrs,  <dig> hrmsnps ,  <dig> snps,  <dig> alp,  <dig> sssnps and  <dig> measnps markers were also used in mapping.
<dig> of these markers exhibited significant deviations  from the expected mendelian ratios and were therefore excluded from further linkage analysis; among the excluded markers were  <dig> gsnps,  <dig> hrmsnps,  <dig> capss,  <dig> rflps,  <dig> ssrs,  <dig> sssnps and  <dig> measnps .
construction of a linkage map for the yi pedigree
of the  <dig> markers that exhibited no segregation distortion,  <dig> could be assigned to specific linkage groups; in total,  <dig> markers were mapped, including  <dig> gsnps,  <dig> hrmsnps,  <dig> capss,  <dig> rflps,  <dig> ssrs,  <dig> sssnps,  <dig> measnps,  <dig> est-ssrs,  <dig> snps and  <dig> alp .
the observed and estimated map lengths were  <dig>  cm  and  <dig>  cm , respectively; the observed and estimated map coverages were estimated to be  <dig> %  and  <dig> % , respectively.
the maximum observed map distance between two adjacent assigned markers in cm  was  <dig>  cm.
significant deviations from the poisson distribution of markers were observed for marker intervals of  <dig> cm,  <dig> cm,  <dig> cm,  <dig> cm,  <dig> cm,  <dig> cm and  <dig> cm , indicating that the  <dig> markers used to construct the linkage map for the yi pedigree were not randomly distributed .
linkage map for the yi pedigree is shown on the treegenes database .
the construction of a partial linkage map around the ms <dig> locus
<dig> ssrs were screened to identify markers linked to the ms <dig> gene.
independence testing was conducted using the chi-squared method; two suitable ssrs were found, both of which are on lg9: cjg0101s and cjg0177_s .
a partial linkage map around the ms <dig> locus was constructed for the to-s pedigree using the markers assigned to lg <dig> in the yi pedigree.
while gsnp markers were not used in the construction of the yi linkage map, those that appeared to be linked to the ms <dig> gene on the basis of the segregation patterns observed in  <dig> male-infertile to-s progeny were also used in the construction of the partial map.
in total,  <dig> markers were ultimately mapped in the vicinity of the ms <dig> locus .
three markers  identified by the candidate gene approach were mapped to lg <dig> but were not closely linked to the ms <dig> gene .
the yi map indicated that most of the sssnp and measnp markers considered were associated with other linkage groups.
the partial linkage map around the ms <dig> locus was constructed for the to-s pedigree is shown on the treegenes database .
the marker most tightly linked to the ms <dig> gene was hrmsnp970_sf, which was separated from it by only  <dig>  cm in the to-s pedigree.
discussion
a high-density linkage map for the yi pedigree
the success rate for snp genotyping  with the goldengate assay was  <dig> % in picea glauca  <cit> ,  <dig> % in p. mariana  <cit>  and  <dig> % in pinus taeda  <cit> ; these values are similar to those we observed for c. japonica , although the evaluation criteria used in this work differed slightly from those used in previous studies.
these values are slightly lower than that obtained in crops, e.g.
<dig> % in barley  <cit> ,  <dig> % in soybean  <cit>  and  <dig> % in maize  <cit> .
the lower conversion rate for conifers compared to food crops may be due to the complexity of conifer genomes, which would be expected to hamper the development of specific probes for the assay, as suggested by pavy et al.
<cit> .
nonetheless, the high success rate achieved in this work suggests that illumina's high-throughput goldengate snp genotyping assay is an efficient method for mapping est-based markers and enriching linkage maps for almost any species.
the goldengate assay was used to construct a high-density linkage map of c. japonica featuring  <dig> markers.
the observed map length  was  <dig>  cm, and the average marker interval was only  <dig>  cm.
a total of  <dig> distinct linkage groups were identified, which corresponds to the haploid number of chromosomes in c. japonica.
the number of mapped markers in c. japonica was higher and the average interval between markers was smaller than those reported for other conifers .
while anonymous markers such as aflps were used extensively in previous conifer linkage maps, most of the mapped markers in c. japonica were highly informative co-dominant markers derived from est.
the incorporation of est-based markers into the linkage map should facilitate comparative mapping between c. japonica and related species such as chamaecyparis obtusa, which is the second most important forestry tree species in japan; it has also been shown that around 30% of c. japonica est-based sts markers are directly applicable to c. obtusa  <cit> .
in addition, when est-based markers are identified in a target trait locus, they are generally more effective than other kinds of markers for identifying genes that affect the relevant traits  <cit> .
the genome length in c. japonica estimated in our study is  <dig>  cm .
the observed and expected genome coverages were  <dig>   and  <dig> % , respectively.
it thus appears that the c. japonica linkage map developed in this work is almost saturated.
as was shown to be the case in p. mariana  <cit> , chi-squared testing indicated that the distribution of markers in the c. japonica genome was non-random.
this suggests that there are marker-rich and marker-poor regions in the c. japonica linkage map.
if markers were distributed equally over the genome, they would be expected to concentrate in regions of suppressed recombination such as centromeric regions, in which the map distance between markers becomes shorter than their physical separation, as reported for barley  <cit>  and maize  <cit> .
feuillet and keller   <cit>  suggested that genes are not distributed randomly and there are gene-rich and gene-poor regions in species with large genomes.
the non-random distribution of markers in the c. japonica linkage map created in this work might thus reflect the distribution of genes in this species, since most of the mapped markers were based on ests.
to fill in the gaps, it would be desirable to add data on markers for non-coding regions such as genomic microsatellite markers or random genetic markers such as aflps.
it seems unlikely that any of the sssnp and measnp markers identified in this work correspond to the ms <dig> gene.
it is possible that this is because the genes associated with male gametophyte development and male sterility in other species are not closely related to the ms <dig> gene in c. japonica.
alternatively, the putative homologs detected in this study might not be orthologous to those involved in male gametophyte development in other species; instead, they may be paralogous, with similar domains.
while one would expect that genes involved in male gametogenesis would exhibit differential expression in male-sterile and male-fertile individuals, and that this difference would be detectable by analysing the microarray expression data, it is possible that the difference may be statistically insignificant owing to the limitations of the methodology.
future studies in this area should aim to address these issues.
in addition to the problems related to the identification of a specific sequence corresponding to ms <dig>  it should be noted that  <dig>  of the  <dig> candidate genes could not be located on the linkage map due to a lack of polymorphism in the parents of the yi pedigree or because the sequence data was very complex.
the efficiency of the mapping could potentially be improved to address these issues by designing primers that span exon/intron junctions.
once suitable candidate genes have been identified, they should be exploited in studies on the to-s pedigree.
a partial linkage map around the ms <dig> locus
although we were unable to assign a specific sequence for ms <dig>  we were able to determine that it is located on lg <dig> and to construct a partial linkage map around the ms <dig> locus .
to our knowledge, this is the first case in which a recessive major gene has been localized on a linkage map in conifers, although they have previously been identified using morphological and biochemical mutants  <cit> .
the closest marker to the ms <dig> locus was hrmsnp970_sf; the map distance between the locus and the marker was only  <dig>  cm.
by using the two closest markers to the ms <dig> locus , the male-sterility or -fertility of  <dig> % of the  <dig> individuals in the to-s pedigree could be accurately determined.
in theory, this will facilitate the selection of individuals that are heterozygous for the ms <dig> gene without needing to create control crosses.
the identification of these two adjacent markers will increase the viability of using mas in the to-s pedigree.
this will make it possible to perform early selection of germinated seedlings, which could be useful in that it would reduce the expenditure of time, labour, and space on the growing of seedlings.
while the two markers will be very useful for to-s pedigree, it will be necessary to identify the target gene itself to do mas in other pedigrees.
to this end, candidate gene approaches may become more useful as the amount of information on the species increases and techniques improve.
an alternative approach for identifying target genes is genome walking using a bac library.
the likelihood of isolating a gene using genome walking depends on the physical distance  in the target species.
on the basis of the estimated genome length calculated from the recombination rates observed in this study , the average physical distance per cm would be roughly  <dig>  mb.
this physical distance per cm in c. japonica suggests that the closest markers we identified may be around  <dig>  mb from ms <dig> locus.
as such, genome walking would be impractical with current methods, but it would be feasible to isolate the ms <dig> gene by bac walking if markers lying within  <dig>  cm of the target could be identified.
we have constructed a bac library for c. japonica that covers  <dig> times as much of the genome as the marker libraries employed in this work and relates to a mapping population with a large number of individuals .
the construction of a more dense linkage map around the ms <dig> gene will greatly facilitate its isolation or that of more tightly-linked markers.
CONCLUSIONS
we have constructed a high-density linkage map for c. japonica using expressed sequence-derived co-dominant snp markers that were primarily genotyped using the goldengate assay.
a total of  <dig> markers were assigned to  <dig> linkage groups with an observed map length of  <dig>  cm and a mean distance between adjacent markers of  <dig>  cm.
the number of linkage groups identified matches the basic chromosome number of c. japonica.
while other conifer linkage maps have largely relied on anonymous markers such as aflps, most of the markers mapped for c. japonica were highly informative co-dominant markers derived from ests.
the expected map coverage rate of this constructed linkage map was very high , indicating that the linkage map developed in this work is almost saturated.
the distribution of the mapped loci on the linkage map for the yi pedigree was not random, as demonstrated by a chi-squared test .
genetic male-sterility in c. japonica is known to be controlled by a major recessive gene .
we mapped the ms <dig> gene to the 9th linkage group and constructed a partial linkage map around the ms <dig> locus using information from the dense map constructed in this work.
a total of  <dig> markers were located on this partial linkage map.
a marker, hrmsnp970_sf that is tightly linked to the ms <dig> gene was identified; the two are separated by only  <dig>  cm.
the markers linked to ms <dig> identified in this work will facilitate the early selection of male-sterile trees, which should prove useful in c. japonica breeding programs.
authors' contributions
ym: preparation of manuscript, construction of linkage map, experimental work and genotyping for est-ssr, hrm and snp markers; tui: snp discovery, assistance with hrm analysis; ku: snp discovery; nf: screening of the candidate gene; ms: development of the to-s mapping population; su: design of primers for kaspar assays; am: assistance with sequencing; nt: provision of segregation data for rflp, caps, ssr and alp markers; ht: discovery of male-sterile tree in c. japonica; ks: providing the funding for the screening and genotyping of the candidate gene; yt: supervising the project, providing the funding for genotyping using the snp array.
all authors read and approved the final manuscript.
supplementary material
additional file 1
candidate markers developed from genes with significant similarity to genes related to male gametophyte development and male sterility in other plant species.
click here for file
additional file 2
candidate markers developed from genes differently expressed between male-fertile and male-sterile individuals.
click here for file
additional file 3
primer information of hrmsnp markers.
the method used for marker development has previously been reported by ujino-ihara et al.
.
click here for file
additional file 4
linkage association between microsatellite markers and a male-sterile gene  in c. japonica.
click here for file
additional file 5
primer information for biomark  <dig>  dynamic array  using kaspar assays.
click here for file
additional file 6
results of marker distribution analysis in each marker interval.
click here for file
acknowledgements
the authors would like to thank m. yano, k. ebana and t. shibaya of the qtl genomics research center at the national institute of agrobiological sciences for their technical advice regarding goldengate snp genotyping and for giving us permission to use their bead array reader.
the authors also thank h. iwata and t. hayashi for their kind advice on linkage analysis, and y. komatsu, m. koshiba and y. taguchi for their help with the laboratory experiments.
this work was partly supported by grant-in-aid  from the forest agency of japan and program for promotion of basic and applied researches for innovations in bio-oriented industry.

 high-density linkage maps facilitate not only the understanding of genome structure and comparative genomic studies, but also quantitative trait loci mapping and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection. mas is an effective method for accelerating the screening of target genes using tightly-linked molecular markers. effective mas for qtls or specific genes has been reported in crops such as barley, rice and tomato. however, it is difficult to employ mas in conifers, primarily because their genomes are very big and complex and are largely uncharacterized. indeed, very few dna markers linked to major genes have been reported in conifers; the only ones of note are some dominant markers linked to a major dominant resistance gene in pinus lambertiana, p. monticola, p. taeda, and p. thunbergii. sugi is an allogamous, diploid, wind-pollinated conifer species with a haploid chromosome number of. using flow cytometry, the dna content of its haploid cells was estimated to be pg/c, which corresponds to a haploid genome size of gb as calculated using the following expression: genome size = × × dna content. c. japonica is frequently used for commercial afforestation in japan, and about 45% of all the man-made forests of japan are composed of this species. however, since the 1970s, the incidence of c. japonica pollinosis in japan has risen in line with the increasing number of man-made c. japonica forests. today, c. japonica pollinosis is a serious social problem in japan, affecting almost 20% of the population. in a genetically male-sterile c. japonica tree whose sterility is determined by a major recessive gene was found in toyama prefecture; this gene is expected play an important role in breeding for reduced pollen dispersal. since the discovery of this male-sterile individual, considerable effort has been expended on characterizing male sterility in c. japonica, identifying male-sterile and plus-trees, creating artificial crosses between male-sterile and plus-trees, and propagating male-sterile trees. male-sterile trees are currently identified by direct inspection of the male strobili using a magnifying glass or a microscope. conversely, plus-trees that are heterozygous for ms have been identified by examining segregation data for the progeny arising from artificial crosses; such trees provide important breeding material for seed production while avoiding the problems associated with inbreeding depression. however, these methods are very time-, labour-, and space-intensive; as such, it would be extremely useful to develop a mas-based method for selecting trees carrying the male-fertile gene. a composite linkage map for c. japonica was constructed using data for two pedigrees, yi and ko. a total of markers were assigned to large linkage groups and some small or non-integrated linkage groups; the total observed map length was cm, and the average marker interval was cm. in general, the most efficient way to study the linkage of a given target gene in a mapping population for which no linkage map is available is to start with a high-density linkage map. in a recent study on spruces, it was found that the golden gate single nucleotide polymorphism assay system developed by illumina greatly facilitates the genetic mapping of species whose genomes have not been extensively studied. to design an snp genotyping array, it is necessary to identify a large number of snps. in the case of c. japonica, a database of expressed sequence tags has been constructed from cdna libraries obtained from seedlings, inner bark, female strobili, male strobili, pollen, leaves, vegetative buds and heartwood; this database is freely-available on the internet. a number of cdna-based sequence-tagged site markers have been also identified using these ests. a library of c. japonica snps suitable for use in a golden gate snp array has been identified, and complements the existing c. japonica est databases, which will also be useful in identifying candidate genes associated with male gametophyte development and male sterility on the basis of sequence similarity and microarray expression analysis. this paper reports a study in which a high-density linkage map for c. japonica was constructed and the mapping population was subjected to the goldengate genotyping assay. using information from the high-density linkage map, we mapped the ms gene and constructed a partial linkage map around the ms locus. this allowed us to identify snp markers that are tightly linked to the ms gene for use in mas. the paper also includes a discussion of the importance of high-density maps and mas markers in tree breeding. methods plant materials and dna extraction a high density linkage map for the yi pedigree of c. japonica was constructed using half-sib progenies from crosses between two f plants × yi ). in addition, to avoid the effects of inbreeding depression, a partial linkage map around the ms locus was constructed for progenies of the to-s pedigree, which was derived from a cross between a to f plant and the elite tree suzu-. the to f plant was a cross between'toyama' and'ohara-' and is thus a male-fertile tree that is heterozygous for the ms gene. suzu- is also male-fertile and heterozygous for the ms gene. of the to-s progeny, were male-fertile and were male-sterile. the actual segregation ratio thus deviated significantly from the expected ratio of 3:; the reason for this is as yet unknown. needle tissue was collected from the parents and progeny of both the yi and to-s pedigrees. genomic dna was extracted from individual needles using a modification of the ctab method. for use in the illumina goldengate assay, the extracted dna was purified using a genomic dna purification kit and its concentration was standardized. genetic markers for the yi pedigree, six kinds of genetic markers were used to construct the linkage map: cleaved amplified polymorphic sequences markers, restriction fragment length polymorphism markers, microsatellite markers, est-derived microsatellite markers, amplicon length polymorphism markers and single nucleotide polymorphisms merkers. the segregation data for capss, rflps, ssrs, alp and snp markers were obtained in the previous study. in this study, we added est-ssr markers and snp markers including gsnps, hrmsnps, sssnps and measnps to the linkage map for the yi pedigree. detailed information on these markers can be found at sugi genome database website. snp genotyping for randomly-developed markers a large set of snp markers was genotyped using the goldengate assay; collectively, this set is referred to as "gsnps". for the yi pedigree, multiplexed genotyping of the gsnp markers was carried out using the 1536-plex goldengate array, in accordance with the manufacturer's protocol. a detailed description of the procedures employed and results obtained in the course of discovering these snps can be found elsewhere. a total of - μg of genomic dna per sample was used in the goldengate assay. to screen the gsnp markers linked to the ms gene, male-sterile progenies and the parents of to-s pedigree were also genotyped using this assay, since the gsnp markers found to be monomorphic in the yi pedigree were excluded from linkage mapping in the to-s pedigree. the goldengate assay employs highly multiplexed allele-specific extension methods and universal pcr amplification reactions. the pcr products, which were fluorescently labeled by the incorporation of 5'-labeled primers p and p, were hybridized to capture probes on the beads in the array. the ratio of the fluorescent signals from allele-specific ligation products was used to determine the sample's genotype. signal intensity data processing, clustering and genotype calling were performed using the genotyping module in the beadstudio software. genotyping was conducted exclusively on the basis of snps with an illumina gentrain score in excess of; the gentrain score provides a measure of the reliability of snp detection based on the distribution of genotypic classes. for each analyzed snp, individual genotypes with an illumina gencall score below were excluded; the gencall score provides a measure of the reliability of an individual snp call relative to the distribution of genotypic classes. high resolution melting analysis was also used to obtain snp genotyping data for linkage map construction; the snps identified in this way are henceforth referred to as "hrmsnps". the development procedure and analyzed condition was reported elsewhere. the linkage map also incorporated est-ssr markers; details on the use of this data have been reported previously. screening and genotyping of candidate genes sequence information for genes related to male gametophyte development and sterility were collected by reviewing the literature on other plant species such as arabidopsis, brassica, oryza, nicotiana and petunia. the sequences from these publications were then compared to the available c. japonica est sequences using tblastn. in addition, genes related to male-sterility were screened by comprehensive expression analysis using a microarray containing around probes derived from tentative consensus sequences obtained from ests. genes that exhibited at least a four-fold difference in expression between male-sterile and male-fertile strobili during the end of september and mid-october were selected for further analysis. these time points were chosen because they represent the periods immediately before and immediately after male gametogenesis was observed in male-sterile strobili. to identify snp markers, pcr primers for the selected candidate genes were designed using the primer software. pcr amplifications were carried out using a model geneamp pcr system in reaction mixtures with a total volume of μl containing mmol/l tris-hcl, mmol/l kcl, mmol/l mgcl mmol/l of each deoxynucleoside triphosphate, μmol/l of each primer, ng template dna, and units of taq polymerase. the following thermal profile was used: min denaturation at 94°c, followed by cycles of s denaturation at 94°c, s annealing at 55-64°c, and s extension at 72°c, with a final extension step of 72°c for min. amplification products were separated by electrophoresis in 2% agarose gels run in × tae buffer. the gels were then stained with ethidium bromide and visualized under uv light. each pcr fragment was sequenced using the bigdye terminator kit and abi prism dna sequencer to identify snps in the parents of the yi pedigree. primers for both the forward and the backward direction were used. snp markers associated with genes having significant similarities to genes from other species that are known to be important in male gametophyte development and/or male-sterility were collectively referred to as sssnps, while snp markers associated with genes exhibiting differential expression between male-fertile and male-infertile individuals were collectively referred to as measnps. the sssnp and measnp markers were genotyped by sequencing. for the mapped measnp markers, proteins with high similarities to their original ests were identified from the ncbi refseq database using blastx construction of the linkage map for the yi pedigree linkage analyses were conducted for all hrmsnp, est-ssr, gsnp, sssnp and measnp markers that exhibited polymorphism in the parents of the yi pedigree. the segregation data for caps, rflp, ssr, snp, and alp markers in the previous study were used together with the genotype data obtained in this work to construct a linkage map. chi-squared tests were performed for each locus to assess its deviation from the expected mendelian segregation ratio. loci exhibiting extreme segregation distortion were excluded from further linkage analysis. all linkage analyses were performed using the joinmap v software with the parameter cp. during the construction of the maps, markers were assigned to tentative linkage groups using logarithm of odds ratio thresholds of to, with increments of; an lod threshold of was ultimately used when defining groups of markers. map distances were calculated using the kosambi mapping function. for the other parameters such as recombination frequency threshold and a ripple value, default settings were used. images of the linkage groups were drawn using the mapchart v software. estimation of genome length and map coverage the observed genome length for the linkage map was calculated as the sum of the sizes of the linkage groups. the expected genome length, ge, was estimated using method of chakravarti et al., in which the total length of the linkage groups is multiplied by the factor /, where m is the number of markers in the linkage groups. the observed map coverage, co, is the ratio of the observed and the estimated genome lengths, i.e. go/ge. the expected genome coverage, ce, was calculated using the equation of bishop et al.: ce=1− here, r is the haploid number of chromosomes, n is the number of positioned loci, x is the maximum observed map distance between two adjacent assigned markers in cm at or above a minimum lod threshold value of, and ge is the estimated genome length. analysis of marker distribution if the markers were randomly distributed and the genome is divided in n intervals, the number of markers per interval would follow a poisson distribution with a mean of μ. to determine whether the markers were randomly distributed, all linkage groups were divided into and cm intervals. the number of intervals that contained markers were counted and the average number of markers per interval was calculated. if the average number of random occurrences per interval is μ, then the probability that x markers will fall within a given interval is p=e-uxx! we compared the actual distribution of markers to that expected for a poisson distribution using the chi-squared test as described by kang et al.. localization of ms gene microsatellite markers were used to identify the linkage group on which the target gene is located because of their high polymorphism and straightforward analysis. thus, to identify the linkage group containing the ms gene, a total of microsatellite markers on the linkage map that were polymorphic in the parents of the to-s pedigree were genotyped for of the male-sterile progeny of the to-s pedigree. if markers were linked to ms they should be significantly deviated from segregation rates expected from parental genotypes. pcr amplifications were carried out using the model geneamp pcr system. a reaction mixture with a total volume of μl was used, consisting of × multiplex pcr master mix, fluorescently-labeled forward primers, reverse primers, and ng of genomic dna. the following thermal profile was used: min at 94°c, then cycles of sec at 94°c, sec at 55-62°c, sec at 72°c, followed by min at 72°c; the results were analyzed using a genetic analyzer. the independence of the segregation of the ssr markers and the ms gene was investigated using chi-square tests to identify markers linked to the ms gene. the construction of a partial linkage map around the ms locus linkage analysis using est-ssr markers indicated that the ms gene was located in the 9th linkage group, which is hereafter referred to as "lg " the markers in lg that exhibited polymorphism in the parents of the to-s pedigree were therefore used to construct a partial linkage map around the ms locus. in addition, gsnp markers that exhibited polymorphism in the parents of the to-s pedigree and whose segregation with the ms gene deviated significantly from that expected in the absence of linkage in male-sterile progenies were also used in the construction of the partial linkage map. the markers of lg- in the to-s pedigree except two microsatellite markers and four caps markers were genotyped on a biomark dynamic array using kaspar assays. primer pairs suitable for the kaspar assays were designed on the basis of the sequences of the relevant markers; see additional file a total of ng of genomic dna per sample was used for specific target amplification; the kaspar reactions were performed using the sta products after dilution by a factor of the primers were designed and the assays were performed as specified by the manufacturer. the data obtained were analyzed using the fluidigm snp genotyping analysis software to obtain genotype calls. linkage analyses for the to-s pedigree were performed using the same conditions as were used for the yi pedigree. loci whose segregation patterns deviated significantly from mendelian ratios were not excluded from the further linkage analysis in the to-s pedigree because distortion of loci linked to the ms gene was expected in this case.  identifying snp markers associated with candidate genes related to male gametophyte development and male sterility on the basis of the sequence similarity results, primer pairs were designed for various candidate genes; of these, generated pcr products that could be separated and identified after electrophoresis on a 2% agarose gel. of these obtained stss, were polymorphic in the parents of the yi pedigree. in the microarray expression analysis, significant differences in expression were observed between trees with male-fertile and male-sterile strobili using probes derived from different tentative consensus sequences. primer pairs were designed on the basis of these sequences and the measnp markers that exhibited polymorphism in the parents of the yi pedigree. genotyping markers a total of gsnp markers from the snp array were successfully genotyped, of which exhibited clear segregation within the yi pedigree. in addition to these gsnp markers, capss, rflps, microsatellites, est-ssrs, hrmsnps, snps, alp, sssnps and measnps markers were also used in mapping. of these markers exhibited significant deviations from the expected mendelian ratios and were therefore excluded from further linkage analysis; among the excluded markers were gsnps, hrmsnps, capss, rflps, ssrs, sssnps and measnps. construction of a linkage map for the yi pedigree of the markers that exhibited no segregation distortion, could be assigned to specific linkage groups; in total, markers were mapped, including gsnps, hrmsnps, capss, rflps, ssrs, sssnps, measnps, est-ssrs, snps and alp. the observed and estimated map lengths were cm and cm, respectively; the observed and estimated map coverages were estimated to be % and %, respectively. the maximum observed map distance between two adjacent assigned markers in cm was cm. significant deviations from the poisson distribution of markers were observed for marker intervals of cm, cm, cm, cm, cm, cm and cm, indicating that the markers used to construct the linkage map for the yi pedigree were not randomly distributed. linkage map for the yi pedigree is shown on the treegenes database. the construction of a partial linkage map around the ms locus ssrs were screened to identify markers linked to the ms gene. independence testing was conducted using the chi-squared method; two suitable ssrs were found, both of which are on lg9: cjg0101s and cjg0177_s. a partial linkage map around the ms locus was constructed for the to-s pedigree using the markers assigned to lg in the yi pedigree. while gsnp markers were not used in the construction of the yi linkage map, those that appeared to be linked to the ms gene on the basis of the segregation patterns observed in male-infertile to-s progeny were also used in the construction of the partial map. in total, markers were ultimately mapped in the vicinity of the ms locus. three markers identified by the candidate gene approach were mapped to lg but were not closely linked to the ms gene. the yi map indicated that most of the sssnp and measnp markers considered were associated with other linkage groups. the partial linkage map around the ms locus was constructed for the to-s pedigree is shown on the treegenes database. the marker most tightly linked to the ms gene was hrmsnp970_sf, which was separated from it by only cm in the to-s pedigree. discussion a high-density linkage map for the yi pedigree the success rate for snp genotyping with the goldengate assay was % in picea glauca, % in p. mariana and % in pinus taeda; these values are similar to those we observed for c. japonica, although the evaluation criteria used in this work differed slightly from those used in previous studies. these values are slightly lower than that obtained in crops, e.g. % in barley, % in soybean and % in maize. the lower conversion rate for conifers compared to food crops may be due to the complexity of conifer genomes, which would be expected to hamper the development of specific probes for the assay, as suggested by pavy et al.. nonetheless, the high success rate achieved in this work suggests that illumina's high-throughput goldengate snp genotyping assay is an efficient method for mapping est-based markers and enriching linkage maps for almost any species. the goldengate assay was used to construct a high-density linkage map of c. japonica featuring markers. the observed map length was cm, and the average marker interval was only cm. a total of distinct linkage groups were identified, which corresponds to the haploid number of chromosomes in c. japonica. the number of mapped markers in c. japonica was higher and the average interval between markers was smaller than those reported for other conifers. while anonymous markers such as aflps were used extensively in previous conifer linkage maps, most of the mapped markers in c. japonica were highly informative co-dominant markers derived from est. the incorporation of est-based markers into the linkage map should facilitate comparative mapping between c. japonica and related species such as chamaecyparis obtusa, which is the second most important forestry tree species in japan; it has also been shown that around 30% of c. japonica est-based sts markers are directly applicable to c. obtusa. in addition, when est-based markers are identified in a target trait locus, they are generally more effective than other kinds of markers for identifying genes that affect the relevant traits. the genome length in c. japonica estimated in our study is cm. the observed and expected genome coverages were and %, respectively. it thus appears that the c. japonica linkage map developed in this work is almost saturated. as was shown to be the case in p. mariana, chi-squared testing indicated that the distribution of markers in the c. japonica genome was non-random. this suggests that there are marker-rich and marker-poor regions in the c. japonica linkage map. if markers were distributed equally over the genome, they would be expected to concentrate in regions of suppressed recombination such as centromeric regions, in which the map distance between markers becomes shorter than their physical separation, as reported for barley and maize. feuillet and keller suggested that genes are not distributed randomly and there are gene-rich and gene-poor regions in species with large genomes. the non-random distribution of markers in the c. japonica linkage map created in this work might thus reflect the distribution of genes in this species, since most of the mapped markers were based on ests. to fill in the gaps, it would be desirable to add data on markers for non-coding regions such as genomic microsatellite markers or random genetic markers such as aflps. it seems unlikely that any of the sssnp and measnp markers identified in this work correspond to the ms gene. it is possible that this is because the genes associated with male gametophyte development and male sterility in other species are not closely related to the ms gene in c. japonica. alternatively, the putative homologs detected in this study might not be orthologous to those involved in male gametophyte development in other species; instead, they may be paralogous, with similar domains. while one would expect that genes involved in male gametogenesis would exhibit differential expression in male-sterile and male-fertile individuals, and that this difference would be detectable by analysing the microarray expression data, it is possible that the difference may be statistically insignificant owing to the limitations of the methodology. future studies in this area should aim to address these issues. in addition to the problems related to the identification of a specific sequence corresponding to ms it should be noted that of the candidate genes could not be located on the linkage map due to a lack of polymorphism in the parents of the yi pedigree or because the sequence data was very complex. the efficiency of the mapping could potentially be improved to address these issues by designing primers that span exon/intron junctions. once suitable candidate genes have been identified, they should be exploited in studies on the to-s pedigree. a partial linkage map around the ms locus although we were unable to assign a specific sequence for ms we were able to determine that it is located on lg and to construct a partial linkage map around the ms locus. to our knowledge, this is the first case in which a recessive major gene has been localized on a linkage map in conifers, although they have previously been identified using morphological and biochemical mutants. the closest marker to the ms locus was hrmsnp970_sf; the map distance between the locus and the marker was only cm. by using the two closest markers to the ms locus, the male-sterility or -fertility of % of the individuals in the to-s pedigree could be accurately determined. in theory, this will facilitate the selection of individuals that are heterozygous for the ms gene without needing to create control crosses. the identification of these two adjacent markers will increase the viability of using mas in the to-s pedigree. this will make it possible to perform early selection of germinated seedlings, which could be useful in that it would reduce the expenditure of time, labour, and space on the growing of seedlings. while the two markers will be very useful for to-s pedigree, it will be necessary to identify the target gene itself to do mas in other pedigrees. to this end, candidate gene approaches may become more useful as the amount of information on the species increases and techniques improve. an alternative approach for identifying target genes is genome walking using a bac library. the likelihood of isolating a gene using genome walking depends on the physical distance in the target species. on the basis of the estimated genome length calculated from the recombination rates observed in this study, the average physical distance per cm would be roughly mb. this physical distance per cm in c. japonica suggests that the closest markers we identified may be around mb from ms locus. as such, genome walking would be impractical with current methods, but it would be feasible to isolate the ms gene by bac walking if markers lying within cm of the target could be identified. we have constructed a bac library for c. japonica that covers times as much of the genome as the marker libraries employed in this work and relates to a mapping population with a large number of individuals. the construction of a more dense linkage map around the ms gene will greatly facilitate its isolation or that of more tightly-linked markers.  we have constructed a high-density linkage map for c. japonica using expressed sequence-derived co-dominant snp markers that were primarily genotyped using the goldengate assay. a total of markers were assigned to linkage groups with an observed map length of cm and a mean distance between adjacent markers of cm. the number of linkage groups identified matches the basic chromosome number of c. japonica. while other conifer linkage maps have largely relied on anonymous markers such as aflps, most of the markers mapped for c. japonica were highly informative co-dominant markers derived from ests. the expected map coverage rate of this constructed linkage map was very high, indicating that the linkage map developed in this work is almost saturated. the distribution of the mapped loci on the linkage map for the yi pedigree was not random, as demonstrated by a chi-squared test. genetic male-sterility in c. japonica is known to be controlled by a major recessive gene. we mapped the ms gene to the 9th linkage group and constructed a partial linkage map around the ms locus using information from the dense map constructed in this work. a total of markers were located on this partial linkage map. a marker, hrmsnp970_sf that is tightly linked to the ms gene was identified; the two are separated by only cm. the markers linked to ms identified in this work will facilitate the early selection of male-sterile trees, which should prove useful in c. japonica breeding programs. authors' contributions ym: preparation of manuscript, construction of linkage map, experimental work and genotyping for est-ssr, hrm and snp markers; tui: snp discovery, assistance with hrm analysis; ku: snp discovery; nf: screening of the candidate gene; ms: development of the to-s mapping population; su: design of primers for kaspar assays; am: assistance with sequencing; nt: provision of segregation data for rflp, caps, ssr and alp markers; ht: discovery of male-sterile tree in c. japonica; ks: providing the funding for the screening and genotyping of the candidate gene; yt: supervising the project, providing the funding for genotyping using the snp array. all authors read and approved the final manuscript. supplementary material additional file 1 candidate markers developed from genes with significant similarity to genes related to male gametophyte development and male sterility in other plant species. click here for file additional file 2 candidate markers developed from genes differently expressed between male-fertile and male-sterile individuals. click here for file additional file 3 primer information of hrmsnp markers. the method used for marker development has previously been reported by ujino-ihara et al.. click here for file additional file 4 linkage association between microsatellite markers and a male-sterile gene in c. japonica. click here for file additional file 5 primer information for biomark dynamic array using kaspar assays. click here for file additional file 6 results of marker distribution analysis in each marker interval. click here for file acknowledgements the authors would like to thank m. yano, k. ebana and t. shibaya of the qtl genomics research center at the national institute of agrobiological sciences for their technical advice regarding goldengate snp genotyping and for giving us permission to use their bead array reader. the authors also thank h. iwata and t. hayashi for their kind advice on linkage analysis, and y. komatsu, m. koshiba and y. taguchi for their help with the laboratory experiments. this work was partly supported by grant-in-aid from the forest agency of japan and program for promotion of basic and applied researches for innovations in bio-oriented industry.
our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene for mas in c. japonica, a species that is important in japanese afforestation but which causes serious social pollinosis problems. we constructed a high-density saturated genetic linkage map for c. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism markers, most of which were genotyped using the goldengate genotyping assay. a total of markers were assigned to linkage groups with an observed map length of cm and a mean distance between two adjacent markers of cm; the number of linkage groups matched the basic chromosome number in c. japonica. the identification of markers that are tightly linked to the ms gene will facilitate the early selection of male-sterile trees, which should expedite c. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity. high-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection. using this map, we located ms on the 9th linkage group and constructed a partial linkage map around the ms locus. using the high-density map, we located the ms gene on the 9th linkage group and constructed a partial linkage map around the ms locus. the map distance between the ms gene and the tightly linked marker was only cm.
 high-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection. mas is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene for mas in c. japonica, a species that is important in japanese afforestation but which causes serious social pollinosis problems.  we constructed a high-density saturated genetic linkage map for c. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism markers, most of which were genotyped using the goldengate genotyping assay. a total of markers were assigned to linkage groups with an observed map length of cm and a mean distance between two adjacent markers of cm; the number of linkage groups matched the basic chromosome number in c. japonica. using this map, we located ms on the 9th linkage group and constructed a partial linkage map around the ms locus. this enabled us to identify a marker that is closely linked to the ms gene, being separated from it by only cm.  using the high-density map, we located the ms gene on the 9th linkage group and constructed a partial linkage map around the ms locus. the map distance between the ms gene and the tightly linked marker was only cm. the identification of markers that are tightly linked to the ms gene will facilitate the early selection of male-sterile trees, which should expedite c. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity. 