BACKGROUND
the genus brassica is one of the core genera within the tribe brassicea.
it comprises a large number of crops with a wide spectrum of morphological variation that can be cultivated under a variety of agro-climatic conditions.
brassicas provide vegetable oil, fresh and preserved vegetables, fodder and condiments, as well as being important sources of dietary fibre, vitamin c and nutritionally beneficial factors including anti-cancer compounds  <cit> .
there are six representative species in the brassica genus including three diploid species b. rapa , b. nigra  and b. oleracea , and three amphidiploids b. juncea , b. napus  and b. carinata .
the genetic relationships among these brassica species are well defined in u's triangle  <cit> .
one of the diploid species, b. rapa, comprises a variety of morphologically diverse cultivated types including chinese cabbage, misuna, aburana, flowering cabbage, turnip, turnip rape, yellow sarson, tatsoi and komatsuna, and these provide leaf heads, leaves, flowering stems, turnips and seeds, the productive organs for economical consumption  <cit> .
furthermore, b. rapa is an excellent model for studying polyploidy genome evolution owing to its paleohexaploid ancestry and its close evolutionary relationships with arabidopsis thaliana  <cit> .
the multi-national b. rapa genome sequencing project  was launched in  <dig> owing to the economical and biological importance of b. rapa, and the a <dig> chromosome was sequenced using traditional sanger technology  <cit> .
in  <dig>  rapid next generation sequencing technology was employed for b. rapa genome sequencing and a high density genetic map based on sequence-tagged markers is necessary to anchor the assembled scaffolds to chromosomes.
several maps concerning b. rapa have been published to be used as reference genetic maps for pseudochromosome assembly   <cit>  despite their coverage only allowing in total  <dig> % of the scaffolds being anchored on to chromosomes .
genetic mapping is important for understanding the origin of and relationships among the genomes of brassica species.
genetic linkage maps can also provide improved insight into genome organization and evolution through comparative mapping, and serve as the basis for genetic studies concerning various agronomic traits through the localization of major genes and quantitative trait loci .
furthermore, they can aid breeding programs with the development of marker assisted selection   <cit> .
more than  <dig> genetic linkage maps have been constructed for b. rapa using a range of marker types including restriction fragment length polymorphisms , random amplified polymorphic dna , amplified fragment length polymorphisms , sequence-related amplified polymorphisms  and simple sequence repeats   <cit> .
however, there are limited published data concerning sequence-tagged pcr-markers, predominantly ssrs, mapped in b. rapa  <cit> , particularly markers that could provide anchors for the b. rapa genome that are transferable to other mapping populations.
recent developments in sequencing technology have simplified and accelerated the discovery of sequence variants, enabling development of sequence-based markers including single nucleotide polymorphisms  and insertion/deletion polymorphisms   <cit> .
indels and snps are the markers of choice for high-resolution genetic mapping and association studies owing to their abundance and distribution throughout the genome  <cit> .
for example, a study investigating genetic variation on human chromosome  <dig> suggested that indels represent 18% of the polymorphisms on this chromosome  <cit> .
studies concerning genetic variation in a. thaliana have demonstrated that indels represent 34% of all genetic polymorphisms  <cit> .
furthermore, indels can contribute directly to a phenotype  <cit> , or can associate with a phenotype as a result of linkage disequilibrium  <cit> .
by re-sequencing  <dig>  sequence-tagged sites  in eight b. rapa genotypes, park et al.
identified and characterized  <dig>  indels in the gene space of the b. rapa genome  <cit> .
indel polymorphisms are the second most frequent type of polymorphism in the genome, and can be genotyped using simple procedures including the analysis of size polymorphisms of polymerase chain reaction  products on agarose gels  <cit> .
another advantage of indel markers is the improbability of two indel mutations being exactly the same length and at the same genomic position.
therefore, shared indels represent identity-by-descent  <cit> .
very few indel markers have been used to construct genetic linkage maps of b. rapa.
with the recent completion of the sequencing of the b. rapa genome , the development of whole genome-wide indel markers based on re-sequencing has become feasible, and this will be a useful resource for the international research community.
in this study, indel and ssr markers, both of which are sequence-tagged pcr markers, were used to construct a high resolution genetic map of b. rapa.
the map was used as a reference linkage map to anchor and orient sequence scaffolds for b. rapa genome assembly.
RESULTS
generation of markers and polymorphism survey
the process of genotyping indel polymorphisms was optimised.
a range of annealing temperatures from 55°c to 63°c for  <dig> primer pairs were tested, and the results demonstrated that annealing at 57°c produced favourable results for all primer pairs.
therefore, amplification of indels was accomplished using a single, uniform set of conditions with a denaturing temperature of 57°c.
the size of amplified dna fragments was within the range of 80- <dig> bp and 4- <dig> bp insertion/deletions were used as markers.
therefore, the pcr products could be separated using page .
a population named rcz16_dh with  <dig> doubled haploid  lines derived from an f <dig> cross between a chinese cabbage dh line  and a rapid cycling inbred line  was used for genetic map construction.
to construct the rcz16_dh map,  <dig> unique pcr-based indel markers for 'z16' and 'l144' were designed.
of these,  <dig>  yielded single pcr fragments and demonstrated polymorphism, eight  did not amplify any products and  <dig>  had no polymorphism.
among the  <dig> polymorphic indels,  <dig> yielded single pcr products with different lengths for the two parental lines, while  <dig> had amplicons in one of the parents only.
these  <dig> primer pairs were discarded to prevent false negatives when carrying out pcr.
an additional  <dig> indel markers were screened, and  <dig> were designed on the basis of the indel variations between 'chiifu-401-42' and 'kenshin';  <dig> pairs were designed on the basis of variations between 'chiifu-401-42' and 'l144'.
in total,  <dig> polymorphic indel primer pairs including  <dig> from 'z16' and 'l144',  <dig> from 'chiifu-401-42' and 'kenshin', and  <dig> from 'chiifu-401-42' and 'l144', were scored and used to generate the rcz16_dh genetic linkage map using  <dig> dh lines.
for assessment of ssrs,  <dig> ssrs from a range of sources were tested  <cit> ;  <dig> presented with polymorphic banding patterns between the parental lines and  <dig> easily scored ssrs were screened for the  <dig> dh lines.
of these, three ssr marker assays  detected more than one segregating locus.
the information concerning all mapped indel and ssr loci is presented in additional file  <dig>
construction of the rcz16_dh linkage map
a total of  <dig> markers including  <dig> indel markers and  <dig> ssr markers were assigned to  <dig> linkage groups  and designated as a01-a <dig>  corresponding to the previously published linkage maps  <cit> .
each of the ten linkage groups contained at least two previously published ssr markers that provided anchors to previously published maps, with the exception of chromosome a <dig> .
anchoring a <dig> by the two indels  located at the scaffolds on which the two bacs  were positioned was confirmed using three ssr markers, locating at a <dig> in the maps of vcs_dh, jwf3p and ckdh .
the linkage map covered a genetic distance of  <dig>  cm, with an average distance of  <dig>  cm between ordered adjacent markers .
the largest linkage group contained the maximum number of markers  for a <dig> and spanned  <dig>  cm, while the smallest contained the minimum number of markers  for a <dig>  with a length of  <dig>  cm.
the distribution of indels along the linkage groups ranged from  <dig> on a <dig> and a <dig> to  <dig> on a <dig>  and the distribution of ssrs ranged from  <dig> on a <dig> to  <dig> on a <dig>  the physical length of the b. rapa genome is approximately  <dig>  mbp .
the map defined herein represents average genetic and physical intervals of  <dig>  cm and  <dig>  kbp per marker, respectively.
therefore, it is currently the most saturated linkage map for b. rapa.
in the final linkage map, 90% of the covered genome had a marker within  <dig> cm.
however, there were still two gaps   <cit>  of  <dig> cm and  <dig> cm on a <dig> and a <dig>  respectively.
linkage
a
b
a adjacent markers >  <dig> cm; b interval distance between adjacent markers ≥ <dig> cm  <cit> .
alignment of the linkage groups to b. rapa pseudochromosomes
the high-resolution rcz16_dh genetic linkage map with  <dig> sequence-based markers was successfully used to anchor and orientate scaffolds for the genome assembly of b. rapa together with the three publicly available b. rapa genetic maps, vcs_dh, jwf3p  and ckdh  <cit> .
the markers of the rcz16_dh genetic linkage map were aligned to the b. rapa genome sequence using their primer sequences;  <dig> ssr markers and the  <dig> indel markers were mapped to a total of  <dig> scaffolds.
among these  <dig> positioned markers, three ssr markers  were detected on more than one segregating locus.
therefore, only  <dig> unique markers were used for anchoring  <dig> scaffolds of b. rapa, covering  <dig> % of the assembled genome.
the uniquely aligned markers used to anchor scaffolds ranged from  <dig> for a <dig> to  <dig> for a <dig>  the number of scaffolds anchored on to the chromosomes by these markers ranged from  <dig> for a <dig> to  <dig> for a <dig>  in  <dig> cases, more than one marker was located on a single scaffold, allowing  <dig> scaffolds to be oriented throughout the  <dig> chromosomes.
the details of the markers used to anchor the scaffolds are presented in table  <dig>
linkage
scaff.lg
a
b
a anchored scaffold length; b anchored scaffold coverage.
to compare the rcz16_dh map to the three publicly available genetic linkage maps, vcs_dh , jwf3p  and ck_dh , the bacs where the markers were located were used as the genetic loci.
these bacs were aligned to scaffold sequences and regarded as common genetic loci if they were within a range of  <dig> kbp in distance to the position of indels or ssrs on the rcz16_dh map.
two hundred  of the  <dig> markers on the rcz16_dh map located common loci on at least one of the three previously published maps.
using vcs_dh, jwf3p and ck_dh maps, there were  <dig> ,  <dig>  and  <dig>  scaffolds anchored to the corresponding chromosomes, respectively.
combining these three maps, the total coverage was  <dig> % of the b. rapa genome.
however, when taken together with the rcz16_dh map, the anchored scaffold number and the total coverage of the b. rapa genome increased to  <dig> and  <dig> %, respectively .
a the number of markers with genetic distance publicly available.
alignment of the rcz16_dh linkage map with the constructed pseudochromosomes verified the accuracy of the scaffold order and orientation .
the indel markers were developed from re-sequencing data, and were selected to develop indel markers from scaffold regions where there had been no marker in previous linkage maps.
as a result, the map enhanced the evaluation of the quality of sequence assembly.
the majority of the markers  were collinear with the sequence assembly.
in several assembly iterations, scaffold misplacement was visible as discontinuity or negative slopes.
discussion
this study concerns the comprehensive linkage analysis of b. rapa.
the map spans  <dig>  cm and is divided into ten linkage groups corresponding to the number of b. rapa chromosomes, with an average distance of  <dig>  cm between adjacent markers.
a conspicuous characteristic of the present map is that  <dig> markers, accounting for  <dig> % of the total mapped markers, are novel indel markers, which increases the number of sequence-based markers for b. rapa.
one of the main purposes of this linkage map is to support the b. rapa genome sequencing project to anchor and orient scaffolds onto the chromosomes.
using publically available genetic linkage maps for b. rapa including vcs_dh , jwf3p    <cit>  and ck_dh   <cit> ,  <dig> ,  <dig>  and  <dig>  scaffolds were anchored to chromosomes, respectively.
however, selecting markers that were evenly distributed along the genome and complementary to the previously reported maps, using the rcz16_dh map,  <dig>  scaffolds were anchored on to the chromosomes.
the rcz16_dh map alone covers more than  <dig> % of the b. rapa genome, indicating the potential of this sequence-based linkage map for linkage and qtl analysis.
in b. rapa, polymorphic dna loci are relatively frequent.
park et al.
demonstrated that the frequency of snps and indels were  <dig>  snps/kb and  <dig>  indels/kb by resequencing  <dig>  stss based on the  <dig> bac sequences of b. rapa ssp.
pekinensis cv chiifu-401- <dig>  <cit> .
among the  <dig>  sequence variants of b. rapa, approximately 24% were indels.
this high frequency of indels has also been reported for other species including maize  <cit> , sugar beet  <cit> , barley  <cit>  and arabidopsis  <cit> .
in the present study, pcr-based indel markers were developed using  <dig> to  <dig> bp pcr products, and the insertion/deletion size varied between  <dig> to  <dig> bp.
the indel polymorphisms were genotyped using a simple procedure that analyzed size polymorphisms of pcr products.
the diploid brassica genomes contain large replicated blocks of collinear segments within and between linkage groups.
these are thought to have derived from a polyploid ancestor, although the exact mechanism by which this occurred is debatable  <cit> .
in the linkage map generated herein, only three ssr markers detected multiple loci and no evidence of conserved blocks of synteny can be deduced from this.
this low level of detection of the replication within the genome could be due to the marker types used to construct the map.
the indel markers were developed directly from scaffold sequences by selecting unique indels, and ambiguous markers were excluded from the marker data set.
furthermore, ssr markers are usually located in non-coding sequences, which are less well conserved between replicated blocks than coding regions.
a total of  <dig> ssr markers are present in the map developed in this study, and  <dig> of these could be aligned to the scaffold sequences using stringent criterion of 100% match of the primer sequences.
among the  <dig> unmapped markers,  <dig> were designed on the basis of est sequences of b. oleracea .
for these markers, mismatches could exist within the primer sequences preventing them from being mapped to the scaffold sequence of b. rapa.
five of the six unmapped ssrs derived from b. rapa sequences were mapped to scaffolds with only one side primer, and this could be due to the gaps in the sequence assembly.
alignment of the rcz16_dh map to pseudochromosomes indicated high collinearity between the genetic and physical distance .
however, the alignment result demonstrated that there were outliers distributed on the linkage groups a <dig>  a <dig>  a <dig>  a <dig> and a <dig>  this could be due to scaffolds being too small or the genomic region having too little recombination to allow precise placements or orientations, as a relatively small mapping population was used in the present study.
the small population size could also lead to the relatively high statistical errors when there were missing marker data for some dh lines.
the other possible reason for the inconsistency of genetic and physical distances could be assembling errors as the indels were designed on the basis of the assembled scaffold sequences.
CONCLUSIONS
this study describes the use of sequence-based and highly polymorphic indel markers to construct a highly resolute reference genetic map of b. rapa.
the result is an improved resource for fine mapping of quantitative trait loci, identifying candidate genes and map-based gene isolation.

 the genus brassica is one of the core genera within the tribe brassicea. it comprises a large number of crops with a wide spectrum of morphological variation that can be cultivated under a variety of agro-climatic conditions. brassicas provide vegetable oil, fresh and preserved vegetables, fodder and condiments, as well as being important sources of dietary fibre, vitamin c and nutritionally beneficial factors including anti-cancer compounds. there are six representative species in the brassica genus including three diploid species b. rapa, b. nigra and b. oleracea, and three amphidiploids b. juncea, b. napus and b. carinata. the genetic relationships among these brassica species are well defined in u's triangle. one of the diploid species, b. rapa, comprises a variety of morphologically diverse cultivated types including chinese cabbage, misuna, aburana, flowering cabbage, turnip, turnip rape, yellow sarson, tatsoi and komatsuna, and these provide leaf heads, leaves, flowering stems, turnips and seeds, the productive organs for economical consumption. furthermore, b. rapa is an excellent model for studying polyploidy genome evolution owing to its paleohexaploid ancestry and its close evolutionary relationships with arabidopsis thaliana. the multi-national b. rapa genome sequencing project was launched in owing to the economical and biological importance of b. rapa, and the a chromosome was sequenced using traditional sanger technology. in rapid next generation sequencing technology was employed for b. rapa genome sequencing and a high density genetic map based on sequence-tagged markers is necessary to anchor the assembled scaffolds to chromosomes. several maps concerning b. rapa have been published to be used as reference genetic maps for pseudochromosome assembly despite their coverage only allowing in total % of the scaffolds being anchored on to chromosomes. genetic mapping is important for understanding the origin of and relationships among the genomes of brassica species. genetic linkage maps can also provide improved insight into genome organization and evolution through comparative mapping, and serve as the basis for genetic studies concerning various agronomic traits through the localization of major genes and quantitative trait loci. furthermore, they can aid breeding programs with the development of marker assisted selection. more than genetic linkage maps have been constructed for b. rapa using a range of marker types including restriction fragment length polymorphisms, random amplified polymorphic dna, amplified fragment length polymorphisms, sequence-related amplified polymorphisms and simple sequence repeats. however, there are limited published data concerning sequence-tagged pcr-markers, predominantly ssrs, mapped in b. rapa, particularly markers that could provide anchors for the b. rapa genome that are transferable to other mapping populations. recent developments in sequencing technology have simplified and accelerated the discovery of sequence variants, enabling development of sequence-based markers including single nucleotide polymorphisms and insertion/deletion polymorphisms. indels and snps are the markers of choice for high-resolution genetic mapping and association studies owing to their abundance and distribution throughout the genome. for example, a study investigating genetic variation on human chromosome suggested that indels represent 18% of the polymorphisms on this chromosome. studies concerning genetic variation in a. thaliana have demonstrated that indels represent 34% of all genetic polymorphisms. furthermore, indels can contribute directly to a phenotype, or can associate with a phenotype as a result of linkage disequilibrium. by re-sequencing sequence-tagged sites in eight b. rapa genotypes, park et al. identified and characterized indels in the gene space of the b. rapa genome. indel polymorphisms are the second most frequent type of polymorphism in the genome, and can be genotyped using simple procedures including the analysis of size polymorphisms of polymerase chain reaction products on agarose gels. another advantage of indel markers is the improbability of two indel mutations being exactly the same length and at the same genomic position. therefore, shared indels represent identity-by-descent. very few indel markers have been used to construct genetic linkage maps of b. rapa. with the recent completion of the sequencing of the b. rapa genome, the development of whole genome-wide indel markers based on re-sequencing has become feasible, and this will be a useful resource for the international research community. in this study, indel and ssr markers, both of which are sequence-tagged pcr markers, were used to construct a high resolution genetic map of b. rapa. the map was used as a reference linkage map to anchor and orient sequence scaffolds for b. rapa genome assembly.  generation of markers and polymorphism survey the process of genotyping indel polymorphisms was optimised. a range of annealing temperatures from 55°c to 63°c for primer pairs were tested, and the results demonstrated that annealing at 57°c produced favourable results for all primer pairs. therefore, amplification of indels was accomplished using a single, uniform set of conditions with a denaturing temperature of 57°c. the size of amplified dna fragments was within the range of 80- bp and 4- bp insertion/deletions were used as markers. therefore, the pcr products could be separated using page. a population named rcz16_dh with doubled haploid lines derived from an f cross between a chinese cabbage dh line and a rapid cycling inbred line was used for genetic map construction. to construct the rcz16_dh map, unique pcr-based indel markers for'z16' and'l144' were designed. of these, yielded single pcr fragments and demonstrated polymorphism, eight did not amplify any products and had no polymorphism. among the polymorphic indels, yielded single pcr products with different lengths for the two parental lines, while had amplicons in one of the parents only. these primer pairs were discarded to prevent false negatives when carrying out pcr. an additional indel markers were screened, and were designed on the basis of the indel variations between'chiifu-401-42' and'kenshin'; pairs were designed on the basis of variations between'chiifu-401-42' and'l144'. in total, polymorphic indel primer pairs including from'z16' and'l144', from'chiifu-401-42' and'kenshin', and from'chiifu-401-42' and'l144', were scored and used to generate the rcz16_dh genetic linkage map using dh lines. for assessment of ssrs, ssrs from a range of sources were tested; presented with polymorphic banding patterns between the parental lines and easily scored ssrs were screened for the dh lines. of these, three ssr marker assays detected more than one segregating locus. the information concerning all mapped indel and ssr loci is presented in additional file construction of the rcz16_dh linkage map a total of markers including indel markers and ssr markers were assigned to linkage groups and designated as a01-a corresponding to the previously published linkage maps. each of the ten linkage groups contained at least two previously published ssr markers that provided anchors to previously published maps, with the exception of chromosome a. anchoring a by the two indels located at the scaffolds on which the two bacs were positioned was confirmed using three ssr markers, locating at a in the maps of vcs_dh, jwf3p and ckdh. the linkage map covered a genetic distance of cm, with an average distance of cm between ordered adjacent markers. the largest linkage group contained the maximum number of markers for a and spanned cm, while the smallest contained the minimum number of markers for a with a length of cm. the distribution of indels along the linkage groups ranged from on a and a to on a and the distribution of ssrs ranged from on a to on a the physical length of the b. rapa genome is approximately mbp. the map defined herein represents average genetic and physical intervals of cm and kbp per marker, respectively. therefore, it is currently the most saturated linkage map for b. rapa. in the final linkage map, 90% of the covered genome had a marker within cm. however, there were still two gaps of cm and cm on a and a respectively. linkage a b a adjacent markers > cm; b interval distance between adjacent markers ≥ cm. alignment of the linkage groups to b. rapa pseudochromosomes the high-resolution rcz16_dh genetic linkage map with sequence-based markers was successfully used to anchor and orientate scaffolds for the genome assembly of b. rapa together with the three publicly available b. rapa genetic maps, vcs_dh, jwf3p and ckdh. the markers of the rcz16_dh genetic linkage map were aligned to the b. rapa genome sequence using their primer sequences; ssr markers and the indel markers were mapped to a total of scaffolds. among these positioned markers, three ssr markers were detected on more than one segregating locus. therefore, only unique markers were used for anchoring scaffolds of b. rapa, covering % of the assembled genome. the uniquely aligned markers used to anchor scaffolds ranged from for a to for a the number of scaffolds anchored on to the chromosomes by these markers ranged from for a to for a in cases, more than one marker was located on a single scaffold, allowing scaffolds to be oriented throughout the chromosomes. the details of the markers used to anchor the scaffolds are presented in table linkage scaff.lg a b a anchored scaffold length; b anchored scaffold coverage. to compare the rcz16_dh map to the three publicly available genetic linkage maps, vcs_dh, jwf3p and ck_dh, the bacs where the markers were located were used as the genetic loci. these bacs were aligned to scaffold sequences and regarded as common genetic loci if they were within a range of kbp in distance to the position of indels or ssrs on the rcz16_dh map. two hundred of the markers on the rcz16_dh map located common loci on at least one of the three previously published maps. using vcs_dh, jwf3p and ck_dh maps, there were, and scaffolds anchored to the corresponding chromosomes, respectively. combining these three maps, the total coverage was % of the b. rapa genome. however, when taken together with the rcz16_dh map, the anchored scaffold number and the total coverage of the b. rapa genome increased to and %, respectively. a the number of markers with genetic distance publicly available. alignment of the rcz16_dh linkage map with the constructed pseudochromosomes verified the accuracy of the scaffold order and orientation. the indel markers were developed from re-sequencing data, and were selected to develop indel markers from scaffold regions where there had been no marker in previous linkage maps. as a result, the map enhanced the evaluation of the quality of sequence assembly. the majority of the markers were collinear with the sequence assembly. in several assembly iterations, scaffold misplacement was visible as discontinuity or negative slopes. discussion this study concerns the comprehensive linkage analysis of b. rapa. the map spans cm and is divided into ten linkage groups corresponding to the number of b. rapa chromosomes, with an average distance of cm between adjacent markers. a conspicuous characteristic of the present map is that markers, accounting for % of the total mapped markers, are novel indel markers, which increases the number of sequence-based markers for b. rapa. one of the main purposes of this linkage map is to support the b. rapa genome sequencing project to anchor and orient scaffolds onto the chromosomes. using publically available genetic linkage maps for b. rapa including vcs_dh, jwf3p and ck_dh,, and scaffolds were anchored to chromosomes, respectively. however, selecting markers that were evenly distributed along the genome and complementary to the previously reported maps, using the rcz16_dh map, scaffolds were anchored on to the chromosomes. the rcz16_dh map alone covers more than % of the b. rapa genome, indicating the potential of this sequence-based linkage map for linkage and qtl analysis. in b. rapa, polymorphic dna loci are relatively frequent. park et al. demonstrated that the frequency of snps and indels were snps/kb and indels/kb by resequencing stss based on the bac sequences of b. rapa ssp. pekinensis cv chiifu-401-. among the sequence variants of b. rapa, approximately 24% were indels. this high frequency of indels has also been reported for other species including maize, sugar beet, barley and arabidopsis. in the present study, pcr-based indel markers were developed using to bp pcr products, and the insertion/deletion size varied between to bp. the indel polymorphisms were genotyped using a simple procedure that analyzed size polymorphisms of pcr products. the diploid brassica genomes contain large replicated blocks of collinear segments within and between linkage groups. these are thought to have derived from a polyploid ancestor, although the exact mechanism by which this occurred is debatable. in the linkage map generated herein, only three ssr markers detected multiple loci and no evidence of conserved blocks of synteny can be deduced from this. this low level of detection of the replication within the genome could be due to the marker types used to construct the map. the indel markers were developed directly from scaffold sequences by selecting unique indels, and ambiguous markers were excluded from the marker data set. furthermore, ssr markers are usually located in non-coding sequences, which are less well conserved between replicated blocks than coding regions. a total of ssr markers are present in the map developed in this study, and of these could be aligned to the scaffold sequences using stringent criterion of 100% match of the primer sequences. among the unmapped markers, were designed on the basis of est sequences of b. oleracea. for these markers, mismatches could exist within the primer sequences preventing them from being mapped to the scaffold sequence of b. rapa. five of the six unmapped ssrs derived from b. rapa sequences were mapped to scaffolds with only one side primer, and this could be due to the gaps in the sequence assembly. alignment of the rcz16_dh map to pseudochromosomes indicated high collinearity between the genetic and physical distance. however, the alignment result demonstrated that there were outliers distributed on the linkage groups a a a a and a this could be due to scaffolds being too small or the genomic region having too little recombination to allow precise placements or orientations, as a relatively small mapping population was used in the present study. the small population size could also lead to the relatively high statistical errors when there were missing marker data for some dh lines. the other possible reason for the inconsistency of genetic and physical distances could be assembling errors as the indels were designed on the basis of the assembled scaffold sequences.  this study describes the use of sequence-based and highly polymorphic indel markers to construct a highly resolute reference genetic map of b. rapa. the result is an improved resource for fine mapping of quantitative trait loci, identifying candidate genes and map-based gene isolation. 
alignment and orientation using ssr markers in common with existing b. rapa linkage maps allowed ten linkage groups to be identified, designated a01-a the total length of the linkage map was cm, with an average distance of cm between adjacent marker loci. pcr-based insertion/deletion markers were developed by re-sequencing the two parental lines. taken together with the previously available linkage maps, scaffolds were anchored on to the chromosomes and the total coverage of the genome was %. the multinational b. rapa genome sequencing project was launched in in next generation sequencing technology was used to sequence the b. rapa genome. this study concerns the construction of a reference genetic linkage map for brassica rapa, forming the backbone for anchoring sequence scaffolds of the b. rapa genome resulting from recent sequencing efforts. using the developed linkage map, scaffolds were anchored on to the chromosomes, encompassing more than % of the b. rapa genome. one hundred and nineteen doubled haploid lines derived from microspore cultures of an f cross between a chinese cabbage dh line and a rapid cycling inbred line were used to construct the linkage map. the development of this linkage map is vital for the integration of genome sequences and genetic information, and provides a useful resource for the international brassica research community. the lengths of linkage groups ranged from cm to cm for a and a respectively.
 brassica rapa is an economically important crop and a model plant for studies concerning polyploidization and the evolution of extreme morphology. the multinational b. rapa genome sequencing project was launched in in next generation sequencing technology was used to sequence the b. rapa genome. several maps concerning b. rapa pseudochromosome assembly have been published but their coverage of the genome is incomplete, anchoring approximately % of the scaffolds on to chromosomes. therefore, a new genetic map to aid pseudochromosome assembly is required.  this study concerns the construction of a reference genetic linkage map for brassica rapa, forming the backbone for anchoring sequence scaffolds of the b. rapa genome resulting from recent sequencing efforts. one hundred and nineteen doubled haploid lines derived from microspore cultures of an f cross between a chinese cabbage dh line and a rapid cycling inbred line were used to construct the linkage map. pcr-based insertion/deletion markers were developed by re-sequencing the two parental lines. the map comprises a total of markers including indels and ssrs. alignment and orientation using ssr markers in common with existing b. rapa linkage maps allowed ten linkage groups to be identified, designated a01-a the total length of the linkage map was cm, with an average distance of cm between adjacent marker loci. the lengths of linkage groups ranged from cm to cm for a and a respectively. using the developed linkage map, scaffolds were anchored on to the chromosomes, encompassing more than % of the b. rapa genome. taken together with the previously available linkage maps, scaffolds were anchored on to the chromosomes and the total coverage of the genome was %.  the development of this linkage map is vital for the integration of genome sequences and genetic information, and provides a useful resource for the international brassica research community. 