BACKGROUND
the genus nelumbo adans.
comprises two living species, n. nucifera gaertn.
and n. lutea pers.
<cit> .
the two species differ in features of their external morphologies, such as plant size, leaf shape, petal shape and color
<cit> , and have significant genetic differences as revealed by dna markers
<cit> .
the species share the same chromosome number  and there is no interspecific reproductive barrier
<cit> .
in asia, lotus has been cultivated as a food crop for its rhizomes and seeds and as an ornamental plant for its magnificent flowers, in addition to its propagation for reasons related to its cultural and religious significance.
although lotus is an important ornamental plant and a commercial crop, knowledge of the genomic constitution of nelumbo species is limited.
however, such information is a prerequisite for the identification of molecular markers linked to agronomic traits to facilitate breeding of improved lotus cultivars.
a genetic linkage map is a powerful research tool for studies on plant genetics.
such a map may provide insights into genome organization, evolution and comparative genomics with related species
<cit> .
from the perspective of improving crops, a genetic map is useful for map-based gene cloning, analysis of quantitative trait loci  underlying traits of economic importance, and molecular breeding using marker-assisted selection
<cit> .
several dna marker types, including random amplified polymorphic dna
<cit> , inter simple sequence repeats
<cit> , simple sequence repeats
<cit>  amplified fragment length polymorphisms
<cit> , and sequence-related amplified polymorphisms
<cit> , have been developed for nelumbo.
the high degree of genetic diversity with nelumbo revealed by these dna markers facilitates the development of genetic linkage maps.
linkage maps have been constructed for many species, but as yet no genetic map has been developed for nelumbo.
simple sequence repeats are tandem repeats of one to six nucleotides present in all eukaryotic genomes
<cit> .
given their codominant inheritance, high polymorphism, and abundant distribution throughout genomes, ssrs have been used widely for genetic mapping, comparative analysis, and qtl analysis in plants
<cit> .
extensive efforts have been made to develop ssr markers in nelumbo through genomic library screening using probes that contain repeated motifs and expressed sequence tags
<cit> .
however, the total number of currently developed ssr markers  is insufficient for genetic analysis in lotus.
moreover, such experimental approaches to developing ssr markers are laborious, time-consuming and costly.
therefore, there is a strong demand for additional ssr markers for lotus genome research and breeding.
with the ever-increasing number of dna sequences available in public databases, genomic sequences provide a more rapid and economic method for develop ssr markers.
based on ssrs developed from the genome sequence, high-density genetic linkage maps can be constructed
<cit> .
recently, we sequenced the genome of an ancient lotus cultivar, ‘chinese antique’, at  <dig> × coverage using a shotgun sequencing strategy, and the dna sequences were assembled into  <dig>  contigs.
the total length of all contigs was  <dig> mb, which represented  <dig> % of the estimated  <dig> mb lotus genome
<cit> .
the availability of lotus genomic sequences provides an excellent opportunity to survey ssr motifs at a genome-wide level.
the ssrs developed from the contig sequences can anchor corresponding contigs onto a genetic map and further establish direct links between genetic, physical, and sequence-based maps
<cit> .
therefore, a ssr-based reference genetic map is essential for assembly of the nelumbo genome sequences and for integration of the genetic and physical maps of nelumbo.
in the study reported herein, we identified ssrs from the contig sequences of the lotus cultivar ‘chinese antique’ and developed a new set of ssr markers to construct two genetic linkage maps.
our objectives were to:  assess the distribution of ssrs in the nelumbo genome;  develop a large number of ssr markers and evaluate ssr polymorphism between n. nucifera ‘chinese antique’ and n. lutea ‘al1’;  construct ssr-based reference linkage maps for asian lotus and american lotus; and  anchor the ssr-associated contigs to the genetic map and facilitate assembly of the contig sequences.
RESULTS
identification and distribution of ssrs in the genome
a total of  <dig>  ssr motifs with a minimum of five contiguous repeating units were identified within the contig sequences.
of the total ssrs identified, di- and tri-nucleotide repeat motifs were the most abundant repeat types, with frequencies of  <dig> % and  <dig> %, respectively .
tetra- and penta-nucleotide repeats were the least frequent repeat types .
the number of each major ssr type identified within the lotus genome is summarized in table
<dig>  the most abundant dinucleotide motifs were ag/ga  and at/ta .
however, rare cg/gc motifs were identified.
among trinucleotide repeats, the aag/aga/gaa motif was the most common , followed by the aat/ata/taa  and atg/gat/tga  motifs.
gc-rich trinucleotide repeats were the least frequent.
of tetra-nucleotide repeats, aaat/taaa/ataa/aata was the most abundant, accounting for  <dig> % of all tetra-nucleotide repeats, and followed by atac/taca/acat/cata .
among pentanucleotide repeats, agaag/gaaga/aagag/agaga/gagaa motif was more common than other combinations.
these data reflected ag/ga and at/ta repeat motifs were the most abundant ssrs in the n. nucifera genome.
the ssr motifs represent variants of both strands of the dna sequence .
development of ssr markers and detection of polymorphism
all microsatellites were selected for ssr marker development.
from the  <dig>  ssr-containing sequences in contigs,  <dig>  non-redundant ssr primer pairs were identified.
priority was given to di- and tri-nucleotide repeats, which accounted for  <dig> % and  <dig> % of the total number of ssr primers, respectively.
among the markers that contained dinucleotide repeats, the largest proportion was for ag/ga, followed by at/ta motifs .
only three markers that contained cg/gc motifs were identified because this motif was very rare in the genome sequences analyzed.
of the markers for trinucleotide repeats, the aag/aga/gaa motif was the most common , followed by the aat/ata/taa  and atg/gat/tga  motifs.
only  <dig> pairs of primers were tested for their amplification potential in the two parents and their six f1progenies.
primer sequence information, repeat motifs, amplicon sizes and polymorphism features for these  <dig> primers are listed in additional file
<dig>  among these primers,  <dig> amplified at least one fragment in the genetic materials, and  <dig> primers that produced ambiguous fragments or smears were excluded from the genotype analysis of the f <dig> lines.
of the successfully amplified primers , the number of alleles per primer ranged from one to seven with a mean of  <dig> , and a majority of them amplified two  or three  alleles.
for  <dig> pairs of primers,  <dig> showed polymorphism among all eight lines, and  <dig> showed polymorphism in the two parents but no segregation in the six lines of the f <dig> population.
finally,  <dig> ssr primers were used to analyze the genotypes of all f <dig> progenies, which produced  <dig> markers with an average of  <dig>  per primer .
analysis of genotypes in the f <dig> population
for  <dig> markers identified using the  <dig> novel ssr primers,  <dig>  segregated in the female parent and  <dig>  segregated in the male parent .
thirty-three out of  <dig> previously published ssr primers were polymorphic among the parents and six f <dig> progenies , which produced  <dig> segregating markers in all seedlings of the mapping population.
the average number of markers per primer was  <dig> .
among these  <dig> ssr markers,  <dig>  segregated in the female parent and  <dig>  segregated in the male parent .
of the  <dig> srap primers tested,  <dig> reproducibly amplified clear polymorphic fragments in the parents and all f <dig> progenies.
a total of  <dig> srap markers were identified in the mapping population .
on average, there were  <dig>  markers per primer combination, and the number of markers ranged from one to eight.
of the  <dig> srap markers,  <dig>  originated from the female parent and the remaining of  <dig> srap markers  originated from the male parent.
the goodness-of-fit of observed-to-expected allelic ratios was analyzed using the χ <dig> test.
of the total of  <dig> markers,  <dig>  showed a significant deviation from mendelian expectations .
<dig> markers deviated toward the female parent and  <dig> markers toward the male parent.
the  <dig> ssr distorted markers derived from the female parent showed no segregation in the f <dig> progenies, which indicated a high level of homozygosity in the female parent.
the  <dig> distorted markers were excluded from the linkage analysis because they could lead to false linkage between some markers or linkage groups.
the remaining  <dig> markers, which comprised  <dig> maternal markers and  <dig> paternal markers, showed the expected 1: <dig> segregation ratio and were used to establish the linkage groups.
construction of two linkage maps
a framework map was constructed using  <dig> ssr markers .
only  <dig> markers were identified in the maternal map and were assigned to eight small linkage groups  with a total length of  <dig>  cm.
the number of markers per linkage group varied from three to seven.
in contrast,  <dig> ssr markers were assigned to the paternal map, which consisted of eight major linkage groups  and three triplets  that covered a total length of  <dig>  cm.
the number of markers in lg1-m to lg8-m varied from eight to  <dig> with an average of  <dig>  .
to increase map coverage, srap markers were added to the framework map to construct an integrated map.
a total of  <dig> markers, including  <dig> novel ssr markers,  <dig> previously published ssr markers, and  <dig> srap markers were available for map construction.
the addition of srap markers to the framework map allowed the ssr markers that were originally unlinked or floating to be placed as accessory markers.
using the ssr marker orders in the framework map as the preferred orders,  <dig> markers composed of  <dig> ssr and  <dig> srap markers were identified in the maternal map.
these were assigned to seven linkage groups , with three to  <dig> markers in each group.
the map spanned  <dig>  cm with an average interval of  <dig>  cm between adjacent markers .
the paternal linkage map consisted of  <dig> markers distributed over  <dig> linkage groups .
there were from three to  <dig> markers per linkage group.
the paternal map spanned  <dig>  cm with an average interval of  <dig>  cm between adjacent markers.
the genetic length of each linkage group varied from  <dig>  cm  to  <dig>  cm  .
discussion
in the study, we analyzed the frequency of microsatellites identified from  <dig>  contig sequences of n. nucifera ‘chinese antique’.
the  <dig> mb of genomic sequence analyzed covers  <dig> % of the lotus genome.
one ssr was detected in every  <dig>  kb in the genome sequences.
the frequency of ssrs in lotus is much lower than that reported in the genome sequences of brassica <cit>  and rice
<cit> , but higher than the estimated frequency of ssrs in the genome of sorghum
<cit> .
in the contig sequences, dinucleotide repeat motifs  were the most frequently detected, followed by tri- , tetra-  and penta-nucleotide  motifs.
the most abundant dinucleotide motif detected was the ag/ga type , followed by at/ta  and ac/ca .
the most common trinucleotide motif detected was the aag/aga/gaa type, followed by aat/ata/taa and atg/gat/tga .
these motif types and their proportions in the lotus genome are in close agreement with the patterns observed in dicots such as arabidopsis <cit> , brassica <cit>  and papaya
<cit> , and in the monocot sorghum
<cit> , in which at and ag combinations of base pair motif types are predominant.
the distribution of ssrs in the lotus genome is different from those observed in humans and drosophila, in which ac is the most frequent dinucleotide repeat motif, followed by at and ag
<cit> .
the gc repeat motif is extremely rare in eukaryotic genomes, except in rice
<cit> , and was absent from the lotus genome.
these data suggested that ag and at motifs were rich in the lotus genome.
the pseudo-testcross strategy is suitable for construction of genetic maps using a f <dig> population and was applied first for genetic mapping in eucalyptus <cit> .
given that lotus is protogynous and cross-pollinated, a high level of heterozygosity is predicted for the genomes of nelumbo species.
with this prediction in mind, we created a f <dig> mapping population to construct genetic maps of nelumbo in the present study.
the two parents, n. nucifera ‘chinese antique’ and n. lutea ‘al1’, diverge strongly in their geographical distributions and important morphological traits, such as plant size, and shape and color of the leaves and petals.
the two parents also show considerable genetic differences
<cit> , which was confirmed here by the high polymorphism  detected using the novel ssr markers .
of  <dig> markers identified in the mapping population, the markers originated from the female parent  were less than those inherited from the male parent .
<dig> markers  showed distorted segregation at the p <  <dig>  level , which was higher than the distorted segregation ratio reported for dendrobium <cit> , strawberry
<cit>  and ryegrass
<cit> .
segregation distortion is a common phenomenon in mapping studies with a f <dig> population
<cit> .
distortion of segregation ratios may result from biological factors, such as genetic isolation mechanisms, chromosome loss, locus duplication, and gamete selection
<cit> .
nonbiological factors, such as scoring errors and sampling errors, also can lead to distortion in segregation ratios
<cit> .
in the present study, both biological factors and technical problems may have caused the observed segregation distortion in the f <dig> population.
the high level of homozygosity of the female parent ‘chinese antique’, which was unexpected, has contributed to the considerable segregation distortion in the f <dig> population.
hence,  <dig> of the markers with distorted segregation ratios derived from the female parent showed no segregation in the f <dig> progenies.
our recent analysis of the f <dig> population estimate heterozygosity to be  <dig> % for ‘chinese antique’, and  <dig>  for ‘al1’ .
thus, the similarly low heterozygosity in the male parent aggravated the distorted segregation in the f <dig> population.
we observed that  <dig> markers skewed to the male parent showed similar segregation ratios and were distributed in a group spanning  <dig>  cm .
molecular marker selection for genetic mapping is crucial for the credibility of linkage maps.
due to their inherited characteristics, ssr markers have many advantages in genetic mapping.
they can also serve as anchor markers during comparative mapping with related species
<cit> .
however, the high level of homozygosity of the female parent precluded the availability of ssr markers assigned to the maternal map.
moreover, using a single type of dna marker to construct a lotus linkage map would easily lead to uneven marker distribution and large intervals between adjacent markers.
therefore, in order to increase the coverage of the linkage map and reduce the gap between markers, srap markers were selected to analyze the genotypes of the f <dig> population.
given that srap primers usually amplify the genomic intron and exon regions of functional genes
<cit> , they complement the use of ssr markers.
when srap markers were added to the framework map constructed using ssr markers, the total length of linkage groups increased from  <dig>  to  <dig>  cm, and the average interval between two adjacent markers decreased from  <dig>  to  <dig>  cm.
moreover, ssr markers in lg4-f and lg6-f of the maternal framework map were incorporated into one linkage group lg1-f in the integrated map.
no large gaps  were detected in these genetic linkage maps .
thus, to some extent, the application of ssr and srap markers increased the length of the linkage map and reduced the distance between two adjacent markers.
using normally segregating markers, genetic linkage maps for nelumbo were constructed successfully .
the integrated linkage maps comprised  <dig> ssr and  <dig> srap markers.
these ssr markers were derived from  <dig> novel ssr primers and  <dig> previously published ssr primers, and were anchored to  <dig> contigs of ‘chinese antique’ .
the contig-based ssr markers can anchor corresponding contigs onto a genetic map and further establish direct links between genetic, physical, and sequence-based maps
<cit> .
this ssr-based linkage maps are important reference linkage maps with which to anchor contigs and assemble the genome sequence of nelumbo.
as a result, these  <dig> contigs were merged into  <dig> scaffolds .
in the following genome assembly work, on the basis of the order of ssr markers in linkage groups and single nucleotide polymorphism  markers identified by restriction-site associated dna sequencing  technology,  <dig>  contigs of lotus were assembled into approximately  <dig>  sequence scaffolds .
in theory, the number of linkage groups should be consistent with the number of haploid chromosomes.
as a genus comprising only diploid species, members of nelumbo have eight pairs of chromosomes.
in the present study,  <dig> and  <dig> linkage groups were detected for the female and male plants, respectively, based on ssr and srap markers .
thus, the numbers of linkage groups did not match with the haploid chromosome number.
it may be inferred that some linkage groups in the paternal map should correspond to one chromosome, but were divided because a large interval existed between the linkage group.
no markers were detected in the eighth linkage group used to create the maternal map.
this may be attributable to the low degree of heterozygosity in the maternal genome.
many preliminary genetic maps for other plant species usually contain a higher number of linkage groups than expected
<cit> .
because the mapping population used in the present study was the f <dig> generation of the cross between ‘chinese antique’ and ‘al1’, no recombination between homoeologous chromosomes was possible
<cit> .
therefore, the markers in the two parental linkage groups could not integrate into one group.
the high quality of the dna markers and the accuracy of genetic mapping were exemplified by the clear separation of linkage groups for ‘chinese antique’ and ‘al1’ .
to improve the linkage map, the number of male and female linkage groups should be supposed equal to the haploid chromosome number, and the density of linkage group should be increased.
the use of more advanced genotyping technology, such as high-throughput sequencing for snp discovery at the whole-genome or -transcriptome scale, would aid the construction of a complete genetic linkage map.
rad-seq of multiple individuals using illumina technology can identify and score thousands of snp markers randomly distributed across the target genome
<cit> .
the platform can effectively generate dense linkage maps for qtl analysis
<cit> .
our recent work involving the rad-seq approach identified  <dig>  snps in the present f <dig> mapping population and enabled construction of a high-density genetic map together with the ssr markers developed in the present study.
this paternal map spanned  <dig>  cm and comprised  <dig>  markers  in nine linkage groups .
using the common ssr markers as anchor markers, comparative analysis identified the collinearity of these linkage maps.
the genetic maps generated in the present study can serve as reference linkage maps of nelumbo species for efficient studies of comparative genetics, qtl analysis of traits of economic importance, and molecular breeding with mas.
CONCLUSIONS
we here present the first report of the construction of genetic linkage maps of nelumbo with ssr markers derived from sequenced contigs.
a total of  <dig>  non-redundant ssr markers were identified at a genome-wide level.
of  <dig> ssr primers that were selected,  <dig>  showed polymorphism among the two parental genotypes and six progenies of their f <dig> population.
using the normally segregating markers, two genetic maps for asian and american lotus were constructed, which comprised  <dig> markers that spanned  <dig>  cm.
ninety-seven contigs were anchored to linkage groups and were integrated into  <dig> scaffolds.
the genetic maps will accelerate the exploitation of genetic resources, qtl analysis, map-based gene cloning, and molecular breeding with mas in nelumbo.

 the genus nelumbo adans. comprises two living species, n. nucifera gaertn. and n. lutea pers.. the two species differ in features of their external morphologies, such as plant size, leaf shape, petal shape and color, and have significant genetic differences as revealed by dna markers. the species share the same chromosome number and there is no interspecific reproductive barrier. in asia, lotus has been cultivated as a food crop for its rhizomes and seeds and as an ornamental plant for its magnificent flowers, in addition to its propagation for reasons related to its cultural and religious significance. although lotus is an important ornamental plant and a commercial crop, knowledge of the genomic constitution of nelumbo species is limited. however, such information is a prerequisite for the identification of molecular markers linked to agronomic traits to facilitate breeding of improved lotus cultivars. a genetic linkage map is a powerful research tool for studies on plant genetics. such a map may provide insights into genome organization, evolution and comparative genomics with related species. from the perspective of improving crops, a genetic map is useful for map-based gene cloning, analysis of quantitative trait loci underlying traits of economic importance, and molecular breeding using marker-assisted selection. several dna marker types, including random amplified polymorphic dna, inter simple sequence repeats, simple sequence repeats amplified fragment length polymorphisms, and sequence-related amplified polymorphisms, have been developed for nelumbo. the high degree of genetic diversity with nelumbo revealed by these dna markers facilitates the development of genetic linkage maps. linkage maps have been constructed for many species, but as yet no genetic map has been developed for nelumbo. simple sequence repeats are tandem repeats of one to six nucleotides present in all eukaryotic genomes. given their codominant inheritance, high polymorphism, and abundant distribution throughout genomes, ssrs have been used widely for genetic mapping, comparative analysis, and qtl analysis in plants. extensive efforts have been made to develop ssr markers in nelumbo through genomic library screening using probes that contain repeated motifs and expressed sequence tags. however, the total number of currently developed ssr markers is insufficient for genetic analysis in lotus. moreover, such experimental approaches to developing ssr markers are laborious, time-consuming and costly. therefore, there is a strong demand for additional ssr markers for lotus genome research and breeding. with the ever-increasing number of dna sequences available in public databases, genomic sequences provide a more rapid and economic method for develop ssr markers. based on ssrs developed from the genome sequence, high-density genetic linkage maps can be constructed. recently, we sequenced the genome of an ancient lotus cultivar, ‘chinese antique’, at × coverage using a shotgun sequencing strategy, and the dna sequences were assembled into contigs. the total length of all contigs was mb, which represented % of the estimated mb lotus genome. the availability of lotus genomic sequences provides an excellent opportunity to survey ssr motifs at a genome-wide level. the ssrs developed from the contig sequences can anchor corresponding contigs onto a genetic map and further establish direct links between genetic, physical, and sequence-based maps. therefore, a ssr-based reference genetic map is essential for assembly of the nelumbo genome sequences and for integration of the genetic and physical maps of nelumbo. in the study reported herein, we identified ssrs from the contig sequences of the lotus cultivar ‘chinese antique’ and developed a new set of ssr markers to construct two genetic linkage maps. our objectives were to: assess the distribution of ssrs in the nelumbo genome; develop a large number of ssr markers and evaluate ssr polymorphism between n. nucifera ‘chinese antique’ and n. lutea ‘al1’; construct ssr-based reference linkage maps for asian lotus and american lotus; and anchor the ssr-associated contigs to the genetic map and facilitate assembly of the contig sequences.  identification and distribution of ssrs in the genome a total of ssr motifs with a minimum of five contiguous repeating units were identified within the contig sequences. of the total ssrs identified, di- and tri-nucleotide repeat motifs were the most abundant repeat types, with frequencies of % and %, respectively. tetra- and penta-nucleotide repeats were the least frequent repeat types. the number of each major ssr type identified within the lotus genome is summarized in table the most abundant dinucleotide motifs were ag/ga and at/ta. however, rare cg/gc motifs were identified. among trinucleotide repeats, the aag/aga/gaa motif was the most common, followed by the aat/ata/taa and atg/gat/tga motifs. gc-rich trinucleotide repeats were the least frequent. of tetra-nucleotide repeats, aaat/taaa/ataa/aata was the most abundant, accounting for % of all tetra-nucleotide repeats, and followed by atac/taca/acat/cata. among pentanucleotide repeats, agaag/gaaga/aagag/agaga/gagaa motif was more common than other combinations. these data reflected ag/ga and at/ta repeat motifs were the most abundant ssrs in the n. nucifera genome. the ssr motifs represent variants of both strands of the dna sequence. development of ssr markers and detection of polymorphism all microsatellites were selected for ssr marker development. from the ssr-containing sequences in contigs, non-redundant ssr primer pairs were identified. priority was given to di- and tri-nucleotide repeats, which accounted for % and % of the total number of ssr primers, respectively. among the markers that contained dinucleotide repeats, the largest proportion was for ag/ga, followed by at/ta motifs. only three markers that contained cg/gc motifs were identified because this motif was very rare in the genome sequences analyzed. of the markers for trinucleotide repeats, the aag/aga/gaa motif was the most common, followed by the aat/ata/taa and atg/gat/tga motifs. only pairs of primers were tested for their amplification potential in the two parents and their six f1progenies. primer sequence information, repeat motifs, amplicon sizes and polymorphism features for these primers are listed in additional file among these primers, amplified at least one fragment in the genetic materials, and primers that produced ambiguous fragments or smears were excluded from the genotype analysis of the f lines. of the successfully amplified primers, the number of alleles per primer ranged from one to seven with a mean of, and a majority of them amplified two or three alleles. for pairs of primers, showed polymorphism among all eight lines, and showed polymorphism in the two parents but no segregation in the six lines of the f population. finally, ssr primers were used to analyze the genotypes of all f progenies, which produced markers with an average of per primer. analysis of genotypes in the f population for markers identified using the novel ssr primers, segregated in the female parent and segregated in the male parent. thirty-three out of previously published ssr primers were polymorphic among the parents and six f progenies, which produced segregating markers in all seedlings of the mapping population. the average number of markers per primer was. among these ssr markers, segregated in the female parent and segregated in the male parent. of the srap primers tested, reproducibly amplified clear polymorphic fragments in the parents and all f progenies. a total of srap markers were identified in the mapping population. on average, there were markers per primer combination, and the number of markers ranged from one to eight. of the srap markers, originated from the female parent and the remaining of srap markers originated from the male parent. the goodness-of-fit of observed-to-expected allelic ratios was analyzed using the χ test. of the total of markers, showed a significant deviation from mendelian expectations. markers deviated toward the female parent and markers toward the male parent. the ssr distorted markers derived from the female parent showed no segregation in the f progenies, which indicated a high level of homozygosity in the female parent. the distorted markers were excluded from the linkage analysis because they could lead to false linkage between some markers or linkage groups. the remaining markers, which comprised maternal markers and paternal markers, showed the expected 1: segregation ratio and were used to establish the linkage groups. construction of two linkage maps a framework map was constructed using ssr markers. only markers were identified in the maternal map and were assigned to eight small linkage groups with a total length of cm. the number of markers per linkage group varied from three to seven. in contrast, ssr markers were assigned to the paternal map, which consisted of eight major linkage groups and three triplets that covered a total length of cm. the number of markers in lg1-m to lg8-m varied from eight to with an average of. to increase map coverage, srap markers were added to the framework map to construct an integrated map. a total of markers, including novel ssr markers, previously published ssr markers, and srap markers were available for map construction. the addition of srap markers to the framework map allowed the ssr markers that were originally unlinked or floating to be placed as accessory markers. using the ssr marker orders in the framework map as the preferred orders, markers composed of ssr and srap markers were identified in the maternal map. these were assigned to seven linkage groups, with three to markers in each group. the map spanned cm with an average interval of cm between adjacent markers. the paternal linkage map consisted of markers distributed over linkage groups. there were from three to markers per linkage group. the paternal map spanned cm with an average interval of cm between adjacent markers. the genetic length of each linkage group varied from cm to cm. discussion in the study, we analyzed the frequency of microsatellites identified from contig sequences of n. nucifera ‘chinese antique’. the mb of genomic sequence analyzed covers % of the lotus genome. one ssr was detected in every kb in the genome sequences. the frequency of ssrs in lotus is much lower than that reported in the genome sequences of brassica and rice, but higher than the estimated frequency of ssrs in the genome of sorghum. in the contig sequences, dinucleotide repeat motifs were the most frequently detected, followed by tri-, tetra- and penta-nucleotide motifs. the most abundant dinucleotide motif detected was the ag/ga type, followed by at/ta and ac/ca. the most common trinucleotide motif detected was the aag/aga/gaa type, followed by aat/ata/taa and atg/gat/tga. these motif types and their proportions in the lotus genome are in close agreement with the patterns observed in dicots such as arabidopsis, brassica and papaya, and in the monocot sorghum, in which at and ag combinations of base pair motif types are predominant. the distribution of ssrs in the lotus genome is different from those observed in humans and drosophila, in which ac is the most frequent dinucleotide repeat motif, followed by at and ag. the gc repeat motif is extremely rare in eukaryotic genomes, except in rice, and was absent from the lotus genome. these data suggested that ag and at motifs were rich in the lotus genome. the pseudo-testcross strategy is suitable for construction of genetic maps using a f population and was applied first for genetic mapping in eucalyptus. given that lotus is protogynous and cross-pollinated, a high level of heterozygosity is predicted for the genomes of nelumbo species. with this prediction in mind, we created a f mapping population to construct genetic maps of nelumbo in the present study. the two parents, n. nucifera ‘chinese antique’ and n. lutea ‘al1’, diverge strongly in their geographical distributions and important morphological traits, such as plant size, and shape and color of the leaves and petals. the two parents also show considerable genetic differences, which was confirmed here by the high polymorphism detected using the novel ssr markers. of markers identified in the mapping population, the markers originated from the female parent were less than those inherited from the male parent. markers showed distorted segregation at the p < level, which was higher than the distorted segregation ratio reported for dendrobium, strawberry and ryegrass. segregation distortion is a common phenomenon in mapping studies with a f population. distortion of segregation ratios may result from biological factors, such as genetic isolation mechanisms, chromosome loss, locus duplication, and gamete selection. nonbiological factors, such as scoring errors and sampling errors, also can lead to distortion in segregation ratios. in the present study, both biological factors and technical problems may have caused the observed segregation distortion in the f population. the high level of homozygosity of the female parent ‘chinese antique’, which was unexpected, has contributed to the considerable segregation distortion in the f population. hence, of the markers with distorted segregation ratios derived from the female parent showed no segregation in the f progenies. our recent analysis of the f population estimate heterozygosity to be % for ‘chinese antique’, and for ‘al1’. thus, the similarly low heterozygosity in the male parent aggravated the distorted segregation in the f population. we observed that markers skewed to the male parent showed similar segregation ratios and were distributed in a group spanning cm. molecular marker selection for genetic mapping is crucial for the credibility of linkage maps. due to their inherited characteristics, ssr markers have many advantages in genetic mapping. they can also serve as anchor markers during comparative mapping with related species. however, the high level of homozygosity of the female parent precluded the availability of ssr markers assigned to the maternal map. moreover, using a single type of dna marker to construct a lotus linkage map would easily lead to uneven marker distribution and large intervals between adjacent markers. therefore, in order to increase the coverage of the linkage map and reduce the gap between markers, srap markers were selected to analyze the genotypes of the f population. given that srap primers usually amplify the genomic intron and exon regions of functional genes, they complement the use of ssr markers. when srap markers were added to the framework map constructed using ssr markers, the total length of linkage groups increased from to cm, and the average interval between two adjacent markers decreased from to cm. moreover, ssr markers in lg4-f and lg6-f of the maternal framework map were incorporated into one linkage group lg1-f in the integrated map. no large gaps were detected in these genetic linkage maps. thus, to some extent, the application of ssr and srap markers increased the length of the linkage map and reduced the distance between two adjacent markers. using normally segregating markers, genetic linkage maps for nelumbo were constructed successfully. the integrated linkage maps comprised ssr and srap markers. these ssr markers were derived from novel ssr primers and previously published ssr primers, and were anchored to contigs of ‘chinese antique’. the contig-based ssr markers can anchor corresponding contigs onto a genetic map and further establish direct links between genetic, physical, and sequence-based maps. this ssr-based linkage maps are important reference linkage maps with which to anchor contigs and assemble the genome sequence of nelumbo. as a result, these contigs were merged into scaffolds. in the following genome assembly work, on the basis of the order of ssr markers in linkage groups and single nucleotide polymorphism markers identified by restriction-site associated dna sequencing technology, contigs of lotus were assembled into approximately sequence scaffolds. in theory, the number of linkage groups should be consistent with the number of haploid chromosomes. as a genus comprising only diploid species, members of nelumbo have eight pairs of chromosomes. in the present study, and linkage groups were detected for the female and male plants, respectively, based on ssr and srap markers. thus, the numbers of linkage groups did not match with the haploid chromosome number. it may be inferred that some linkage groups in the paternal map should correspond to one chromosome, but were divided because a large interval existed between the linkage group. no markers were detected in the eighth linkage group used to create the maternal map. this may be attributable to the low degree of heterozygosity in the maternal genome. many preliminary genetic maps for other plant species usually contain a higher number of linkage groups than expected. because the mapping population used in the present study was the f generation of the cross between ‘chinese antique’ and ‘al1’, no recombination between homoeologous chromosomes was possible. therefore, the markers in the two parental linkage groups could not integrate into one group. the high quality of the dna markers and the accuracy of genetic mapping were exemplified by the clear separation of linkage groups for ‘chinese antique’ and ‘al1’. to improve the linkage map, the number of male and female linkage groups should be supposed equal to the haploid chromosome number, and the density of linkage group should be increased. the use of more advanced genotyping technology, such as high-throughput sequencing for snp discovery at the whole-genome or -transcriptome scale, would aid the construction of a complete genetic linkage map. rad-seq of multiple individuals using illumina technology can identify and score thousands of snp markers randomly distributed across the target genome. the platform can effectively generate dense linkage maps for qtl analysis. our recent work involving the rad-seq approach identified snps in the present f mapping population and enabled construction of a high-density genetic map together with the ssr markers developed in the present study. this paternal map spanned cm and comprised markers in nine linkage groups. using the common ssr markers as anchor markers, comparative analysis identified the collinearity of these linkage maps. the genetic maps generated in the present study can serve as reference linkage maps of nelumbo species for efficient studies of comparative genetics, qtl analysis of traits of economic importance, and molecular breeding with mas.  we here present the first report of the construction of genetic linkage maps of nelumbo with ssr markers derived from sequenced contigs. a total of non-redundant ssr markers were identified at a genome-wide level. of ssr primers that were selected, showed polymorphism among the two parental genotypes and six progenies of their f population. using the normally segregating markers, two genetic maps for asian and american lotus were constructed, which comprised markers that spanned cm. ninety-seven contigs were anchored to linkage groups and were integrated into scaffolds. the genetic maps will accelerate the exploitation of genetic resources, qtl analysis, map-based gene cloning, and molecular breeding with mas in nelumbo. 
a genetic linkage map is an essential resource for plant genetic studies and crop improvement but has not been generated for nelumbo. the present study reports the first construction of genetic linkage maps for nelumbo, which can serve as reference linkage maps to accelerate characterization germplasm, genetic mapping for traits of economic interest, and molecular breeding with marker-assisted selection. we aimed to develop genomic simple sequence repeat markers from the genome sequence and construct two genetic maps for nelumbo to assist genome assembly and integration of a genetic map with the genome sequence. the normally segregating markers, which comprised newly developed ssrs, previously published ssrs and sequence-related amplified polymorphism markers, were used for genetic map construction. genetic mapping of ssr markers derived from sequenced contigs in nelumbo enabled the associated contigs to be anchored in the linkage map and facilitated assembly of the genome sequences of ‘chinese antique’. the map for american lotus was cm, and contained markers distributed in genetic linkage groups. ag/ga repeats constituted % of dinucleotide repeat motifs, followed by at/ta. the map for asian lotus was cm with markers distributed in seven linkage groups. of ssr primers tested, produced scorable alleles with an average of per primer, and showed polymorphism among two parental genotypes, n. nucifera ‘chinese antique’ and n. lutea ‘al1’, and six progenies of their f population. a total of ssr motifs were identified from the genome sequences. the number of markers per linkage group ranged from three to with an average genetic distance of cm between adjacent markers. moreover, ssr markers contained in linkage groups were anchored to genomic dna sequence contigs of ‘chinese antique’. di- and tri-nucleotide repeat motifs were the most abundant, and accounted for % and % of all ssrs, respectively.
 the genus nelumbo adans. comprises two living species, n. nucifera gaertan. and n. lutea pers.. a genetic linkage map is an essential resource for plant genetic studies and crop improvement but has not been generated for nelumbo. we aimed to develop genomic simple sequence repeat markers from the genome sequence and construct two genetic maps for nelumbo to assist genome assembly and integration of a genetic map with the genome sequence.  a total of ssr motifs were identified from the genome sequences. di- and tri-nucleotide repeat motifs were the most abundant, and accounted for % and % of all ssrs, respectively. ag/ga repeats constituted % of dinucleotide repeat motifs, followed by at/ta. of ssr primers tested, produced scorable alleles with an average of per primer, and showed polymorphism among two parental genotypes, n. nucifera ‘chinese antique’ and n. lutea ‘al1’, and six progenies of their f population. the normally segregating markers, which comprised newly developed ssrs, previously published ssrs and sequence-related amplified polymorphism markers, were used for genetic map construction. the map for asian lotus was cm with markers distributed in seven linkage groups. the map for american lotus was cm, and contained markers distributed in genetic linkage groups. the number of markers per linkage group ranged from three to with an average genetic distance of cm between adjacent markers. moreover, ssr markers contained in linkage groups were anchored to genomic dna sequence contigs of ‘chinese antique’. the contigs were merged into scaffolds.  genetic mapping of ssr markers derived from sequenced contigs in nelumbo enabled the associated contigs to be anchored in the linkage map and facilitated assembly of the genome sequences of ‘chinese antique’. the present study reports the first construction of genetic linkage maps for nelumbo, which can serve as reference linkage maps to accelerate characterization germplasm, genetic mapping for traits of economic interest, and molecular breeding with marker-assisted selection. genetic linkage mapgenome sequencenelumbosrapssr 