BACKGROUND
chicken is the first major agricultural species for which the complete genome sequencing was undertaken.
this is partly due to its position as a model species in various fields of biology including embryo development, oncology, immunology and evolution  <cit> .
moreover, as it is the only bird species for which the genome study is so advanced, very much is expected from its use in comparative genome analyses for annotation, including that of the human genome, by detection of conserved sequences  <cit> .
its intermediate phylogenetic position between mammals and fishes will also certainly provide valuable information on the evolution of vertebrate karyotypes.
radiation hybrid maps have a resolution power intermediate to that of the genetic and bac contig maps and are also a powerful tool for the mapping of ests and genes by simple pcr.
they are thus useful at two levels: first, they can be used constructively as scaffolds for a correct genome assembly or for detecting and correcting misassembled portions of the genome; second, before obtaining whole annotated genome sequences, they are very efficient tools for inter-species comparative genome analyses through the easy mapping of genes and ests  <cit> .
the successful production of a rh panel in chicken is quite recent  <cit> , and therefore rh maps are only available for a limited number of chromosomes  <cit> .
having identified qtl  for fatness on chicken chromosome  <dig>  <cit> , our objective was to build a high-resolution and gene-rich rh map for this chromosome, as a basis for high precision comparative mapping with human and for the development of new polymorphic markers.
the available human/chicken comparative mapping data indicated conservation of synteny between gga <dig> and portions of hsa <dig>  hsa <dig> and hsa <dig>  in addition, two genes from hsa <dig> had also been shown to be located on gga <dig>  <cit> .
this information was used to develop markers from chicken est sequence data orthologous to genes in these human regions, in addition to the existing markers from the chicken chromosome  <dig> genetic map.
while in the process of finishing our map, the first draft sequence assembly of the chicken genome was released .
the quality of both the gga <dig> rh map and of the sequence assembly was therefore checked by alignment of all the markers by blast searches.
RESULTS
development of est markers
in addition to the  <dig> microsatellite markers from the genetic map, and  <dig> primer pairs chosen either from available primer data in the literature or designed using the gene sequence deposited in genbank/embl,  <dig> primer pairs were chosen from chicken est markers selected on the basis of the known conservations of synteny between human and chicken using the iccare  software .
constraints on the design of primers were to avoid presence of long introns, whose position and length was predicted on the basis of the orthologous human gene structure, and to design primers in the most divergent regions of the human/chicken alignment, to limit cross-amplification with the hamster dna present in the hybrids.
one hundred and thirty nine primer pairs out of  <dig>  enabled a successful amplification and the subsequent mapping of the corresponding genes, confirming the high success rate obtained when using the iccare software for designing chicken pcr primers based on est data  <cit> .
construction of the gga <dig> rh map
altogether, genotyping data was obtained for a total of  <dig> markers, comprising  <dig> gene fragments  and  <dig> microsatellites from the gga <dig> genetic map.
two-point analysis using a lod threshold of  <dig> enabled to constitute a group of  <dig> markers, including all the microsatellite markers from the genetic map.
the remaining  <dig> markers correspond to the external boundaries of the regions of conserved synteny with human, from which ests were chosen for marker development and map either to other chromosomes for which rh maps were developed  or to unknown regions .
after multipoint analysis, a 1000: <dig> framework map  <dig>  cr <dig> long, comprising a total of  <dig> markers including  <dig> microsatellites and  <dig> ests was obtained.
the remaining  <dig> markers are located relative to the framework map within confidence intervals, to build a comprehensive map .
to compare the rh and the genetic maps, the best possible position of the non-framework common markers had to be estimated.
that of the markers on the rh map was computed by the carthagene program and is indicated in addition to the confidence interval.
for the genetic map, the central position of the marker's confidence interval was used as their most probable position.
as a result, the order of the markers on the rh map matches exactly that of the same markers on genetic map  <cit> , with only one notable discrepancy concerning the position of brf <dig> .
however, when the position of this gene was checked on the sequence assembly, the agreement was with the rh map, suggesting the position of this gene on the genetic map is erroneous.
an average retention frequency of  <dig> % was observed for the  <dig> gga <dig> markers studied here, although with a high variation, with values ranging from  <dig> % to  <dig> %.
this finding is within the range observed in other studies reported on this panel:  <dig> % overall retention using  <dig> markers chosen genome-wise  <cit> ,  <dig> % for gga <dig>  <cit> ,  <dig>  for gga <dig>  <cit>  and 18% for gga <dig>  <cit> .
as already noticed for several species including human  <cit>  or cow  <cit> , but also for chicken chromosomes  <dig> and  <dig>  <cit> , a centromeric effect is detected when observing retention frequencies of markers along the map, with a higher retention of markers in the region between  <dig> to  <dig> cr, in which the retention culminates at a value of  <dig> %, whereas it is around 15% for the rest of the chromosome .
alignment of the rh map to the genomic sequence
a preliminary data set based on the first draft chicken genome assembly has been deposited into public databases by a team led by r. wilson and w. warren, from the washington university school of medicine in st. louis .
we compared our data with the gga <dig> sequence, by using blastn searches and sequence alignments.
the agreement between the rh framework map and the sequence orders is almost perfect , although with a few discrepancies, most of them suggesting possible improvements to be made in the sequence assembly.
first, a group of markers , that we developed on the basis of the conservation of synteny between gga <dig> and hsa <dig>  is assigned to gga <dig> in the sequence assembly.
three of these markers are on the framework map and for all four, the rh genotypings obtained are very similar to those obtained with the flanking rh framework markers slc17a <dig> and arntl , both located on the gga <dig> sequence assembly.
furthermore, when two-point analysis of the four markers was computed with the flanking markers loc <dig>  and slc22a <dig>  suggested in the gga <dig> sequence assembly, lod scores were equal to zero.
this part of the genome assigned to a wrong chromosome on the sequence assembly covers a region at least  <dig> cr long, corresponding to a distance of  <dig> to  <dig> mb, as estimated from the cr to mb ratio.
indeed, the length of the sequence between the two extreme markers cstf <dig> and gpr <dig> on the gga <dig> assembly is  <dig>  mb.
the retention frequency of these four markers is amongst the highest of all, suggesting that their location is close to the centromere and that the possible sequence assembly problems are related to this proximity, perhaps due to repetitive sequences.
second, we observed an inversion of the gene order for a segment in the upper part of the chromosome  adjacent to the group we described as wrongly assigned to gga <dig> in the sequence assembly.
as the difference of likelihood between our 1000: <dig> framework map and the map order in this area suggested from the assembly is higher than  <dig>  we considered that order of the rh map is the correct one.
this could also be due to assembly difficulties close to the centromere region.
third, several markers absent in the sequence assembly could be localised on the rh map .
most of these markers belong to regions for which sequence information is available, but that couldn't be incorporated in the sequence assembly at all  or that could be assigned to gga <dig>  but without a clear location .
in addition, one gene  also appeared to belong to a region with no sequence available .
this gene had previously been located on the cytogenetic map to the short arm of gga <dig>  <cit> , so we consider our data as a confirmation.
fourth, we observed a discrepancy in the local order of the two framework map markers mcw <dig> and gtf2a <dig>  however, the difference of likelihood between our framework map and the same map with an inversion of these two markers is only  <dig> .
it is therefore difficult to conclude as to which between the sequence and the rh map presents the correct order.
from these data we conclude that radiation hybrid maps can be useful to help detect errors in the draft sequence assembly and for mapping genes either absent or of unknown location in the assembly.
comparison cr6000/cm/kb
the average cr/cm ratio is  <dig>  when calculated over the whole map length.
this relatively high value, as compared to the  <dig> cr/cm obtained for gga <dig>  <cit> , must be inflected by the disparity observed along the chromosome .
this heterogeneity actually reflects disparities in the recombination rate along the chromosome, with recombination events more frequent at the end of the long arm.
the agreement between the gene order found on rh map and the sequence assembly is very high.
considering only the q arm of the chromosome, the cr/mb ratio is  <dig> , or  <dig>  kb per cr.
this ratio, similar to that obtained for gga <dig> , is quite lower than the  <dig> kb/cr and  <dig> kb/cr values obtained for gga <dig>  <cit>  and gga <dig>  <cit>  respectively, suggesting a higher resolution for the larger chromosomes.
this result can have two origins: first, the kb/cr ratio is not constant from one chromosome to another, regardless of their physical length  <cit> ; second, the previous calculations were based on physical length values estimated from cytogenetic studies:  <dig> mb for gga <dig>  <cit>  and  <dig> mb for gga <dig>  <cit> .
if we consider the actual chromosome length based on sequence assembly, these chromosomes are shorter than previously estimated, with values of  <dig>  and  <dig>  mb , the ratio is thus now closer to the value we obtain here for gga <dig>
comparative mapping
CONCLUSIONS
we have built a high resolution rh map of chicken chromosome  <dig> using the chickrh <dig> panel.
in doing this, we fulfilled our objective of obtaining a detailed comparative map of gga <dig>  providing jointly a source of potential polymorphic markers and of candidate genes for qtl mapping on this chromosome.
at the end of our work, the first draft chicken genome assembly was released and we aligned it to our gga <dig> rh map.
although we detected a few errors to correct, this allowed us to demonstrate the high quality of the sequence assembly, which may have benefited from a low frequency of repeated elements.
in the near future, the chickrh <dig> panel will be used to assist in improving the chicken genome assembly.
this is clearly needed in the regions for which the genetic map is still not complete, such as some microchromosomes, but also for parts of macrochromosomes, as shown in this study.

 chicken is the first major agricultural species for which the complete genome sequencing was undertaken. this is partly due to its position as a model species in various fields of biology including embryo development, oncology, immunology and evolution. moreover, as it is the only bird species for which the genome study is so advanced, very much is expected from its use in comparative genome analyses for annotation, including that of the human genome, by detection of conserved sequences. its intermediate phylogenetic position between mammals and fishes will also certainly provide valuable information on the evolution of vertebrate karyotypes. radiation hybrid maps have a resolution power intermediate to that of the genetic and bac contig maps and are also a powerful tool for the mapping of ests and genes by simple pcr. they are thus useful at two levels: first, they can be used constructively as scaffolds for a correct genome assembly or for detecting and correcting misassembled portions of the genome; second, before obtaining whole annotated genome sequences, they are very efficient tools for inter-species comparative genome analyses through the easy mapping of genes and ests. the successful production of a rh panel in chicken is quite recent, and therefore rh maps are only available for a limited number of chromosomes. having identified qtl for fatness on chicken chromosome, our objective was to build a high-resolution and gene-rich rh map for this chromosome, as a basis for high precision comparative mapping with human and for the development of new polymorphic markers. the available human/chicken comparative mapping data indicated conservation of synteny between gga and portions of hsa hsa and hsa in addition, two genes from hsa had also been shown to be located on gga. this information was used to develop markers from chicken est sequence data orthologous to genes in these human regions, in addition to the existing markers from the chicken chromosome genetic map. while in the process of finishing our map, the first draft sequence assembly of the chicken genome was released. the quality of both the gga rh map and of the sequence assembly was therefore checked by alignment of all the markers by blast searches.  development of est markers in addition to the microsatellite markers from the genetic map, and primer pairs chosen either from available primer data in the literature or designed using the gene sequence deposited in genbank/embl, primer pairs were chosen from chicken est markers selected on the basis of the known conservations of synteny between human and chicken using the iccare software. constraints on the design of primers were to avoid presence of long introns, whose position and length was predicted on the basis of the orthologous human gene structure, and to design primers in the most divergent regions of the human/chicken alignment, to limit cross-amplification with the hamster dna present in the hybrids. one hundred and thirty nine primer pairs out of enabled a successful amplification and the subsequent mapping of the corresponding genes, confirming the high success rate obtained when using the iccare software for designing chicken pcr primers based on est data. construction of the gga rh map altogether, genotyping data was obtained for a total of markers, comprising gene fragments and microsatellites from the gga genetic map. two-point analysis using a lod threshold of enabled to constitute a group of markers, including all the microsatellite markers from the genetic map. the remaining markers correspond to the external boundaries of the regions of conserved synteny with human, from which ests were chosen for marker development and map either to other chromosomes for which rh maps were developed or to unknown regions. after multipoint analysis, a 1000: framework map cr long, comprising a total of markers including microsatellites and ests was obtained. the remaining markers are located relative to the framework map within confidence intervals, to build a comprehensive map. to compare the rh and the genetic maps, the best possible position of the non-framework common markers had to be estimated. that of the markers on the rh map was computed by the carthagene program and is indicated in addition to the confidence interval. for the genetic map, the central position of the marker's confidence interval was used as their most probable position. as a result, the order of the markers on the rh map matches exactly that of the same markers on genetic map, with only one notable discrepancy concerning the position of brf. however, when the position of this gene was checked on the sequence assembly, the agreement was with the rh map, suggesting the position of this gene on the genetic map is erroneous. an average retention frequency of % was observed for the gga markers studied here, although with a high variation, with values ranging from % to %. this finding is within the range observed in other studies reported on this panel: % overall retention using markers chosen genome-wise, % for gga, for gga and 18% for gga. as already noticed for several species including human or cow, but also for chicken chromosomes and, a centromeric effect is detected when observing retention frequencies of markers along the map, with a higher retention of markers in the region between to cr, in which the retention culminates at a value of %, whereas it is around 15% for the rest of the chromosome. alignment of the rh map to the genomic sequence a preliminary data set based on the first draft chicken genome assembly has been deposited into public databases by a team led by r. wilson and w. warren, from the washington university school of medicine in st. louis. we compared our data with the gga sequence, by using blastn searches and sequence alignments. the agreement between the rh framework map and the sequence orders is almost perfect, although with a few discrepancies, most of them suggesting possible improvements to be made in the sequence assembly. first, a group of markers, that we developed on the basis of the conservation of synteny between gga and hsa is assigned to gga in the sequence assembly. three of these markers are on the framework map and for all four, the rh genotypings obtained are very similar to those obtained with the flanking rh framework markers slc17a and arntl, both located on the gga sequence assembly. furthermore, when two-point analysis of the four markers was computed with the flanking markers loc and slc22a suggested in the gga sequence assembly, lod scores were equal to zero. this part of the genome assigned to a wrong chromosome on the sequence assembly covers a region at least cr long, corresponding to a distance of to mb, as estimated from the cr to mb ratio. indeed, the length of the sequence between the two extreme markers cstf and gpr on the gga assembly is mb. the retention frequency of these four markers is amongst the highest of all, suggesting that their location is close to the centromere and that the possible sequence assembly problems are related to this proximity, perhaps due to repetitive sequences. second, we observed an inversion of the gene order for a segment in the upper part of the chromosome adjacent to the group we described as wrongly assigned to gga in the sequence assembly. as the difference of likelihood between our 1000: framework map and the map order in this area suggested from the assembly is higher than we considered that order of the rh map is the correct one. this could also be due to assembly difficulties close to the centromere region. third, several markers absent in the sequence assembly could be localised on the rh map. most of these markers belong to regions for which sequence information is available, but that couldn't be incorporated in the sequence assembly at all or that could be assigned to gga but without a clear location. in addition, one gene also appeared to belong to a region with no sequence available. this gene had previously been located on the cytogenetic map to the short arm of gga, so we consider our data as a confirmation. fourth, we observed a discrepancy in the local order of the two framework map markers mcw and gtf2a however, the difference of likelihood between our framework map and the same map with an inversion of these two markers is only. it is therefore difficult to conclude as to which between the sequence and the rh map presents the correct order. from these data we conclude that radiation hybrid maps can be useful to help detect errors in the draft sequence assembly and for mapping genes either absent or of unknown location in the assembly. comparison cr6000/cm/kb the average cr/cm ratio is when calculated over the whole map length. this relatively high value, as compared to the cr/cm obtained for gga, must be inflected by the disparity observed along the chromosome. this heterogeneity actually reflects disparities in the recombination rate along the chromosome, with recombination events more frequent at the end of the long arm. the agreement between the gene order found on rh map and the sequence assembly is very high. considering only the q arm of the chromosome, the cr/mb ratio is, or kb per cr. this ratio, similar to that obtained for gga, is quite lower than the kb/cr and kb/cr values obtained for gga and gga respectively, suggesting a higher resolution for the larger chromosomes. this result can have two origins: first, the kb/cr ratio is not constant from one chromosome to another, regardless of their physical length; second, the previous calculations were based on physical length values estimated from cytogenetic studies: mb for gga and mb for gga. if we consider the actual chromosome length based on sequence assembly, these chromosomes are shorter than previously estimated, with values of and mb, the ratio is thus now closer to the value we obtain here for gga comparative mapping  we have built a high resolution rh map of chicken chromosome using the chickrh panel. in doing this, we fulfilled our objective of obtaining a detailed comparative map of gga providing jointly a source of potential polymorphic markers and of candidate genes for qtl mapping on this chromosome. at the end of our work, the first draft chicken genome assembly was released and we aligned it to our gga rh map. although we detected a few errors to correct, this allowed us to demonstrate the high quality of the sequence assembly, which may have benefited from a low frequency of repeated elements. in the near future, the chickrh panel will be used to assist in improving the chicken genome assembly. this is clearly needed in the regions for which the genetic map is still not complete, such as some microchromosomes, but also for parts of macrochromosomes, as shown in this study. 
moreover, once framework rh maps of a genome have been constructed, a quick location of markers by simple pcr on the rh panel is possible. the remaining markers were placed alongside the framework markers within confidence intervals. the chicken chickrh panel recently produced was used here to construct a high resolution rh map of chicken gga to confirm the validity of the map and to provide valuable comparative mapping information, both markers from the genetic map and a high number of ests were used. a total of markers were typed on the chickrh rh panel, of which were assigned to gga the final map is composed of framework markers extending over a cr distance. finally, this rh map was used for testing the accuracy of the chicken genome assembly for chromosome the high resolution framework map obtained in this study has markers covering the entire chicken chromosome and reveals the existence of a high number of rearrangements when compared to the human genome. only two discrepancies were observed in relation to the sequence assembly recently reported for this chromosome.
 the resolution of radiation hybrid maps is intermediate between that of the genetic and bac contig maps. moreover, once framework rh maps of a genome have been constructed, a quick location of markers by simple pcr on the rh panel is possible. the chicken chickrh panel recently produced was used here to construct a high resolution rh map of chicken gga to confirm the validity of the map and to provide valuable comparative mapping information, both markers from the genetic map and a high number of ests were used. finally, this rh map was used for testing the accuracy of the chicken genome assembly for chromosome  a total of markers were typed on the chickrh rh panel, of which were assigned to gga the final map is composed of framework markers extending over a cr distance. the remaining markers were placed alongside the framework markers within confidence intervals.  the high resolution framework map obtained in this study has markers covering the entire chicken chromosome and reveals the existence of a high number of rearrangements when compared to the human genome. only two discrepancies were observed in relation to the sequence assembly recently reported for this chromosome. 