BACKGROUND
in october  <dig>  the initial draft of the bovine genome sequence using a whole genome shotgun strategy has been released  <cit> .
preliminary assemblies of the current bovine genome sequence update representing a 6× coverage have been established using whole genome shotgun sequences  and bac end sequences  on bovine chromosomes and were announced in october  <dig>  and ncbi bos taurus genome mapview   <cit> ).
a high-resolution physical map will provide a basic framework to assist the final high-quality sequence assembly and annotation process of the bovine genome for anchoring genes, anonymous loci, wgs, bes, and preliminary contigs along bovine chromosomes.
subsequently, the availability of the complete architecture of bovine chromosomes will facilitate positional cloning efforts focusing on targeted identification of the causative sequence variation underlying quantitative trait loci   <cit> .
in order to construct a high-resolution map for a specific chromosome or targeted chromosomal region, comparative mapping information from maps of the annotated human and mouse genomes can be utilized efficiently.
the location of bovine loci that are homologues of human genes may be predicted from the current knowledge about the conservation of synteny between genomes, but has to be actually proven by direct mapping on the bovine genome.
however, the successful use of genome information from sequence-ready maps relies upon the precise identification and characterization of segments of conserved synteny, gene order, and evolutionary breakpoints in the bovine genome.
radiation hybrid  mapping has been shown to be a powerful tool to integrate comparative genome data with information from existing bovine genetic and physical maps to generate high-resolution maps .
considering all rh panels available for the bovine genome, the  <dig>  rad whole genome rh panel  has been shown to have the high mapping power required to create accurate maps of a highly elevated resolution in defined small chromosomal regions  <cit> .
in our study, bovine chromosome  <dig>  was chosen as a candidate chromosome, because a number of different qtl for various phenotypic traits, including milk production, functional, and conformation traits in dairy cattle as well as growth and body composition traits in meat cattle, have been mapped consistently in the middle region of this chromosome .
although, initial results focusing on the candidate genes underlying the qtl for milk production traits at the molecular level have been reported recently  <cit> , further structural and functional research is required to dissect the different qtl at the gene-based level.
previous comparative studies based on fluorescence in situ hybridization  and rh mapping  revealed that the region associated to the indicated qtl on bta <dig> corresponds to two segments of conserved synteny on human chromosome  <dig> .
however, multiple inter- and intrachromosomal micro-rearrangements with respect to orthologous human chromosomes as reported for several bovine chromosomes  could not be excluded given the data currently available for bta <dig>  consequently, to enable fine mapping of qtl and final identification of causal genes underlying the qtl within small chromosome segments of bta <dig>  the content and order of genes within the syntenic regions need to be defined and characterized precisely.
hence, the objective of this study was to construct a high-resolution "gene-rich" rh map for the targeted chromosomal region of bta <dig>  based on our first bta <dig> rh map  <cit>  and by exploiting the available human and bovine genome sequence information, rh mapping was utilized to link both microsatellite markers and genes into an improved comprehensive, high-resolution map.
in order to increase the number of comparative anchor loci on the physical map of bta <dig> and to refine boundaries for blocks of conserved synteny, a large number of new genes, which have been derived from the orthologous region of the human genome was mapped onto bta <dig>  in addition, novel targeted isolated anonymous markers and bes were integrated into the rh map of the bta <dig> region.
RESULTS
framework and comprehensive rh map construction
a total number of  <dig> loci  including  <dig> genes and expressed sequence tags  were typed on the cattle-hamster  <dig> k wg-rh panel  <cit> .
hundred eleven genes and ests have human orthologous loci in the syntenic chromosomal region on hsa <dig>  no significant sequence similarity to human genes and ests was found for  <dig> bovine ests .
for the  <dig> loci tested,  <dig> different retention patterns were observed.
a total of  <dig> loci displayed unique retention patterns, whereas  <dig> shared identical retention patterns with at least one other locus indicating no obligate chromosome breaks between them .
the retention frequencies of loci varied from  <dig>  % for cssm <dig> and ptpn <dig> to  <dig>  % for manba, and averaged  <dig>  % for all loci.
the distribution of the retention frequencies across the chromosomal region covered is shown in figure  <dig>  retention pattern did not show a consistent bias along the chromosomal region.
the chromosomal region analyzed is represented by one contiguous rh linkage group at twopoint lodscore  criterion  <dig> comprising a total of  <dig> loci and by  <dig> singletons.
the positions of the singletons  on bta <dig> were retrieved from published bovine maps  <cit> .
at twopoint lod threshold  <dig>  the large twopoint lod 4-linkage group is fragmented into one major group containing  <dig> loci, and one further linkage group consisting of  <dig> loci.
maximum likelihood multipoint linkage analysis integrated  <dig> loci  with unique retention pattern into a framework map at multipoint lod  <dig> as described in materials and methods.
on the basis of this framework map encompassing the region between manba and antrx <dig>  we sequentially integrated the remaining loci to generate a comprehensive map with the most likely order .
the resulting marker order of the comprehensive map spanning  <dig>  cr <dig>  as determined by the maximum likelihood program maxlik is in agreement with the one determined by calculating the order with the minimal number of obligate breaks option  and by subjecting the proposed order to a ripple procedure  along the chromosomal region.
the respective chromosomal region of bta <dig> we focused in this study includes the segment between bm <dig> as the most proximal marker and bm <dig> as the anchor marker in the distal part.
according to the comprehensive genetic map  <cit> , this segment comprises a distance of about  <dig>  cm.
in comparison to our rh map, a genome ratio of  <dig>  cr <dig> /cm was calculated, which corresponded to the value obtained in our initial study .
considering the preliminary bovine sequence assemblies of bta <dig> provided at the ensembl and ncbi databases , a ratio of  <dig>  cr <dig> /mb was inferred for this chromosomal region.
the average inter-loci interval on the targeted bta <dig> region covered on our rh map is  <dig>  cr <dig>  corresponding to approximately  <dig> kb.
identification of whole genome contig sequences specific to bta6
the comprehensive comparison of our high-resolution rh map with the genome sequence map of bta <dig> required the identification of additional sequence contig scaffolds of the existing 6× coverage draft of the bovine genome sequence specific to our bta <dig> region.
therefore, all sequences  integrated in our high-resolution bta <dig> rh map but not placed on the current ncbi sequence assembly map of bta <dig>  were used to screen the bovine genome sequence resources  <cit> .
a total of  <dig> loci could be assigned to sequence contigs, which were already assigned to bta <dig> .
for  <dig> loci, we detected corresponding sequence contigs, which were not yet placed on the bovine genome .
furthermore,  <dig> loci  were found on sequence contigs, which had been assigned to other chromosomes than bta <dig> .
for a total of  <dig> loci, no sequence contigs were found within the available bovine sequence resources.
generation of the comparative map
with the aim to construct a high-resolution multi-species comparative rh map, we started with  <dig> genes and ests previously assigned to bta <dig>  <cit> .
furthermore, we added bovine genes inferred from their human orthologs localized in the syntenic chromosome segment of hsa4p <dig> -hsa4q <dig>  for which corresponding bovine mrna sequences were known  <cit> .
to further increase the number of mapped coding sequences on bta <dig> as comparative anchor loci, we started again from the syntenic human chromosomal region of hsa <dig> by screening sequences of known and predicted human genes from this region using the blastn algorithm against the cattle est database  <cit> .
this comparative in silico mapping approach is the targeted version of the compass mapping procedure , which utilizes the human-cattle comparative mapping knowledge for predictive positioning of unknown bovine ests on the bovine genome.
following this targeted mapping approach, we identified a total of  <dig> new bovine gene loci  derived from human gene sequences with known positions on hsa <dig>  and verified and localized them on the comprehensive rh map of bta <dig>  in addition,  <dig> bovine chromosome region-specific ests  were integrated on our high-resolution rh map of bta <dig>
a further approach focused on linking  <dig> anonymous loci mapped on our rh map of bta <dig> , bes, and ests) with the syntenic hsa <dig> region by merging sequence and mapping information.
therefore, these sequences were applied for direct similarity search with the human genome sequence using the blastn program  <cit>  to find positions of orthology on the syntenic chromosome hsa <dig>  for  <dig> out of these anonymous markers, orthologous sequences localized on hsa <dig> were identified .
placement of these  <dig> anonymous sequences on hsa <dig> was derived from the positions of human bac contigs harboring sequence motifs homologous to the targeted bovine sequences .
homology search of all  <dig> anonymous sequences against the whole human genome did not reveal any unique and significant homologous region on other chromosomes than hsa <dig>
to better define the bta6-hsa <dig> comparative map, the bovine genomic sequences  containing the anonymous marker sequences were used as intermediary sequences between bovine anonymous loci and the human genomic sequence as described in methods.
thus, comparative positions of anonymous markers without direct human alignments were established by performing sequence similarity search on the human genome sequence with bovine wgs contig sequences containing the targeted marker sequences.
including the information from the intermediary alignment procedure,  <dig> additional anchor points could be localized on hsa <dig> .
ten marker sequences could not be anchored on the human genome sequence by this in silico mapping procedure.
finally, the cattle-human comparative map  of the defined chromosomal region on bta <dig> was generated by aligning the rh map established in this study with the orthologous region of hsa <dig> taking the human genome sequence assembly as a reference.
physical map positions of human genes and ests for the construction of the comparative chromosomal map based on the coordinates of the human genome sequence .
for comparison of the chromosomal organization of bta <dig> to equivalent chromosomes from other species, gene mapping information of genes on orthologous chromosomal regions  was retrieved from the species genome sequence maps available at the ncbi mapviewer database  <cit> .
the comparative map displaying the gene order in the orthologous chromosomal regions cross-linking our bovine comprehensive  <dig> k wg-rh map of bta <dig> and corresponding chromosomes from multi-species genomes is presented in figure  <dig>
discussion
the high-resolution  <dig> k wg-rh map for bta <dig> and comparison with genetic and physical maps
high-resolution comparative mapping in combination with sequencing information can be utilized efficiently not only to create a sequence-ready chromosome map, but also to contribute to the selection of positional and functional candidate genes within chromosomal regions containing genetic variability affecting phenotypes of physiological and economic importance in cattle.
for targeted comparative mapping studies in species with lacking or incompletely annotated genomes like the bovine, the availability of the complete architecture of human and mouse genomes provides an excellent resource employing the information from gene-rich maps of the human and mouse genomes in combination with high-resolution synteny rh mapping.
this strategy was applied in our study on bta <dig> to powerfully accelerate the research on genes associated with qtl for lactation and growth related traits mapped on this chromosome.
the resolution and gene density of the current whole-genome cattle rh maps  <cit>  is often inadequate either to support the assembly of the bovine genome sequence or for the molecular dissection of complex traits in cattle.
on bta <dig>  the proportion of bovine genes that have been mapped is relatively small and unevenly distributed.
to overcome these limitations and to construct an improved gene-based map, our aim was to enrich previous marker-dominated maps by integrating available information from anchored genes and by filling in with targeted selected loci to bridge existing gaps.
therefore, comparative alignment of the human sequence of the orthologous chromosome was utilized for in silico identification of bovine ests specific for the selected chromosome region of bta <dig>  furthermore, an increase in density of loci on the bta <dig> rh map was achieved by targeted integration of novel markers from a microdissection library  <cit>  and the high-density bovine genetic map  <cit> , particularly in intervals with poor marker coverage.
out of a total of  <dig> loci,  <dig> loci were used to construct an integrated rh map of high-resolution for the targeted chromosomal region of bta <dig> using a  <dig> k wg-rh panel.
the average retention frequency of  <dig> bta <dig> loci typed was  <dig>  %.
this value is in the same magnitude as the frequencies of  <dig> % found for bta <dig> loci in the bovine  <dig> k wg- rh panel  <cit>  and  <dig>  % reported in the  <dig> k wg-sunbrh panel  <cit> .
using rh analysis we have included known genes and ests from currently available bta <dig> maps and added a total of  <dig> novel genes and ests accurately in relation to the position of known loci onto the map.
hence, the density of genes and ests integrated on our rh map exceeds the density on available bovine genetic and physical maps of the respective region of bta <dig> substantially.
the order of loci determined in our high-resolution, gene-rich map for the targeted bta <dig> region is generally consistent with that reported on previous published rh  <cit>  and linkage maps  <cit> .
compared to the sunbrh map  <cit> , local inversions of loci pairs were found only for  <dig> placements within small chromosomal segments .
these discrepancies observed between adjacent loci in very close positions could be possibly due to the resolution limit of the rh panels used.
major differences were only detected when comparing the order of loci in our rh map to that in the second bta <dig> linkage group of the second generation comprehensive map  <cit> .
in contrast to the latter map, the interval including spp <dig>  abcg <dig>  herc <dig>  and fam13a <dig> is positioned proximal to ibsp in a reverse order in our rh map.
this placement is in agreement with recently published data  <cit> .
taken together, these two reports and our data provide strong support for the proposed genomic structure of the chromosomal region of bta <dig>  the genomic structure of this chromosomal region is of special interest, because recently, two different candidate genes for a qtl with effects on milk production traits have been reported there  <cit> , which requires further detailed structural and functional analysis of the corresponding chromosomal area.
to connect our high-resolution  <dig> k wg-rh map for bta <dig> with the existing 6× coverage bovine genome sequence assembly, we compared this map with the sequence map available for bta <dig>  <cit>  and conducted additional blast sequence similarity search with sequences of loci that were not placed there.
a total of  <dig> new placements of loci onto bta <dig> were identified providing  <dig> new assignments onto known bta <dig> contigs and  <dig> placements to sequence contigs from the pool of unassigned bovine sequence contigs in the ncbi archive.
in summary, we found  <dig> % of all sequences mapped on our rh map to be presented in the current 6× coverage draft of the bovine genome sequence exemplarily underlining its high potential as a framework sequence resource to represent the molecular architecture of the bovine chromosomes.
comparing the locus order on our  <dig> k wg-rh map of bta <dig> with the currently available 6× coverage sequence assembly of bta <dig>  <cit>  as demonstrated in figure  <dig>  we found a good overall agreement of both maps, although a number of discordances were observed.
these differences in locus order focused as followed indicate regions in the currently available 6× assembly, which have to be considered critically in the course of the curation process to establish the final sequence assembly on bta <dig>
generally, the order of multiple loci on a sequence contig agrees with the order obtained by our rh map .
however, orientation of the contigs within the sequence assembly is sometimes opposite to the order of loci in our rh map .
substantial differences in locus order were observed on the current sequence assembly, when the position of sequence contigs is arranged in strong contrast to the placement of the corresponding loci by rh mapping .
the position of these loci on our rh map is highly supported, because they entered the rh map as framework markers.
the arrangement of sequence contigs nw_ <dig> , nw_ <dig> , and nw_ <dig>  observed in the segment between  <dig> and  <dig> mb on the sequence assembly  is in contrast not only to our rh map but also to published results based on bac contigs  <cit>  as we discussed above when comparing the existing rh maps.
furthermore, some loci are represented by more than one assignment onto different sequence contigs in the sequence assembly .
there are  <dig> loci placed on our rh map , which are currently assigned to other than bta <dig> specific sequence contigs.
these positions of loci with questionable placements indicating problem areas in the current assembly have to be refined in further assembly versions.
screening the resources of bovine genome sequence contigs with sequences mapped on bta <dig> but without a placement onto bta6-specific sequence contigs on the current sequence assembly, we identified  <dig> new bovine sequence contigs carrying loci, which were mapped on bta <dig> .
these new assignments have predominantly filled gaps within subchromosomal regions poorly covered with genes  and could be helpful to guide the validation of the sequence assembly.
comparing both, our rh map and the sequence assembly map of bta <dig> to the loci order on hsa <dig>  the bta <dig> rh map shows a lower number of interchromosomal rearrangements with hsa <dig> than the current sequence contig assembly.
as demonstrated in additional file  <dig>  the order of loci on the rh map is highly consistent within both chromosomal segments syntenic to hsa4: the inversed segment comprising loci from manba to ibsp as well as the segment comprising the loci from lap <dig> to dmp <dig>  there are only two regions in our rh map indicating rearrangements , which have to be reanalyzed in further investigations.
in contrast, there appeared a significant difference in the conservation of gene order, when the bta <dig> sequence assembly map is compared with hsa <dig>  considering the high degree of conservation of gene order in syntenic blocks of human and bovine chromosomes as well as in comparison with syntenic blocks in other species , the elevated number of intrachromosomal rearrangements suggested by the bta <dig> sequence assembly map seems less likely than the gene order determined by our rh map.
however, aligning the positions of loci on our rh map to the preliminary genome sequence assembly of bta <dig> revealed an adequate overall agreement in locus order and distances between loci, although a number of inconsistencies have been pointed up because of misassigned contigs and rearrangements.
this is supported by a correlation of  <dig>  between positions of loci given in cr and mb .
based on our  <dig> k wg-rh map and the genetic map  <cit> , a mean genome ratio of  <dig>  cr <dig> /cm was calculated.
the comparison of the resolution of our rh map with the preliminary bovine sequence assembly of bta <dig> revealed an estimated ratio of about  <dig>  cr <dig> /mb.
this indicates that one cr on our high-resolution rh map is covered by  <dig>  kb on the contig map of bta <dig>  aligning the genetic map with the present contig map, a ratio of approximately  <dig> kb/cm could be deduced, a value deviating from the generally expected ratio  <dig> mb/cm.
the multi-species comparative map
in comparison to previous published bovine-human comparative physical maps of bta <dig>  we were able to increase the number of anchor points of genes and markers between species.
this was the prerequisite to close physical gaps between linkage groups depicted in the comprehensive second generation map  <cit>  as well as the gap between ppargc1a and ibsp in the sunbrh map  <cit> .
those inter-species breakpoints between human and bovine, which were already reported in our initial map  <cit> , could be refined exactly to be between lap <dig> and qdpr as well as between ibsp and dmp <dig>  as shown in figure  <dig>  a highly conserved segment comprising the interval from lap <dig> to dmp <dig> on hsa <dig>  is completely maintained on bta <dig> without significant intrachromosomal rearrangements.
the locus adjacent to lap <dig> on hsa <dig>  qdpr , which is closely positioned with ldb <dig> and fgfbp <dig> , was anchored on the distal part of bta <dig> closely to bm <dig> .
the breakpoint on hsa <dig> and the chromosomal interval, which is attached to qdpr, could be inferred by exploiting the currently available bovine genome sequence assembly.
for this purpose, the bovine genomic reference sequence  <cit>  was screened with the sequence of bm <dig>  which detected homology to the bovine bta <dig> contig nw_ <dig>  this contig is located adjacent to the contig nw_ <dig>  which is predicted to contain man2b <dig> .
man2b <dig> is located at  <dig>  mb on hsa <dig>  thus, considering the comparative in silico mapping information, it can be concluded that on bta <dig>  the chromosomal block containing man2b <dig> is attached closely to the breakpoint-containing segment including qdpr, ldb <dig>  and fgfbp <dig>  further chromosomal rearrangements compared to hsa <dig> were observed in the extreme telomeric part of bta <dig> around msx <dig>  acox <dig>  and lrpap <dig> .
in order to resolve this chromosomal region accurately and define the breakpoints, additional loci have to be integrated in the map.
comparative cross-species analysis of the gene order indicates a highly conserved organization across the human-bovine breakpoints within the interval from fgbp1-qdpr-lap <dig> to dmp1-ibsp-spp1-pkd <dig> on hsa <dig>  mmu <dig>  and rno <dig>  as outlined above, on bta <dig> this interval is shortened to the chromosome segment ranging from lap <dig> to dmp <dig> due to interchromosomal breaks and rearrangements between lap <dig> and qdpr as well as dmp <dig> and ibsp.
the general gene order, however, within this ancestral interval lap <dig> to dmp <dig> seems to be maintained across species.
comparing the gene positions on the mouse and rat chromosomes syntenic to hsa <dig>  the interval at the breakpoint distal from pkd <dig> is broken again and placed on different chromosomes: the segment from abcg <dig> to smarcad <dig>  on mmu <dig> and rno <dig>  respectively, and the segment beginning with bmpr1b on mmu <dig> and rno <dig>  respectively.
in chicken the whole chromosomal region of the hsa4p <dig> -4q <dig> is maintained on chromosome  <dig> , although, there is a number of intrachromosomal rearrangements resulting in several individual gene blocks and micro-rearrangements along the chromosome.
when compared to hsa <dig> and bta <dig>  the syntenic segment on gga <dig> revealing the longest stretch of orthologous genes in identical order encompasses the region from kiaa <dig> to paics.
comparing the chromosomal region analyzed in our study to the canine genome sequence, the genes orthologous to hsa <dig> and bta <dig> were discovered on canine chromosomes  <dig>   <dig>  and  <dig> .
however, several breakpoints differing from other species were found.
the interval ranging from near nup <dig> to cenpe/flj <dig> on hsa <dig> is completely located on cfa <dig> in an identical order.
this interval also represents the longest syntenic gene interval transmitted without any breaks.
an additional break was observed near kiaa <dig>  which was also detected on chicken gga <dig>  the segment involving genes from kiaa <dig> to lbd <dig> is located on cfa <dig> without any break between lap <dig> and qdpr.
this is analogous to the mouse and rat chromosomes mmu <dig> and rno <dig>  but in contrast to bta <dig>  in addition, the interval comprising mlr <dig> to ugdh was inversely mapped on cfa <dig>  the fragment from kiaa <dig> to il <dig>  which is attached to ugdh on hsa <dig>  appeared on cfa <dig>
from zoo-fish experiments, genetic, and rh mapping studies, it is known that the porcine chromosome syntenic to hsa <dig> and bta <dig> is chromosome  <dig> , whereas in horse the corresponding chromosomal region was identified on chromosomes  <dig> and  <dig> .
due to the lower degree of details in gene-based mapping information from the currently available porcine and equine maps, accurate subchromosomal breakpoints and boundaries could not be retrieved for the construction of corresponding comparative maps.
the proportion of genes mapped on the orthologous chromosomes is relatively small and, consequently, not sufficient to pursue the positions of the genes that have been mapped on our rh map of bta <dig>  for pig, the information we retrieved from several rh and linkage maps indicates that most of the genes from hsa <dig> are conserved on ssc <dig>  in the recently published bac-based comparative human-porcine physical map  <cit> ,  <dig> conserved syntenic regions from hsa <dig> were placed on ssc <dig> indicating that there a significant higher number of rearrangements in gene order exists compared to the human- bovine comparative map.
the analysis of the homologous chromosomal regions between species based on available sequence data and annotation of genes revealed the occurrence of gene deserts defined as megabase-size genomic segments devoid of protein-coding genes.
on hsa <dig>  which is populated with gene deserts corresponding to approximately  <dig> % of the length of the whole chromosome  <cit> , there exists one outstanding region spanning about  <dig>  mb on hsa4p <dig> , only separated by pcdh <dig>  the second gene desert interval is located near the pericentromeric segment on hsa4q12-q <dig>  where lphn <dig> is flanked on both sides by gaps of about  <dig> and  <dig> mb, respectively, without annotated protein-coding genes.
when compared to genome sequences from dog, mouse, rat and chicken, the overall architecture of the gene desert region around pcdh <dig> was found to be conserved in mammals and birds including the flanking genes  <cit> .
the complete genome sequence assembly of the bovine syntenic region of bta <dig> is not yet available.
therefore, our high-resolution gene-based rh map of the targeted bta <dig> was covered with a high density of anonymous loci in these gene-poor regions.
anchoring of a substantial number of the sequences of these anonymous loci onto hsa <dig>  enabled us to infer that the overall architecture of these gene deserts seems to be maintained also on bta <dig>
taken together, most of the rearrangements detected between the human hsa <dig> region and the chromosomal region on bovine bta <dig>  chicken gga <dig>  and most likely porcine ssc <dig> are intrachromosomal.
in contrast, the respective chromosomal region in rodent, canine, and equine genomes, is transferred onto two or three chromosomes.
thus, our observations have suggested a higher number of conserved segments between the bovine, human, chicken and pig genomes than between the bovine and the second species group.
this implies that the organization of the human genome regarding chromosomal synteny is closer to that of the cattle, chicken and pig than that of the mouse, rat, dog, and horse.
CONCLUSIONS
in conclusion, the high-resolution rh map for the defined region of bta <dig> presented here integrates anonymous markers, ests, and genes from currently available bovine linkage and rh maps as well as a high number of comparative anchor loci derived from orthologous hsa <dig>  although a number of links to the currently existing genetic, cytogenetic, and rh maps were possible, a multitude of contigs and scaffolds of the available bovine genome sequence resources still have to be anchored and/or oriented on the chromosomes.
in this context, our high-resolution gene-enriched rh map will enable a great improvement of genome sequence assembly of bta <dig> by linking the physical mapping data with sequence information from the preliminary sequence contig assembly of this chromosome.
with a resolution of  <dig> locus/ <dig> kb, our rh map will provide a valuable platform to guide the comprehensive high-quality assembly of the genome sequence and gene annotation on bta <dig>
furthermore, mapping a large number of genes on bta <dig> and cross-referencing them with map locations in corresponding syntenic multi-species chromosome segments; our rh map will refine synteny segments at a high-resolution level to decipher mammalian chromosome evolution.
finally, the connections between available physical and linkage maps, including cross-species comparative maps and bovine genome sequence provide a resource for gene-based fine mapping of qtl by increasing efficiency of identifying genes and causal mutations affecting animal performance.
connecting animal phenotypes associated with the qtl anchored on genomic sequence level with putative underlying genes would accelerate the identification of sequence polymorphisms and gene variants and the development of snp markers for validation of association substantially.

 in october the initial draft of the bovine genome sequence using a whole genome shotgun strategy has been released. preliminary assemblies of the current bovine genome sequence update representing a 6× coverage have been established using whole genome shotgun sequences and bac end sequences on bovine chromosomes and were announced in october and ncbi bos taurus genome mapview ). a high-resolution physical map will provide a basic framework to assist the final high-quality sequence assembly and annotation process of the bovine genome for anchoring genes, anonymous loci, wgs, bes, and preliminary contigs along bovine chromosomes. subsequently, the availability of the complete architecture of bovine chromosomes will facilitate positional cloning efforts focusing on targeted identification of the causative sequence variation underlying quantitative trait loci. in order to construct a high-resolution map for a specific chromosome or targeted chromosomal region, comparative mapping information from maps of the annotated human and mouse genomes can be utilized efficiently. the location of bovine loci that are homologues of human genes may be predicted from the current knowledge about the conservation of synteny between genomes, but has to be actually proven by direct mapping on the bovine genome. however, the successful use of genome information from sequence-ready maps relies upon the precise identification and characterization of segments of conserved synteny, gene order, and evolutionary breakpoints in the bovine genome. radiation hybrid mapping has been shown to be a powerful tool to integrate comparative genome data with information from existing bovine genetic and physical maps to generate high-resolution maps. considering all rh panels available for the bovine genome, the rad whole genome rh panel has been shown to have the high mapping power required to create accurate maps of a highly elevated resolution in defined small chromosomal regions. in our study, bovine chromosome was chosen as a candidate chromosome, because a number of different qtl for various phenotypic traits, including milk production, functional, and conformation traits in dairy cattle as well as growth and body composition traits in meat cattle, have been mapped consistently in the middle region of this chromosome. although, initial results focusing on the candidate genes underlying the qtl for milk production traits at the molecular level have been reported recently, further structural and functional research is required to dissect the different qtl at the gene-based level. previous comparative studies based on fluorescence in situ hybridization and rh mapping revealed that the region associated to the indicated qtl on bta corresponds to two segments of conserved synteny on human chromosome. however, multiple inter- and intrachromosomal micro-rearrangements with respect to orthologous human chromosomes as reported for several bovine chromosomes could not be excluded given the data currently available for bta consequently, to enable fine mapping of qtl and final identification of causal genes underlying the qtl within small chromosome segments of bta the content and order of genes within the syntenic regions need to be defined and characterized precisely. hence, the objective of this study was to construct a high-resolution "gene-rich" rh map for the targeted chromosomal region of bta based on our first bta rh map and by exploiting the available human and bovine genome sequence information, rh mapping was utilized to link both microsatellite markers and genes into an improved comprehensive, high-resolution map. in order to increase the number of comparative anchor loci on the physical map of bta and to refine boundaries for blocks of conserved synteny, a large number of new genes, which have been derived from the orthologous region of the human genome was mapped onto bta in addition, novel targeted isolated anonymous markers and bes were integrated into the rh map of the bta region.  framework and comprehensive rh map construction a total number of loci including genes and expressed sequence tags were typed on the cattle-hamster k wg-rh panel. hundred eleven genes and ests have human orthologous loci in the syntenic chromosomal region on hsa no significant sequence similarity to human genes and ests was found for bovine ests. for the loci tested, different retention patterns were observed. a total of loci displayed unique retention patterns, whereas shared identical retention patterns with at least one other locus indicating no obligate chromosome breaks between them. the retention frequencies of loci varied from % for cssm and ptpn to % for manba, and averaged % for all loci. the distribution of the retention frequencies across the chromosomal region covered is shown in figure retention pattern did not show a consistent bias along the chromosomal region. the chromosomal region analyzed is represented by one contiguous rh linkage group at twopoint lodscore criterion comprising a total of loci and by singletons. the positions of the singletons on bta were retrieved from published bovine maps. at twopoint lod threshold the large twopoint lod 4-linkage group is fragmented into one major group containing loci, and one further linkage group consisting of loci. maximum likelihood multipoint linkage analysis integrated loci with unique retention pattern into a framework map at multipoint lod as described in materials and methods. on the basis of this framework map encompassing the region between manba and antrx we sequentially integrated the remaining loci to generate a comprehensive map with the most likely order. the resulting marker order of the comprehensive map spanning cr as determined by the maximum likelihood program maxlik is in agreement with the one determined by calculating the order with the minimal number of obligate breaks option and by subjecting the proposed order to a ripple procedure along the chromosomal region. the respective chromosomal region of bta we focused in this study includes the segment between bm as the most proximal marker and bm as the anchor marker in the distal part. according to the comprehensive genetic map, this segment comprises a distance of about cm. in comparison to our rh map, a genome ratio of cr /cm was calculated, which corresponded to the value obtained in our initial study. considering the preliminary bovine sequence assemblies of bta provided at the ensembl and ncbi databases, a ratio of cr /mb was inferred for this chromosomal region. the average inter-loci interval on the targeted bta region covered on our rh map is cr corresponding to approximately kb. identification of whole genome contig sequences specific to bta6 the comprehensive comparison of our high-resolution rh map with the genome sequence map of bta required the identification of additional sequence contig scaffolds of the existing 6× coverage draft of the bovine genome sequence specific to our bta region. therefore, all sequences integrated in our high-resolution bta rh map but not placed on the current ncbi sequence assembly map of bta were used to screen the bovine genome sequence resources. a total of loci could be assigned to sequence contigs, which were already assigned to bta. for loci, we detected corresponding sequence contigs, which were not yet placed on the bovine genome. furthermore, loci were found on sequence contigs, which had been assigned to other chromosomes than bta. for a total of loci, no sequence contigs were found within the available bovine sequence resources. generation of the comparative map with the aim to construct a high-resolution multi-species comparative rh map, we started with genes and ests previously assigned to bta. furthermore, we added bovine genes inferred from their human orthologs localized in the syntenic chromosome segment of hsa4p -hsa4q for which corresponding bovine mrna sequences were known. to further increase the number of mapped coding sequences on bta as comparative anchor loci, we started again from the syntenic human chromosomal region of hsa by screening sequences of known and predicted human genes from this region using the blastn algorithm against the cattle est database. this comparative in silico mapping approach is the targeted version of the compass mapping procedure, which utilizes the human-cattle comparative mapping knowledge for predictive positioning of unknown bovine ests on the bovine genome. following this targeted mapping approach, we identified a total of new bovine gene loci derived from human gene sequences with known positions on hsa and verified and localized them on the comprehensive rh map of bta in addition, bovine chromosome region-specific ests were integrated on our high-resolution rh map of bta a further approach focused on linking anonymous loci mapped on our rh map of bta, bes, and ests) with the syntenic hsa region by merging sequence and mapping information. therefore, these sequences were applied for direct similarity search with the human genome sequence using the blastn program to find positions of orthology on the syntenic chromosome hsa for out of these anonymous markers, orthologous sequences localized on hsa were identified. placement of these anonymous sequences on hsa was derived from the positions of human bac contigs harboring sequence motifs homologous to the targeted bovine sequences. homology search of all anonymous sequences against the whole human genome did not reveal any unique and significant homologous region on other chromosomes than hsa to better define the bta6-hsa comparative map, the bovine genomic sequences containing the anonymous marker sequences were used as intermediary sequences between bovine anonymous loci and the human genomic sequence as described in methods. thus, comparative positions of anonymous markers without direct human alignments were established by performing sequence similarity search on the human genome sequence with bovine wgs contig sequences containing the targeted marker sequences. including the information from the intermediary alignment procedure, additional anchor points could be localized on hsa. ten marker sequences could not be anchored on the human genome sequence by this in silico mapping procedure. finally, the cattle-human comparative map of the defined chromosomal region on bta was generated by aligning the rh map established in this study with the orthologous region of hsa taking the human genome sequence assembly as a reference. physical map positions of human genes and ests for the construction of the comparative chromosomal map based on the coordinates of the human genome sequence. for comparison of the chromosomal organization of bta to equivalent chromosomes from other species, gene mapping information of genes on orthologous chromosomal regions was retrieved from the species genome sequence maps available at the ncbi mapviewer database. the comparative map displaying the gene order in the orthologous chromosomal regions cross-linking our bovine comprehensive k wg-rh map of bta and corresponding chromosomes from multi-species genomes is presented in figure discussion the high-resolution k wg-rh map for bta and comparison with genetic and physical maps high-resolution comparative mapping in combination with sequencing information can be utilized efficiently not only to create a sequence-ready chromosome map, but also to contribute to the selection of positional and functional candidate genes within chromosomal regions containing genetic variability affecting phenotypes of physiological and economic importance in cattle. for targeted comparative mapping studies in species with lacking or incompletely annotated genomes like the bovine, the availability of the complete architecture of human and mouse genomes provides an excellent resource employing the information from gene-rich maps of the human and mouse genomes in combination with high-resolution synteny rh mapping. this strategy was applied in our study on bta to powerfully accelerate the research on genes associated with qtl for lactation and growth related traits mapped on this chromosome. the resolution and gene density of the current whole-genome cattle rh maps is often inadequate either to support the assembly of the bovine genome sequence or for the molecular dissection of complex traits in cattle. on bta the proportion of bovine genes that have been mapped is relatively small and unevenly distributed. to overcome these limitations and to construct an improved gene-based map, our aim was to enrich previous marker-dominated maps by integrating available information from anchored genes and by filling in with targeted selected loci to bridge existing gaps. therefore, comparative alignment of the human sequence of the orthologous chromosome was utilized for in silico identification of bovine ests specific for the selected chromosome region of bta furthermore, an increase in density of loci on the bta rh map was achieved by targeted integration of novel markers from a microdissection library and the high-density bovine genetic map, particularly in intervals with poor marker coverage. out of a total of loci, loci were used to construct an integrated rh map of high-resolution for the targeted chromosomal region of bta using a k wg-rh panel. the average retention frequency of bta loci typed was %. this value is in the same magnitude as the frequencies of % found for bta loci in the bovine k wg- rh panel and % reported in the k wg-sunbrh panel. using rh analysis we have included known genes and ests from currently available bta maps and added a total of novel genes and ests accurately in relation to the position of known loci onto the map. hence, the density of genes and ests integrated on our rh map exceeds the density on available bovine genetic and physical maps of the respective region of bta substantially. the order of loci determined in our high-resolution, gene-rich map for the targeted bta region is generally consistent with that reported on previous published rh and linkage maps. compared to the sunbrh map, local inversions of loci pairs were found only for placements within small chromosomal segments. these discrepancies observed between adjacent loci in very close positions could be possibly due to the resolution limit of the rh panels used. major differences were only detected when comparing the order of loci in our rh map to that in the second bta linkage group of the second generation comprehensive map. in contrast to the latter map, the interval including spp abcg herc and fam13a is positioned proximal to ibsp in a reverse order in our rh map. this placement is in agreement with recently published data. taken together, these two reports and our data provide strong support for the proposed genomic structure of the chromosomal region of bta the genomic structure of this chromosomal region is of special interest, because recently, two different candidate genes for a qtl with effects on milk production traits have been reported there, which requires further detailed structural and functional analysis of the corresponding chromosomal area. to connect our high-resolution k wg-rh map for bta with the existing 6× coverage bovine genome sequence assembly, we compared this map with the sequence map available for bta and conducted additional blast sequence similarity search with sequences of loci that were not placed there. a total of new placements of loci onto bta were identified providing new assignments onto known bta contigs and placements to sequence contigs from the pool of unassigned bovine sequence contigs in the ncbi archive. in summary, we found % of all sequences mapped on our rh map to be presented in the current 6× coverage draft of the bovine genome sequence exemplarily underlining its high potential as a framework sequence resource to represent the molecular architecture of the bovine chromosomes. comparing the locus order on our k wg-rh map of bta with the currently available 6× coverage sequence assembly of bta as demonstrated in figure we found a good overall agreement of both maps, although a number of discordances were observed. these differences in locus order focused as followed indicate regions in the currently available 6× assembly, which have to be considered critically in the course of the curation process to establish the final sequence assembly on bta generally, the order of multiple loci on a sequence contig agrees with the order obtained by our rh map. however, orientation of the contigs within the sequence assembly is sometimes opposite to the order of loci in our rh map. substantial differences in locus order were observed on the current sequence assembly, when the position of sequence contigs is arranged in strong contrast to the placement of the corresponding loci by rh mapping. the position of these loci on our rh map is highly supported, because they entered the rh map as framework markers. the arrangement of sequence contigs nw_, nw_, and nw_ observed in the segment between and mb on the sequence assembly is in contrast not only to our rh map but also to published results based on bac contigs as we discussed above when comparing the existing rh maps. furthermore, some loci are represented by more than one assignment onto different sequence contigs in the sequence assembly. there are loci placed on our rh map, which are currently assigned to other than bta specific sequence contigs. these positions of loci with questionable placements indicating problem areas in the current assembly have to be refined in further assembly versions. screening the resources of bovine genome sequence contigs with sequences mapped on bta but without a placement onto bta6-specific sequence contigs on the current sequence assembly, we identified new bovine sequence contigs carrying loci, which were mapped on bta. these new assignments have predominantly filled gaps within subchromosomal regions poorly covered with genes and could be helpful to guide the validation of the sequence assembly. comparing both, our rh map and the sequence assembly map of bta to the loci order on hsa the bta rh map shows a lower number of interchromosomal rearrangements with hsa than the current sequence contig assembly. as demonstrated in additional file the order of loci on the rh map is highly consistent within both chromosomal segments syntenic to hsa4: the inversed segment comprising loci from manba to ibsp as well as the segment comprising the loci from lap to dmp there are only two regions in our rh map indicating rearrangements, which have to be reanalyzed in further investigations. in contrast, there appeared a significant difference in the conservation of gene order, when the bta sequence assembly map is compared with hsa considering the high degree of conservation of gene order in syntenic blocks of human and bovine chromosomes as well as in comparison with syntenic blocks in other species, the elevated number of intrachromosomal rearrangements suggested by the bta sequence assembly map seems less likely than the gene order determined by our rh map. however, aligning the positions of loci on our rh map to the preliminary genome sequence assembly of bta revealed an adequate overall agreement in locus order and distances between loci, although a number of inconsistencies have been pointed up because of misassigned contigs and rearrangements. this is supported by a correlation of between positions of loci given in cr and mb. based on our k wg-rh map and the genetic map, a mean genome ratio of cr /cm was calculated. the comparison of the resolution of our rh map with the preliminary bovine sequence assembly of bta revealed an estimated ratio of about cr /mb. this indicates that one cr on our high-resolution rh map is covered by kb on the contig map of bta aligning the genetic map with the present contig map, a ratio of approximately kb/cm could be deduced, a value deviating from the generally expected ratio mb/cm. the multi-species comparative map in comparison to previous published bovine-human comparative physical maps of bta we were able to increase the number of anchor points of genes and markers between species. this was the prerequisite to close physical gaps between linkage groups depicted in the comprehensive second generation map as well as the gap between ppargc1a and ibsp in the sunbrh map. those inter-species breakpoints between human and bovine, which were already reported in our initial map, could be refined exactly to be between lap and qdpr as well as between ibsp and dmp as shown in figure a highly conserved segment comprising the interval from lap to dmp on hsa is completely maintained on bta without significant intrachromosomal rearrangements. the locus adjacent to lap on hsa qdpr, which is closely positioned with ldb and fgfbp, was anchored on the distal part of bta closely to bm. the breakpoint on hsa and the chromosomal interval, which is attached to qdpr, could be inferred by exploiting the currently available bovine genome sequence assembly. for this purpose, the bovine genomic reference sequence was screened with the sequence of bm which detected homology to the bovine bta contig nw_ this contig is located adjacent to the contig nw_ which is predicted to contain man2b. man2b is located at mb on hsa thus, considering the comparative in silico mapping information, it can be concluded that on bta the chromosomal block containing man2b is attached closely to the breakpoint-containing segment including qdpr, ldb and fgfbp further chromosomal rearrangements compared to hsa were observed in the extreme telomeric part of bta around msx acox and lrpap. in order to resolve this chromosomal region accurately and define the breakpoints, additional loci have to be integrated in the map. comparative cross-species analysis of the gene order indicates a highly conserved organization across the human-bovine breakpoints within the interval from fgbp1-qdpr-lap to dmp1-ibsp-spp1-pkd on hsa mmu and rno as outlined above, on bta this interval is shortened to the chromosome segment ranging from lap to dmp due to interchromosomal breaks and rearrangements between lap and qdpr as well as dmp and ibsp. the general gene order, however, within this ancestral interval lap to dmp seems to be maintained across species. comparing the gene positions on the mouse and rat chromosomes syntenic to hsa the interval at the breakpoint distal from pkd is broken again and placed on different chromosomes: the segment from abcg to smarcad on mmu and rno respectively, and the segment beginning with bmpr1b on mmu and rno respectively. in chicken the whole chromosomal region of the hsa4p -4q is maintained on chromosome, although, there is a number of intrachromosomal rearrangements resulting in several individual gene blocks and micro-rearrangements along the chromosome. when compared to hsa and bta the syntenic segment on gga revealing the longest stretch of orthologous genes in identical order encompasses the region from kiaa to paics. comparing the chromosomal region analyzed in our study to the canine genome sequence, the genes orthologous to hsa and bta were discovered on canine chromosomes and. however, several breakpoints differing from other species were found. the interval ranging from near nup to cenpe/flj on hsa is completely located on cfa in an identical order. this interval also represents the longest syntenic gene interval transmitted without any breaks. an additional break was observed near kiaa which was also detected on chicken gga the segment involving genes from kiaa to lbd is located on cfa without any break between lap and qdpr. this is analogous to the mouse and rat chromosomes mmu and rno but in contrast to bta in addition, the interval comprising mlr to ugdh was inversely mapped on cfa the fragment from kiaa to il which is attached to ugdh on hsa appeared on cfa from zoo-fish experiments, genetic, and rh mapping studies, it is known that the porcine chromosome syntenic to hsa and bta is chromosome, whereas in horse the corresponding chromosomal region was identified on chromosomes and. due to the lower degree of details in gene-based mapping information from the currently available porcine and equine maps, accurate subchromosomal breakpoints and boundaries could not be retrieved for the construction of corresponding comparative maps. the proportion of genes mapped on the orthologous chromosomes is relatively small and, consequently, not sufficient to pursue the positions of the genes that have been mapped on our rh map of bta for pig, the information we retrieved from several rh and linkage maps indicates that most of the genes from hsa are conserved on ssc in the recently published bac-based comparative human-porcine physical map, conserved syntenic regions from hsa were placed on ssc indicating that there a significant higher number of rearrangements in gene order exists compared to the human- bovine comparative map. the analysis of the homologous chromosomal regions between species based on available sequence data and annotation of genes revealed the occurrence of gene deserts defined as megabase-size genomic segments devoid of protein-coding genes. on hsa which is populated with gene deserts corresponding to approximately % of the length of the whole chromosome, there exists one outstanding region spanning about mb on hsa4p, only separated by pcdh the second gene desert interval is located near the pericentromeric segment on hsa4q12-q where lphn is flanked on both sides by gaps of about and mb, respectively, without annotated protein-coding genes. when compared to genome sequences from dog, mouse, rat and chicken, the overall architecture of the gene desert region around pcdh was found to be conserved in mammals and birds including the flanking genes. the complete genome sequence assembly of the bovine syntenic region of bta is not yet available. therefore, our high-resolution gene-based rh map of the targeted bta was covered with a high density of anonymous loci in these gene-poor regions. anchoring of a substantial number of the sequences of these anonymous loci onto hsa enabled us to infer that the overall architecture of these gene deserts seems to be maintained also on bta taken together, most of the rearrangements detected between the human hsa region and the chromosomal region on bovine bta chicken gga and most likely porcine ssc are intrachromosomal. in contrast, the respective chromosomal region in rodent, canine, and equine genomes, is transferred onto two or three chromosomes. thus, our observations have suggested a higher number of conserved segments between the bovine, human, chicken and pig genomes than between the bovine and the second species group. this implies that the organization of the human genome regarding chromosomal synteny is closer to that of the cattle, chicken and pig than that of the mouse, rat, dog, and horse.  in conclusion, the high-resolution rh map for the defined region of bta presented here integrates anonymous markers, ests, and genes from currently available bovine linkage and rh maps as well as a high number of comparative anchor loci derived from orthologous hsa although a number of links to the currently existing genetic, cytogenetic, and rh maps were possible, a multitude of contigs and scaffolds of the available bovine genome sequence resources still have to be anchored and/or oriented on the chromosomes. in this context, our high-resolution gene-enriched rh map will enable a great improvement of genome sequence assembly of bta by linking the physical mapping data with sequence information from the preliminary sequence contig assembly of this chromosome. with a resolution of locus/ kb, our rh map will provide a valuable platform to guide the comprehensive high-quality assembly of the genome sequence and gene annotation on bta furthermore, mapping a large number of genes on bta and cross-referencing them with map locations in corresponding syntenic multi-species chromosome segments; our rh map will refine synteny segments at a high-resolution level to decipher mammalian chromosome evolution. finally, the connections between available physical and linkage maps, including cross-species comparative maps and bovine genome sequence provide a resource for gene-based fine mapping of qtl by increasing efficiency of identifying genes and causal mutations affecting animal performance. connecting animal phenotypes associated with the qtl anchored on genomic sequence level with putative underlying genes would accelerate the identification of sequence polymorphisms and gene variants and the development of snp markers for validation of association substantially. 
a number of different quantitative trait loci for various phenotypic traits, including milk production, functional, and conformation traits in dairy cattle as well as growth and body composition traits in meat cattle, have been mapped consistently in the middle region of bovine chromosome. mapping a large number of genes on bta and cross-referencing them with map locations in corresponding syntenic multi-species chromosome segments achieved a refined accurate alignment of conserved segments and evolutionary breakpoints across the species included. the gene-anchored high-resolution rh map for the targeted region of bta presented here will provide a valuable platform to guide high-quality assembling and annotation of the currently existing bovine genome sequence draft to establish the final architecture of bta hence, a sequence-based map will provide a key resource to facilitate prospective continued efforts for the selection and validation of relevant positional and functional candidates underlying qtl for milk production and growth-related traits mapped on bta and on similar chromosomal regions from evolutionary closely related species like sheep and goat. screening the available bovine genome sequence resources, a total of loci could be assigned to sequence contigs, which were already identified as specific for bta for loci, corresponding sequence contigs, which were not yet placed on the bovine genome assembly, were identified. a comprehensive high-resolution gene-rich map linking densely spaced bovine markers and genes to the annotated human genome sequence is required as a framework to facilitate this approach for the region on bta carrying the qtl. in addition, the improved potential of this high-resolution rh map for bta with respect to comparative mapping was demonstrated. dense genetic and physical maps and, ultimately, a fully annotated genome sequence as well as their mutual connections are required to efficiently identify genes and gene variants responsible for genetic variation of phenotypic traits. therefore, we constructed a high-resolution radiation hybrid map for the qtl containing chromosomal region of bta this new rh map with a total of loci including genes and ests displays a substantial increase in loci density compared to existing physical bta maps. furthermore, the high-resolution sequence-referenced bta map will enable precise identification of multi-species conserved chromosome segments and evolutionary breakpoints in mammalian phylogenetic studies.
 a number of different quantitative trait loci for various phenotypic traits, including milk production, functional, and conformation traits in dairy cattle as well as growth and body composition traits in meat cattle, have been mapped consistently in the middle region of bovine chromosome. dense genetic and physical maps and, ultimately, a fully annotated genome sequence as well as their mutual connections are required to efficiently identify genes and gene variants responsible for genetic variation of phenotypic traits. a comprehensive high-resolution gene-rich map linking densely spaced bovine markers and genes to the annotated human genome sequence is required as a framework to facilitate this approach for the region on bta carrying the qtl.  therefore, we constructed a high-resolution radiation hybrid map for the qtl containing chromosomal region of bta this new rh map with a total of loci including genes and ests displays a substantial increase in loci density compared to existing physical bta maps. screening the available bovine genome sequence resources, a total of loci could be assigned to sequence contigs, which were already identified as specific for bta for loci, corresponding sequence contigs, which were not yet placed on the bovine genome assembly, were identified. in addition, the improved potential of this high-resolution rh map for bta with respect to comparative mapping was demonstrated. mapping a large number of genes on bta and cross-referencing them with map locations in corresponding syntenic multi-species chromosome segments achieved a refined accurate alignment of conserved segments and evolutionary breakpoints across the species included.  the gene-anchored high-resolution rh map for the targeted region of bta presented here will provide a valuable platform to guide high-quality assembling and annotation of the currently existing bovine genome sequence draft to establish the final architecture of bta hence, a sequence-based map will provide a key resource to facilitate prospective continued efforts for the selection and validation of relevant positional and functional candidates underlying qtl for milk production and growth-related traits mapped on bta and on similar chromosomal regions from evolutionary closely related species like sheep and goat. furthermore, the high-resolution sequence-referenced bta map will enable precise identification of multi-species conserved chromosome segments and evolutionary breakpoints in mammalian phylogenetic studies. 