BACKGROUND
a linkage map is an essential framework for mapping traits of interest and is often the first step towards understanding genome evolution and aids genome assembly  <cit> .
with the advent of molecular genetic techniques and sophisticated statistical tools for linkage analysis, linkage maps have been constructed for some economically important aquaculture species, such as channel catfish, tilapia, asian seabass, european sea bass, shrimps, and oysters  <cit> .
until last decade, dominant dna markers such as aflp and rapd were used to construct linkage maps that are now replaced by co-dominant markers such as microsatellites and snps.
microsatellites have been extensively used in construction of genetic maps for some aquaculture fishes recently, e.g., brown trout, salmo trutta  <cit> , nile tilapia, oreochromis niloticus  <cit> , channel catfish, ictalurus punctatus  <cit> , asian seabass, lates calcarifer  <cit>  and blacklip abalone, haliotis rubra  <cit> .
with the rapid development of dna sequencing and genotyping technologies, single nucleotide polymorphism   <cit> , the most frequent polymorphism in genome, became the most favored dna markers for linkage mapping and whole genome association studies in humans  <cit>  and model organisms  <cit> .
however, the application of snps in linkage mapping of aquacultured fish is just in its infancy  <cit> .
comparative mapping contributes largely to solve issues in the evolution of individual species, differences and similarities between various species and to characterize functions of genomes by providing a detailed analysis of conservation among orthologous intervals in different species  <cit> .
this would not only help to reduce cost and increase efficiency in genetic research across species through offering the possibility to transfer genomic information available from model species to nonmodel organisms, but also aids whole genome sequence assembly as well as molecular breeding  <cit> .
the availability of an increasing number of fish whole genome sequences  offer opportunities to accelerate fish genomic comparative studies, such as, transferring genomic information from model species to food fish.
genetic mapping for non-model species with sequence-based molecular markers could provide a suitable support for the knowledge of genome organization and evolutionary studies by comparative map analysis.
it represents a key towards integrating known genome data from model species into identifying genes in the species of interest  <cit> .
the grass carp  is one of the largest members of the family cyprinidae  <cit> .
it is an herbivorous freshwater fish of great commercial value and is extensively cultivated in eastern asia for food.
cultured grass carp may grow up fast reaching up to  <dig> kg in the first year, growing approximately 2- <dig> kg/year in temperate areas and  <dig>  kg/year in tropical areas  <cit> .
grass carp also has extensive ecological adaptability, of the  <dig> countries in which grass carp have been introduced, at least  <dig>  appear to have self-sustaining populations  <cit> .
in addition, due to their ability to be cultured easily, hardiness and effective biological controls on wide variety of aquatic vegetation; this species has been extensively used for aquatic weed control purposes in rivers, fish ponds and reservoirs  <cit> .
in recent years, commercial harvesting of grass carp has increased in many countries.
according to the statistics of food and agriculture organization of the united nations  <cit> , the global annual production of grass carp summed up to  <dig> , <dig> metric tons in  <dig> with a 30% increase for past decades and was listed as the 3rd biggest contributor to the world's aquaculture production.
the revenue generated by aquaculture of this species was over  <dig> billion us dollars in  <dig>  <cit> .
therefore, the species represents one of the most promising species in world aquaculture  <cit> .
however, research on grass carp genomics remained at slow pace.
although to date around  <dig> microsatellite sequences of grass carp are available in genbank database, and some microsatellite markers have been applied in genetic diversity and comparative analysis of grass carp  <cit> , no genetic map is available in grass carp.
the aim of this study is to construct a first generation genetic map of grass carp using microsatellite and snp markers to supply a basis for genome-wide search of qtl for economically important traits and to conduct a comparative mapping analysis to provide new insights into the evolution of fish genomes and to aid future genome assembly.
RESULTS
markers
a total of  <dig> microsatellites and  <dig> snp markers were informative in the two mapping families and could be used for linkage analysis.
four microsatellite markers  were scored as duplicates.
linkage analysis
two grass carp families containing  <dig> progenies in each were scored for  <dig> informative loci.
a total of  <dig> markers  were mapped to  <dig> linkage groups  whereas  <dig> remained  unmapped.
there is no significant deviation of mendelian segregation for mapped markers after bonferroni correction.
using linkage analysis and lod ≥  <dig> threshold, for male segregation data,  <dig> loci were finally assembled into  <dig> linkage groups  in both families, and for female data,  <dig> loci were assembled into  <dig> linkage groups in both families .
among them,  <dig> loci were located in both maps which could be used to identify homologous pairs of linkage groups in both sexes.
the merged sex-specific maps in both families showed different length.
the map length for female and male was  <dig>  cm and  <dig>  cm respectively.
the average spacing between loci in male map  was lower than that in the female map , indicating that sex-specific differences in recombination rates existed in grass carp.
comparison of recombination differences between the parents of two mapping families revealed that the average recombination ratio  across all pairwise comparisons was  <dig>   for family  <dig> and  <dig>   for family  <dig>  therefore, a significantly higher recombination rate was evident in the female map but no significant family-specific difference in overall recombination ratio for both sexes was detected .
the distributions of recombination ratio between both parents in two mapping families were given in additional file 2: figure s <dig>
the resulting sex-averaged map composed of  <dig> linkage groups  contained  <dig> loci.
six  of the duplicate loci were assigned to  <dig> linkage groups  and the remaining two were unmapped .
the marker order in the sex-averaged map was the same as in the sex-specific maps.
the map spanned  <dig>  cm with individual linkage groups ranging from  <dig>  to  <dig>  cm .
the average resolution of the map was  <dig>  ±  <dig>  cm ranging from  <dig>  cm  to  <dig>  cm  for individual linkage groups.
the average number of loci per linkage group was  <dig>  ±  <dig>  with a range varying from  <dig>  to  <dig> loci  per linkage group.
only  <dig>  out of the  <dig> intervals were larger than  <dig> cm.
overall statistics including number of markers, linkage length and map length for the sex-specific and sex-averaged maps are given in table  <dig> and  <dig>
ald, average inter-locus distance; '1' the homologous zebrafish chromosome that share at least two markers in common with a given grass carp linkage group; '2', the number of syntenic loci in one pair of grass carp lg and homologous zebrafish chromosome; r, the ratio of syntenic region to displayed region of reference chromosome  in nih genetic sequence database http://blast.ncbi.nlm.nih.gov/blast.cgi; length of syntenic region  was calculated based on the distance between the longest syntenic pair of loci in each homologous zebrafish chromosome.
comparative mapping
the sequences of  <dig> mapped microsatellites and  <dig> snps in ests and gene loci were used for similarity searches against genome sequences of four model fish species.
our results showed that  <dig> %  to  <dig> %  of the blasted sequences were significantly conserved between grass carp and model fish species .
between grass carp and fugu/medaka, no microsatellites with more than  <dig> or more repeat motif were conserved, whereas between grass carp and zebrafish,  <dig> % of microsatellite loci were conserved.
when compared to microsatellite data, much higher conservation  of homologous ests and gene loci between grass carp and model species was detected.
of the total  <dig> loci with significant hits ,  <dig> loci  matched at least  <dig> model species and  <dig>  matched all the  <dig> model species.
by syntenic detection, some blocks with very high conservation were also found.
for example, a syntenic block defined by markers snp <dig> and cid <dig> on cid_lg <dig> was conserved among at least  <dig> species.
the detailed comparative mapping results were presented in additional files 3- <dig> tables s <dig> and s <dig>
'a', shows the conservation of orthologs, e.g., type  <dig> indicates unique orthologs identified for each model species, type  <dig> shows orthologs identified only in two model species , type  <dig> shows orthologs identified in three model species , etc; 'b', ratio of all orthologous markers to the total informative markers used for similarity searches.
the grass carp genetic map was utilized in the establishment of gene orthology relationships based on conserved syntenies among species.
the zebrafish genome with  <dig> chromosomes was fairly characterized.
one hundred and sixty-four  markers were common in grass carp linkage map and zebrafish genome map.
the marker density in the comparative map ranged from  <dig>  to  <dig>  markers with an average of  <dig>  ±  <dig>  per lg .
these allow us to evaluate large-scale synteny patterns between the two species.
"d1-25" refer to the  <dig> zebrafish chromosomes and "c1-24" refer to the  <dig> grass carp linkage groups, respectively; 'a', the zebrafish chromosome number that shows synteny to a given grass carp linkage group.
all  <dig> grass carp lgs were clearly associated with zebrafish chromosomes .
in most cases, the grass carp lg showed extensive synteny towards a particular zebrafish chromosome, sharing two or more loci with zebrafish chromosome.
the average number of syntenic loci in one pair of grass carp lg and homologous zebrafish chromosome was  <dig>  ±  <dig>  with a range varying from  <dig>  to  <dig>  per chromosome .
in total,  <dig> of the  <dig> grass carp lgs  mapped each to a single zebrafish chromosome,  <dig> yielded hits with  <dig> chromosomes,  <dig> with  <dig> chromosomes and one  mapped to  <dig> chromosomes .
each zebrafish chromosome was mainly syntenic to one grass carp lg and shared ≥50% orthologous markers of the chromosome in common .
the zebrafish genome map covered by these syntenic markers summed up to  <dig>  mb with a range of  <dig>  mb  to  <dig>  mb  per chromosome, accounting for 63% of the displayed regions of zebrafish reference genome .
a large amount of syntenic blocks could be defined by two or more syntenic loci in this study.
this showed substantial macrosynteny relationship existed between the two species.
the extensive colinearity was very evident for some syntenic chromosomes/linkage pairs e.g.
cid_lg3-dr_lg <dig>  cid_lg5-dr_lg <dig>  cid_lg8-dr_lg <dig>  cid_lg9-dr_lg <dig>  cid_lg10-dr_lg <dig> but partial collinearity was more frequently observed for other chromosomes/linkage pairs, indicating large-scale chromosomal rearrangements were rare but small inversions and translocations were prevailing.
however, there were few exceptions e.g.
both of the zebrafish chromosomes, dr_lg <dig> and dr_lg <dig>  were mainly syntenic to one grass carp linkage group, cid_lg <dig> and shared ≥50% orthologous markers of the chromosome.
in addition,  <dig> snp markers mapped to  <dig> grass carp lgs were assigned to  <dig> chromosomes of the zebrafish genome map of which,  <dig>  were found in syntenic regions .
however, for the  <dig> mapped microsatellite markers, only  <dig> % were found in syntenic regions.
this data indicates a strong conservation of functional genes in the two species.
discussion
dna markers and genetic maps
genetic maps are essential tools for fish genomic studies  <cit> .
development of a large number of sequence-based genetic markers including microsatellites and snps is necessary for mapping qtls for traits of interest and carrying out marker-assisted selection .
using  <dig> informative dna makers, we have constructed a first generation linkage map of grass carp including  <dig> microsatellites and  <dig> snps.
due to difficulty in scoring,  <dig> markers were not assigned to the linkage map.
two hundred and seventy-nine loci were arranged into  <dig> linkage groups.
grass carp is a diploid fish with 2n =  <dig> chromosomes  <cit> , therefore the linkage group number is corresponding to the haploid chromosome number of grass carp.
according to our best knowledge, this map is the first linkage map of a food fish species in the family cyprinidae exclusively based on co-dominant markers .
the genome size of grass carp estimated with a haploid c-value of 1- <dig>  pg  <cit>  and the conversion formulas   <cit>  was 978- <dig> mb.
the length of the grass carp linkage map was  <dig>  cm.
therefore, the ratio between physical and linkage distance was  <dig> -  <dig> kb/cm.
this figure is similar to some other fishes, such as rainbow trout  <cit> , but higher than that of turbot  <cit>  and tiger pufferfish   <cit> .
the length of the grass carp map is far less than the linkage map of the common carp  <cit> , but similar to the length of silver carp  and bighead carp  genetic maps  <cit> .
compared to density of first-generation linkage maps of other species, such as the turbot   <cit> , tiger pufferfish   <cit> , barramundi   <cit>  and olive flounder   <cit> , the average resolution of our map was slightly better with an average distance of  <dig>   cm between loci.
therefore, such a map would be dense enough for the initial mapping of interesting agriculture traits and facilitate future comparative genomic analyses for understanding genome evolution.
chinese scientists are planning to sequence the complete genome of grass carp.
therefore, this linkage map could be complementary to a genome sequencing project.
sex recombination ratio
it is important to know the relative rates of recombination for both sexes of any species  <cit> .
significant differences in sex recombination ratios have been reported for many fishes such as rainbow trout   <cit> , zebrafish   <cit> , atlantic halibut   <cit> , european sea bass, dicentrarchus labrax   <cit> , turbot   <cit> , barramundi, lates calcarifer   <cit> .
family-specific recombination rates were also demonstrated in some salmon species  <cit> .
these studies have shown that reduced male recombination rates might exist extensively in teleost fishes although it varied greatly among chromosomes and sub-chromosomal regions.
the female:male recombination rates  for the parents of two families in this study are similar to the ratios that were reported previously.
however, we observed some exceptions for some marker intervals with higher recombination rates in male, which have also been reported in some fishes, such as zebrafish, trout and barramundi  <cit> .
previous studies suggested many factors such as pericentromeric suppression, cpg islands, gc content, polya/polyt content, simple repeats, line, sine elements and other sequence features would influence recombination rate, however the molecular mechanisms responsible for the difference between the two sexes are still not well understood  <cit> .
comparative mapping
both grass carp and zebrafish belong to the family cyprinidae with the divergence time of  <dig> ±  <dig> million years   <cit>  while the time of divergence between zebrafish and takifugu was  <dig> ±  <dig> mya  <cit> .
the similarity searches against  <dig> model fish species reference genomes showed that  <dig> % grass carp sequences used were considered to be significantly similar to zebrafish genome sequences.
however, much lower significant hits ranging from  <dig> % to  <dig> % in tetraodon, fugu and medaka was found.
the result is in agreement with phylogenetic data  <cit> , showing zebrafish was more closely related to grass carp than other model species.
karyotype evolution in teleosts occurred mainly by inversion and not by fusion or fission of chromosomes  <cit> .
since grass carp  and zebrafish  have different karyotyes and genome sizes   <cit> , the strict pairwise synteny of chromosome pairs between grass carp and zebrafish was not expected.
our study demonstrated extensive colinearity for some syntenic chromosome/linkage pairs.
for example, each zebrafish chromosome was mainly syntenic to one grass carp lg and shared ≥50% othologous markers of the chromosome in common.
this study also indicated that substantial macrosynteny relationship, inversions and translocations existed between grass carp and zebrafish.
therefore, our results provide further support to the studies of karyotype evolution in teleosts by jaillon et al.
<cit>  and kai et al.
<cit> .
however, due to the precision in marker order on the current grass carp linkage map was not high enough for some markers, and reference sequences were not free of assembly error  <cit> , it was possible that some of the disagreements in marker order between grass carp and zebrafish were not accurate, which can be confirmed by further cytogenetic studies, such as fish experiments.
comparative genomics had shown the existence of several blocks of synteny between zebrafish and human gene maps  <cit> .
our study showed a strong conservation of gene order between grass carp and zebrafish and other model species.
for example,  <dig> % of the gene-based snp markers and  <dig> % of the flank sequence-based microsatellite markers were found in syntenic regions of grass carp to zebrafish; and  <dig> % of the loci with significant hits matched at least  <dig> model species and  <dig> % matched  <dig> model species.
however, the conservation was different for most of the compared chromosomes between grass carp and zebrafish.
this could be reflected by differentiated homologous zebrafish chromosome number that shows synteny to different grass carp linkage group .
some studies had reported that chromosomes might be functionally partitioned  <cit>  and large-scale genomic rearrangements were nonrandom with nucleotide variation  <cit> .
bourque et al.
<cit>  also suggested that the total number of microrearrangements was much higher for anonymous sequence-based data than for gene-based data, as many of the microrearrangements in sequence-based synteny blocks lay outside exons.
these studies demonstrated that conservation of synteny might reflect important functional relationships at chromosomal levels.
synteny among species or genera might also aid initial qtl experiments with candidate gene approaches  <cit> .
in this study,  <dig> snps derived from functional genes or ests related to complex genetic basis for interesting quantitative traits in productions such as muscle myosin heavy chain , tnf-related apoptosis inducing ligand  and c-fos  were mapped on to the linkage map at high confidence level.
most of them were also assigned to the conserved synteny regions of grass carp linkage map to zebrafish genome.
the orthologous markers closely linked to these genes on conserved chromosomes of related model fish species might be considered to identify candidate genes and carry out mas in further breeding strategies of the grass carp.
CONCLUSIONS
we constructed a first generation linkage map containing  <dig> dna markers .
the marker density was  <dig>  cm/locus.
recombination rate was higher in females than in males.
this linkage map will provide important base for mapping qtl affecting economically important traits and could be complementary to grass carp genome sequencing project.
comparative mapping between the grass carp and other model organisms revealed that substantial macrosynteny relationship and extensive colinearity existed between grass carp and zebrafish.
comparative mapping will facilitate understanding genome evolution.
in near future the identification of qtl associated traits, such as related to disease and parasite resistance and growth rate, would be conducted with the markers on the map.
our study also showed strong syntenic relationships and a number of conserved colineality existed between grass carp and zebrafish genome.
therefore, gene prediction directly from zebrafish to grass carp was possible.
further studies focusing on the transferring of genomic information available from zebrafish to the selection of grass carp had been put forward.
a high density map with gene-based markers was also planned, which would provide a detailed analysis of conservation among orthologous intervals in related species and a basis for location of genes and mas of grass carp or other cyprinidae.

 a linkage map is an essential framework for mapping traits of interest and is often the first step towards understanding genome evolution and aids genome assembly. with the advent of molecular genetic techniques and sophisticated statistical tools for linkage analysis, linkage maps have been constructed for some economically important aquaculture species, such as channel catfish, tilapia, asian seabass, european sea bass, shrimps, and oysters. until last decade, dominant dna markers such as aflp and rapd were used to construct linkage maps that are now replaced by co-dominant markers such as microsatellites and snps. microsatellites have been extensively used in construction of genetic maps for some aquaculture fishes recently, e.g., brown trout, salmo trutta, nile tilapia, oreochromis niloticus, channel catfish, ictalurus punctatus, asian seabass, lates calcarifer and blacklip abalone, haliotis rubra. with the rapid development of dna sequencing and genotyping technologies, single nucleotide polymorphism, the most frequent polymorphism in genome, became the most favored dna markers for linkage mapping and whole genome association studies in humans and model organisms. however, the application of snps in linkage mapping of aquacultured fish is just in its infancy. comparative mapping contributes largely to solve issues in the evolution of individual species, differences and similarities between various species and to characterize functions of genomes by providing a detailed analysis of conservation among orthologous intervals in different species. this would not only help to reduce cost and increase efficiency in genetic research across species through offering the possibility to transfer genomic information available from model species to nonmodel organisms, but also aids whole genome sequence assembly as well as molecular breeding. the availability of an increasing number of fish whole genome sequences offer opportunities to accelerate fish genomic comparative studies, such as, transferring genomic information from model species to food fish. genetic mapping for non-model species with sequence-based molecular markers could provide a suitable support for the knowledge of genome organization and evolutionary studies by comparative map analysis. it represents a key towards integrating known genome data from model species into identifying genes in the species of interest. the grass carp is one of the largest members of the family cyprinidae. it is an herbivorous freshwater fish of great commercial value and is extensively cultivated in eastern asia for food. cultured grass carp may grow up fast reaching up to kg in the first year, growing approximately 2- kg/year in temperate areas and kg/year in tropical areas. grass carp also has extensive ecological adaptability, of the countries in which grass carp have been introduced, at least appear to have self-sustaining populations. in addition, due to their ability to be cultured easily, hardiness and effective biological controls on wide variety of aquatic vegetation; this species has been extensively used for aquatic weed control purposes in rivers, fish ponds and reservoirs. in recent years, commercial harvesting of grass carp has increased in many countries. according to the statistics of food and agriculture organization of the united nations, the global annual production of grass carp summed up to, metric tons in with a 30% increase for past decades and was listed as the 3rd biggest contributor to the world's aquaculture production. the revenue generated by aquaculture of this species was over billion us dollars in. therefore, the species represents one of the most promising species in world aquaculture. however, research on grass carp genomics remained at slow pace. although to date around microsatellite sequences of grass carp are available in genbank database, and some microsatellite markers have been applied in genetic diversity and comparative analysis of grass carp, no genetic map is available in grass carp. the aim of this study is to construct a first generation genetic map of grass carp using microsatellite and snp markers to supply a basis for genome-wide search of qtl for economically important traits and to conduct a comparative mapping analysis to provide new insights into the evolution of fish genomes and to aid future genome assembly.  markers a total of microsatellites and snp markers were informative in the two mapping families and could be used for linkage analysis. four microsatellite markers were scored as duplicates. linkage analysis two grass carp families containing progenies in each were scored for informative loci. a total of markers were mapped to linkage groups whereas remained unmapped. there is no significant deviation of mendelian segregation for mapped markers after bonferroni correction. using linkage analysis and lod ≥ threshold, for male segregation data, loci were finally assembled into linkage groups in both families, and for female data, loci were assembled into linkage groups in both families. among them, loci were located in both maps which could be used to identify homologous pairs of linkage groups in both sexes. the merged sex-specific maps in both families showed different length. the map length for female and male was cm and cm respectively. the average spacing between loci in male map was lower than that in the female map, indicating that sex-specific differences in recombination rates existed in grass carp. comparison of recombination differences between the parents of two mapping families revealed that the average recombination ratio across all pairwise comparisons was for family and for family therefore, a significantly higher recombination rate was evident in the female map but no significant family-specific difference in overall recombination ratio for both sexes was detected. the distributions of recombination ratio between both parents in two mapping families were given in additional file 2: figure s the resulting sex-averaged map composed of linkage groups contained loci. six of the duplicate loci were assigned to linkage groups and the remaining two were unmapped. the marker order in the sex-averaged map was the same as in the sex-specific maps. the map spanned cm with individual linkage groups ranging from to cm. the average resolution of the map was ± cm ranging from cm to cm for individual linkage groups. the average number of loci per linkage group was ± with a range varying from to loci per linkage group. only out of the intervals were larger than cm. overall statistics including number of markers, linkage length and map length for the sex-specific and sex-averaged maps are given in table and ald, average inter-locus distance;'1' the homologous zebrafish chromosome that share at least two markers in common with a given grass carp linkage group;'2', the number of syntenic loci in one pair of grass carp lg and homologous zebrafish chromosome; r, the ratio of syntenic region to displayed region of reference chromosome in nih genetic sequence database http://blast.ncbi.nlm.nih.gov/blast.cgi; length of syntenic region was calculated based on the distance between the longest syntenic pair of loci in each homologous zebrafish chromosome. comparative mapping the sequences of mapped microsatellites and snps in ests and gene loci were used for similarity searches against genome sequences of four model fish species. our results showed that % to % of the blasted sequences were significantly conserved between grass carp and model fish species. between grass carp and fugu/medaka, no microsatellites with more than or more repeat motif were conserved, whereas between grass carp and zebrafish, % of microsatellite loci were conserved. when compared to microsatellite data, much higher conservation of homologous ests and gene loci between grass carp and model species was detected. of the total loci with significant hits, loci matched at least model species and matched all the model species. by syntenic detection, some blocks with very high conservation were also found. for example, a syntenic block defined by markers snp and cid on cid_lg was conserved among at least species. the detailed comparative mapping results were presented in additional files 3- tables s and s'a', shows the conservation of orthologs, e.g., type indicates unique orthologs identified for each model species, type shows orthologs identified only in two model species, type shows orthologs identified in three model species, etc;'b', ratio of all orthologous markers to the total informative markers used for similarity searches. the grass carp genetic map was utilized in the establishment of gene orthology relationships based on conserved syntenies among species. the zebrafish genome with chromosomes was fairly characterized. one hundred and sixty-four markers were common in grass carp linkage map and zebrafish genome map. the marker density in the comparative map ranged from to markers with an average of ± per lg. these allow us to evaluate large-scale synteny patterns between the two species. "d1-25" refer to the zebrafish chromosomes and "c1-24" refer to the grass carp linkage groups, respectively;'a', the zebrafish chromosome number that shows synteny to a given grass carp linkage group. all grass carp lgs were clearly associated with zebrafish chromosomes. in most cases, the grass carp lg showed extensive synteny towards a particular zebrafish chromosome, sharing two or more loci with zebrafish chromosome. the average number of syntenic loci in one pair of grass carp lg and homologous zebrafish chromosome was ± with a range varying from to per chromosome. in total, of the grass carp lgs mapped each to a single zebrafish chromosome, yielded hits with chromosomes, with chromosomes and one mapped to chromosomes. each zebrafish chromosome was mainly syntenic to one grass carp lg and shared ≥50% orthologous markers of the chromosome in common. the zebrafish genome map covered by these syntenic markers summed up to mb with a range of mb to mb per chromosome, accounting for 63% of the displayed regions of zebrafish reference genome. a large amount of syntenic blocks could be defined by two or more syntenic loci in this study. this showed substantial macrosynteny relationship existed between the two species. the extensive colinearity was very evident for some syntenic chromosomes/linkage pairs e.g. cid_lg3-dr_lg cid_lg5-dr_lg cid_lg8-dr_lg cid_lg9-dr_lg cid_lg10-dr_lg but partial collinearity was more frequently observed for other chromosomes/linkage pairs, indicating large-scale chromosomal rearrangements were rare but small inversions and translocations were prevailing. however, there were few exceptions e.g. both of the zebrafish chromosomes, dr_lg and dr_lg were mainly syntenic to one grass carp linkage group, cid_lg and shared ≥50% orthologous markers of the chromosome. in addition, snp markers mapped to grass carp lgs were assigned to chromosomes of the zebrafish genome map of which, were found in syntenic regions. however, for the mapped microsatellite markers, only % were found in syntenic regions. this data indicates a strong conservation of functional genes in the two species. discussion dna markers and genetic maps genetic maps are essential tools for fish genomic studies. development of a large number of sequence-based genetic markers including microsatellites and snps is necessary for mapping qtls for traits of interest and carrying out marker-assisted selection. using informative dna makers, we have constructed a first generation linkage map of grass carp including microsatellites and snps. due to difficulty in scoring, markers were not assigned to the linkage map. two hundred and seventy-nine loci were arranged into linkage groups. grass carp is a diploid fish with 2n = chromosomes, therefore the linkage group number is corresponding to the haploid chromosome number of grass carp. according to our best knowledge, this map is the first linkage map of a food fish species in the family cyprinidae exclusively based on co-dominant markers. the genome size of grass carp estimated with a haploid c-value of 1- pg and the conversion formulas was 978- mb. the length of the grass carp linkage map was cm. therefore, the ratio between physical and linkage distance was - kb/cm. this figure is similar to some other fishes, such as rainbow trout, but higher than that of turbot and tiger pufferfish. the length of the grass carp map is far less than the linkage map of the common carp, but similar to the length of silver carp and bighead carp genetic maps. compared to density of first-generation linkage maps of other species, such as the turbot, tiger pufferfish, barramundi and olive flounder, the average resolution of our map was slightly better with an average distance of cm between loci. therefore, such a map would be dense enough for the initial mapping of interesting agriculture traits and facilitate future comparative genomic analyses for understanding genome evolution. chinese scientists are planning to sequence the complete genome of grass carp. therefore, this linkage map could be complementary to a genome sequencing project. sex recombination ratio it is important to know the relative rates of recombination for both sexes of any species. significant differences in sex recombination ratios have been reported for many fishes such as rainbow trout, zebrafish, atlantic halibut, european sea bass, dicentrarchus labrax, turbot, barramundi, lates calcarifer. family-specific recombination rates were also demonstrated in some salmon species. these studies have shown that reduced male recombination rates might exist extensively in teleost fishes although it varied greatly among chromosomes and sub-chromosomal regions. the female:male recombination rates for the parents of two families in this study are similar to the ratios that were reported previously. however, we observed some exceptions for some marker intervals with higher recombination rates in male, which have also been reported in some fishes, such as zebrafish, trout and barramundi. previous studies suggested many factors such as pericentromeric suppression, cpg islands, gc content, polya/polyt content, simple repeats, line, sine elements and other sequence features would influence recombination rate, however the molecular mechanisms responsible for the difference between the two sexes are still not well understood. comparative mapping both grass carp and zebrafish belong to the family cyprinidae with the divergence time of ± million years while the time of divergence between zebrafish and takifugu was ± mya. the similarity searches against model fish species reference genomes showed that % grass carp sequences used were considered to be significantly similar to zebrafish genome sequences. however, much lower significant hits ranging from % to % in tetraodon, fugu and medaka was found. the result is in agreement with phylogenetic data, showing zebrafish was more closely related to grass carp than other model species. karyotype evolution in teleosts occurred mainly by inversion and not by fusion or fission of chromosomes. since grass carp and zebrafish have different karyotyes and genome sizes, the strict pairwise synteny of chromosome pairs between grass carp and zebrafish was not expected. our study demonstrated extensive colinearity for some syntenic chromosome/linkage pairs. for example, each zebrafish chromosome was mainly syntenic to one grass carp lg and shared ≥50% othologous markers of the chromosome in common. this study also indicated that substantial macrosynteny relationship, inversions and translocations existed between grass carp and zebrafish. therefore, our results provide further support to the studies of karyotype evolution in teleosts by jaillon et al. and kai et al.. however, due to the precision in marker order on the current grass carp linkage map was not high enough for some markers, and reference sequences were not free of assembly error, it was possible that some of the disagreements in marker order between grass carp and zebrafish were not accurate, which can be confirmed by further cytogenetic studies, such as fish experiments. comparative genomics had shown the existence of several blocks of synteny between zebrafish and human gene maps. our study showed a strong conservation of gene order between grass carp and zebrafish and other model species. for example, % of the gene-based snp markers and % of the flank sequence-based microsatellite markers were found in syntenic regions of grass carp to zebrafish; and % of the loci with significant hits matched at least model species and % matched model species. however, the conservation was different for most of the compared chromosomes between grass carp and zebrafish. this could be reflected by differentiated homologous zebrafish chromosome number that shows synteny to different grass carp linkage group. some studies had reported that chromosomes might be functionally partitioned and large-scale genomic rearrangements were nonrandom with nucleotide variation. bourque et al. also suggested that the total number of microrearrangements was much higher for anonymous sequence-based data than for gene-based data, as many of the microrearrangements in sequence-based synteny blocks lay outside exons. these studies demonstrated that conservation of synteny might reflect important functional relationships at chromosomal levels. synteny among species or genera might also aid initial qtl experiments with candidate gene approaches. in this study, snps derived from functional genes or ests related to complex genetic basis for interesting quantitative traits in productions such as muscle myosin heavy chain, tnf-related apoptosis inducing ligand and c-fos were mapped on to the linkage map at high confidence level. most of them were also assigned to the conserved synteny regions of grass carp linkage map to zebrafish genome. the orthologous markers closely linked to these genes on conserved chromosomes of related model fish species might be considered to identify candidate genes and carry out mas in further breeding strategies of the grass carp.  we constructed a first generation linkage map containing dna markers. the marker density was cm/locus. recombination rate was higher in females than in males. this linkage map will provide important base for mapping qtl affecting economically important traits and could be complementary to grass carp genome sequencing project. comparative mapping between the grass carp and other model organisms revealed that substantial macrosynteny relationship and extensive colinearity existed between grass carp and zebrafish. comparative mapping will facilitate understanding genome evolution. in near future the identification of qtl associated traits, such as related to disease and parasite resistance and growth rate, would be conducted with the markers on the map. our study also showed strong syntenic relationships and a number of conserved colineality existed between grass carp and zebrafish genome. therefore, gene prediction directly from zebrafish to grass carp was possible. further studies focusing on the transferring of genomic information available from zebrafish to the selection of grass carp had been put forward. a high density map with gene-based markers was also planned, which would provide a detailed analysis of conservation among orthologous intervals in related species and a basis for location of genes and mas of grass carp or other cyprinidae. 
the aim of this study is to construct a first generation genetic map of grass carp using microsatellites and snps to generate a new resource for mapping qtl for economically important traits and to conduct a comparative mapping analysis to shed new insights into the evolution of fish genomes. this map provides a valuable resource for mapping phenotypic variations and serves as a reference to approach comparative genomics and understand the evolution of fish genomes and could be complementary to grass carp genome sequencing project. the average resolution of the map was cm/locus. a linkage map is an essential framework for mapping traits of interest and is often the first step towards understanding genome evolution. we constructed a first generation linkage map of grass carp with a mapping panel containing two f families including progenies. the linkage map of grass carp presented here is the first linkage map of a food fish species based on co-dominant markers in the family cyprinidae. grass carp belongs to the family cyprinidae which includes more than fish species. the number of lgs was corresponding to the haploid chromosome number of grass carp. blast searches of sequences of mapped markers of grass carp against the whole genome sequence of zebrafish revealed substantial macrosynteny relationship and extensive colinearity of markers between grass carp and zebrafish. it is one of the most important freshwater food fish species in world aquaculture.
 grass carp belongs to the family cyprinidae which includes more than fish species. it is one of the most important freshwater food fish species in world aquaculture. a linkage map is an essential framework for mapping traits of interest and is often the first step towards understanding genome evolution. the aim of this study is to construct a first generation genetic map of grass carp using microsatellites and snps to generate a new resource for mapping qtl for economically important traits and to conduct a comparative mapping analysis to shed new insights into the evolution of fish genomes.  we constructed a first generation linkage map of grass carp with a mapping panel containing two f families including progenies. sixteen snps in genes and microsatellite markers were mapped to twenty-four linkage groups. the number of lgs was corresponding to the haploid chromosome number of grass carp. the sex-specific map was and cm long in females and males respectively whereas the sex-averaged map spanned cm. the average resolution of the map was cm/locus. blast searches of sequences of mapped markers of grass carp against the whole genome sequence of zebrafish revealed substantial macrosynteny relationship and extensive colinearity of markers between grass carp and zebrafish.  the linkage map of grass carp presented here is the first linkage map of a food fish species based on co-dominant markers in the family cyprinidae. this map provides a valuable resource for mapping phenotypic variations and serves as a reference to approach comparative genomics and understand the evolution of fish genomes and could be complementary to grass carp genome sequencing project. 