BACKGROUND
chromosome  <dig> contains a region of the porcine genome in which the highest number of quantitative trait loci  have been mapped, affecting most notably growth, fat deposition and carcass composition  <cit> .
among all qtl detected on this chromosome, many have been mapped in an interval spanning the sla complex in position 7p12-q <dig>  development of a high resolution porcine gene map and comparison with the human map is a major step towards the identification of the genes responsible for qtl effects.
the development of two radiation hybrid panels imprh and imnprh <dig> constructed after irradiation of porcine cells at  <dig>  and  <dig>  rads  <cit>  provides tools of choice for such comparative mapping studies at  <dig> complementary resolutions : 1) one breakage could be observed on at least one hybrid of the panel for markers at an average distance of > <dig> and  <dig> kb, 2) the order of a set of markers can be unambiguously determined, provided that the distance between adjacent markers range from  <dig>  to  <dig>  mb on imprh panel and from  <dig> to  <dig> kb on imnprh <dig> panel  <cit> .
at first glance, the chromosomal region in which qtl have been mapped on ssc <dig> seemed identical to the p arm of hsa <dig>  <cit> .
a more detailed study showed that a fragment of ~ <dig>  mb found at a pericentromeric location on hsa <dig> is situated at  <dig> mb from the expected location on the porcine genome, precisely in the region of interest  <cit> .
this fragment was defined by three genes  located close to the centromere at  <dig> mb on hsa6p <dig> -6p <dig>  and mapped on ssc7q <dig> close to rab2l and daxx genes localized at  <dig> mb on hsa6p <dig> .
a preliminary physical map of this region was proposed by barbosa  <cit> , but  <dig> limited bac contigs were built in this rearranged region.
however, it failed to establish a complete comparative map of this region and to ensure that no additional fragments found elsewhere on the human genome are located in the porcine qtl region.
we now report the building of a complete bac map covering the whole region.
this was achieved by taking advantage of the fingerprinting of a  <dig> × bac library , undertaken to establish primary bac contigs.
we monitored the assembly of the complete contig by mapping genes and markers on the high resolution imnprh <dig> panel, as previously done in the halothane region  <cit> .
sequence comparison of bac ends from this contig with the human sequence allowed us to identify  <dig> bac containing the four synteny breaking points detected in this region.
a local bovine rh map was also established in this region to determine if the rearrangements observed on the pig genome are specific of suines or if they occurred in an ancestral species.
a multispecies comparison of this region was finally performed.
RESULTS
development of a preliminary porcine gene map
to study the complete rearrangements that occurred in this region of an estimated  <dig>  mb and containing the  <dig> genes lano , bmp <dig> and bag <dig>  <cit> , it was necessary to: 1) define the chromosomal blocks arranged differently between hsa <dig> pericentromeric region and ssc7; 2) determine if additional human fragments moved in this porcine region.
in the first approach, we increased the density of genes mapped in this region of the imnprh <dig> panel  <cit> .
using the iccare browser  <cit> , we identified genes for which porcine est were available.
we chose  <dig> genes located on hsa6p <dig>  and  <dig> genes located on hsa6p <dig> -6p <dig> : col11a <dig> , hsd17b <dig> , ring <dig> , vps <dig> , rps <dig> , gclc , tinag , hcrtr <dig> , col21a <dig>  and dst .
no gene was selected below prim2a since no porcine sequence was available for the few genes located in this region very close to the centromere of hsa <dig>  when possible, primers were developed in the 3' utr sequence of selected genes .
for gclc only, we had to choose primers in two consecutive exons framing an intron.
markers developed for these  <dig> genes, as well as markers previously developed  <cit>  for rxrb , elovl <dig>  and prim2a , were analyzed on the  <dig> clones of imnprh <dig> panel.
in addition to markers produced in the frame of this project, we included in the analysis markers already mapped in this region on imprh and imnprh <dig> panels, in particular hke <dig> , rab2l , lano  ,bmp <dig>  and bag <dig>   <cit> .
an average retention frequency of 40% was observed, ranging from 27% for prim2a to 55% for rab2l and hke <dig>  a draft 1000: <dig> framework map was then built with carthagene software  <cit> , using a stepwise locus adding strategy .
the robustness of the map was tested by comparison of the likelihood of the reference map to the likelihood of maps derived from the reference map by all possible permutations in a window of  <dig> markers and by inversion of parts of the map using the simulated annealing algorithm.
the  <dig> additional genes  were mapped at their most likely location relatively to markers of this preliminary framework map, considering the distance between framework markers as fixed.
to compare the order of genes between ssc <dig> and hsa <dig>  we determined the location of these genes on the ncbi build  <dig> assembly of the human genome available at ucsc  <cit> .
we confirmed the insertion in pig of a large fragment between ring <dig> and vps <dig> genes , which are only  <dig> kb apart on the human genome .
the fragment found in pig between these two genes is located on human genome close to the centromere on the hsa6p arm.
the mapping of these genes on the porcine rh map allowed us to distinguish two main conserved fragments: one fragment containing bmp <dig> – hctr <dig> – sw <dig> – tinag – lano – gclc , and a second fragment containing dst, bag <dig> and prim2a .
the order of genes is conserved inside each fragment; however, the  <dig> blocks are oppositely oriented in the porcine and human genomes.
a large distance is observed between gclc and bag <dig>  suggesting that another fragment is inserted between them, possibly a fragment called "6" in the human genome.
this additional porcine fragment is particularly interesting because it contains sw <dig>  a marker mapped at the most likely location of the qtl of interest.
at that step, it was not possible to determine whether the rearranged fragment includes a short fragment located on the q arm of hsa <dig>  the first gene found on hsa6q arm also mapped on the porcine genome, being flj <dig> located on ssc <dig>  <cit> .
markers in bold belong to the framework rh map, whereas markers in italic are positioned at the most likely location.
other markers have been developed in the frame of the contig building.
the arrows on the side represent the fragments conserved between the pig and human genomes.
primer sequences, retention fraction, rh map location on porcine map and human location are indicated.
bac contig construction
in order to confirm the rearrangements previously observed between hsa <dig>  and ssc <dig> , we built a complete bac contig of this region.
as in previous studies, an almost complete bac contig of the sla region has been developed  <cit> , but a gap remained to be filled precisely in this region that was very different between the human and pig genomes.
we screened by pcr the inra bac library  <cit>  using primers defined for microsatellites sw <dig> and sw <dig>  and for the  <dig> following genes ring <dig>  hsd17b <dig>  bmp <dig>  hcrtr <dig>  tinag, lano, gclc, prim2a, bag <dig> and dst.
for each of these  <dig> genes or  <dig> markers , we identified one positive bac .
to accelerate the establishment of a complete bac contig, we took advantage of the availability of preliminary bac contigs built after the fingerprinting of  <dig>  <dig> bac of the inra library .
it appeared that  <dig> of these  <dig> bac were mapped in  <dig> different inra contigs , containing from  <dig> to  <dig> bac .
in the frame of the inra physical mapping project,  <dig>  <dig> inra bac regularly dispersed on the inra contigs had been selected.
the sequence of their extremities were determined at cns  and compared with the human sequence .
analysis of  <dig> porcine bes anchored on the human region between  <dig> and  <dig> mb, or between  <dig> and  <dig> mb, allowed us to identify  <dig> bac located on the pig genome in the chosen region.
among these bac,  <dig> belonged to one of the  <dig> inra contigs already identified.
<dig> additional bac allowed us to identify  <dig> others inra contigs  located in this same region, and the last  <dig> clones correspond to bac that had not been fingerprinted.
to fill several gaps, the inra bac library was screened with markers derived from bes, and  <dig> additional inra bac  were recruited.
for bac of interest whose bes had not been sequenced by the cns, we sequenced the extremities in order to get on with the construction of the contig; these sequences were submitted to embl nucleotide sequence database .
to fill the remaining gaps, we selected  <dig> additional bac  on the international bac map .
the building of the complete bac contig was also monitored regularly by the mapping of bac end- markers on the imnprh <dig> rh panel, verifying that the order of the markers and the distances between them were consistent with the bac contig results.
in particular, markers found at the extremities of the  <dig> inra contigs were mapped on the rh panel.
for these  <dig> markers, an average retention frequency of 35% was observed.
a 1000: <dig> framework map was then established, including previously mapped genes and  <dig> markers derived from bes.
the  <dig> others bac ends were mapped at their most likely location relatively to markers of the framework.
this rh map covering  <dig> cr is in total agreement with the bac contig covering the whole region .
analysis of bac contig
the availability of bes from bac located between prim2a  and gclc  permitted characterization of the intermediate fragment in which no genes were mapped .
all the significant homologies detected with these bes show that this fragment corresponds to fragment  <dig> located on hsa <dig> in the interval  <dig>  –  <dig>  mb framing the centromere.
among bes anchored at this human region, bi0666e01f is highly homologous to an intron of khdrbs <dig>  demonstrating the presence of the gene in this porcine region.
in addition, the comparison of porcine bes produced from bac bi0222c <dig>  bi0223c <dig> and bi0819c <dig> revealed that these sequences share significant homology with  <dig> human fragments at a distance of  <dig> kb, suggesting the presence of duplication in the human genome.
a dot-plot of the human region  <dig>  mb- <dig>  mb on itself was performed with dotter, using its default settings  <cit> .
the dot-plot showed that this region contains many large repeated sequences.
a fragment of  <dig> kb located at  <dig>  mb- <dig>  mb is duplicated in position  <dig>  mb- <dig>  mb, and a  <dig> kb fragment from  <dig>  mb to  <dig>  mb is duplicated and turned over in position  <dig>  mb- <dig>  mb on hsa <dig> .
in the pig, this fragment covers ~ <dig> cr, which corresponds to ~ <dig> kb suggesting that a single copy is present in the pig genome.
finally, for bac bi0688h <dig> and bi0046e <dig> mapped in the same region, hits obtained for the two bes of both clones were  <dig> kb apart, which is not compatible with the size of a bac.
analysis of this human region on ensembl database  <cit>  indicates that no porcine bes matches the human genome between  <dig>  and  <dig>  mb, whereas bes were anchored in the surrounding region on average each  <dig> kb.
altogether these results suggest that a  <dig> kb region  is missing in the pig genome.
from this analysis, an estimate can be made of the size of the rearranged region on the pig genome – considering that the size of this region on the porcine genome is similar to the size of human region also found on the porcine genome.
the sizes of the three rearranged fragments are: ~ <dig>  mb for fragment  <dig>   <dig>  mb for fragment  <dig> and  <dig>  mb for fragment  <dig>  the whole rearranged fragment covers thus ~ <dig>  mb of the porcine genome.
to confirm the reality of the rearrangements identified during the building of the bac contig, we tried to identify a porcine bac spanning each of the  <dig> breakpoints, with bes anchored on the  <dig> expected locations on human genome .
in order to increase the probability of identifying these bac, we also selected from the international map  <cit>  bac overlapping inra bac close to synteny breaking points.
for bac with no significant homology, we used fasta to detect any potential homology, using less stringent parameters.
in pig, one bac anchored on the  <dig> expected human locations was identified, for the  <dig> breaking points a-b, c-d, e-f and g-h , bt0141e <dig> , bi0222c <dig>  and rpci44_0428o <dig> ) .
analysis of the anchorage on the human map of all bes available in these regions, allowed us to estimate more precisely the location of the  <dig> breakpoints on the human genome : a/h breakpoint is located at  <dig>  –  <dig>  mb, i/c at  <dig>  –  <dig>  mb, b/g at  <dig>  –  <dig> , f/d after  <dig>  mb and before  <dig>  mb , e point after  <dig>  mb.
multispecies comparison of the organization of the studied region
to compare the order of genes in this region between various species, we identified the location on the genome of sequenced species of several genes representing each chromosomal fragment described previously: fragment  <dig> , fragment  <dig> , fragment  <dig> , fragment  <dig> , fragment  <dig> , fragment  <dig>  and fragment  <dig> .
their locations were determined by blasting the porcine sequences against current genome assemblies: mouse sequence build  <dig> assembly from ncbi, dog whole genome shotgun  assembly v <dig>  , and chicken galgal <dig> assembly.
to enrich the set of data available for the chicken genome, we developed markers for vps <dig> and rab2l which were not identified on the sequence of chicken genome with the ests available for these genes.
we mapped these  <dig> markers on chickrh panel  <cit>  and analyzed the results on chickrh server  <cit> .
it was also important to include a species phylogenetically close to the pig in the comparison.
we selected cattle, a species for which both detailed comparative mapping studies have been performed  <cit>  and a partial genomic sequence is available.
however, in the region of interest, the assembly of the bovine genome is by no means complete  and available rh maps are insufficiently detailed.
this led us to develop a bovine rh map in this region.
we used the iccare browser  <cit> , to select bovine est available for  <dig> genes of this region: rxrb, ring <dig>  hke <dig>  hsd17b <dig>  vps <dig>  rab2l, elovl <dig>  tinag, lano, col21a <dig>  dst, bag <dig>  prim2a, khdrbs <dig>  phf <dig> and ptp4a <dig> .
we developed pairs of primers for each gene and we mapped them on the rh <dig> bovine panel  <cit> .
an average retention frequency of 36% was observed, ranging from 26% for glo <dig> to 50% for ptp4a <dig>  vectors obtained for the  <dig> new genes were analyzed together with  <dig> markers previously available for bta <dig>  <cit> .
a 1000: <dig> framework map including  <dig> genes, was built with carthagene software  <cit>  in the region from prim2a to vps <dig> .
in addition gene khdrbs <dig> is linked with a lod score  <dig>  at the top of this map.
four additional genes hsd17b <dig>  rxrb, ring <dig>  and hke <dig> were found totally linked and mapped at their most likely location relatively to markers of the framework .
elovl <dig> was found linked to markers previously mapped in a distal position.
phf <dig> and ptp4a <dig> were found significantly linked together , but totally unlinked to other genes.
in the dog genome, all the selected genes are grouped into two large fragments  located on chromosome  <dig> .
in the mouse genome, the same region is similarly split into  <dig> conserved fragments : fragments  <dig>   <dig> and  <dig> are located on mmu1; fragments  <dig> and  <dig> on mmu9; and fragments  <dig> and  <dig> on mmu <dig>  in chicken, a conserved fragment containing fragments  <dig>   <dig>   <dig>   <dig> and  <dig> is located on gga <dig>  two other genes, vps <dig> and rab2l , are mapped in a linkage group that also contains markers derived from chicken mhc region located on gga <dig> .
no information is available for rxrb and ring <dig> .
if the order of genes is thus globally conserved in these species, a very different situation is observed on the bovine genome :  <dig> of the  <dig> selected genes are localized on bta <dig>  but in an order very different from the generally conserved order, and  <dig> others genes  are located on chromosome  <dig>
the analysis of these results shows that the whole fragment  inserted on the pig genome between fragments  <dig> and  <dig>  seems very well conserved in chicken, dog, mouse and human.
in cattle and pig, belonging both to the clade of cetartiodactyles, fragments  <dig>   <dig> and  <dig> are found at a different location.
however, in this  <dig>  mb large fragment, a total conservation of gene order was observed between human and cattle genomes.
a global analysis of the chromosomal rearrangements that occurred during evolution was carried out using grimm.
<cit> .
the results obtained are summarized on figure  <dig>
markers in bold belong to the framework rh map whereas markers in italic are positionned with the best likelihood.
primer sequences, retention fraction, rh map location on bovine map and human location are indicated.
discussion
the study of porcine chromosome  <dig> is of particular interest due to the presence of many qtl affecting traits of economic importance and of mhc  <cit> .
one approach to study this chromosome is to identify all the genes located on this chromosome and to compare their order relatively to that in the human genome.
previously, a strong conservation of gene order was found between a large fragment of porcine ssc <dig> and human hsa <dig> p arm  <cit> .
we had highlighted the existence of a fragment estimated to be less than  <dig> mb rearranged between ssc <dig> and hsa <dig>  <cit> .
this fragment defined by  <dig> genes  located on hsa6p <dig> -6p <dig>  is found in the pig genome between genes located on hsa6p <dig>   <cit> .
the building of a bac contig in the chromosomal region surrounding sla complex provided additional information, even if the contig remained incomplete  <cit> .
this study describes a situation that is even more complex, suggesting the presence of vps <dig>  within the rearranged region.
in the pig genome, this rearranged chromosomal region is included in the region where the qtl of interest is mapped, between markers sw <dig> and nfy .
in order to identify all candidate genes that may be responsible for the qtl effects, it was particularly important to establish a detailed comparative mapping analysis of this region.
we first built a dense porcine rh map  using the high resolution imnprh212000rad panel, allowing us to order unambiguously genes at distances of  <dig> to  <dig> kb  <cit> .
our data confirmed the shift of the rearranged fragment and its precise location in the pig genome between ring <dig> and vps <dig> genes  .
in contrast to barbosa et al  <cit> , we show that vps <dig> is located close to rab2l in both species.
we also determined more precisely the extent of the fragment rearranged between the human and pig genomes.
the upper frontier of this fragment is located between elovl <dig>  and gclc, located respectively at  <dig>  and  <dig>  mb on hsa <dig>  the lower boundary of the fragment is defined by the first gene located on hsa6q also mapped on ssc <dig>  this first gene was previously designated as flj <dig>  and was located at  <dig>  mb on hsa <dig>  <cit> .
we showed that gluld <dig>  located at  <dig>  mb on hsa <dig>  also mapped on ssc <dig> and constitutes the limit currently identified in this fragment.
the gene-based rh map  that we built allowed us to define  <dig> sub-fragments of this region arranged differently on the pig and human genomes.
two of these sub-fragments are delimited by genes: bmp <dig> and gclc for the first one , and prim2a and col21a <dig> for the second .
no genes were identified in the third fragment , which contains the anonymous microsatellite sw <dig>  this map greatly refines the internal rearrangements suggested by our previous work  <cit> .
the sum of the sizes of fragments  <dig> and  <dig> is ~ <dig>  mb and is similar to the orthologous human region.
fragment  <dig> covers  <dig>  mb in the human map, whereas the size of this fragment in the porcine rh map suggests a size of approximately  <dig> kb which would indicate that a fragment of  <dig> kb is absent in the porcine genome.
the fragment containing sw <dig> on the pig genome might thus be orthologous to part of the region flanking the centromere of hsa <dig>  but at that step it remained to be proved .
to confirm the order of genes suggested by rh mapping and study this region in more detail, we built a contig of bac covering the entire region .
the availability of elementary contigs resulting from the fingerprinting of clones from the inra bac library and sequences of extremities of  <dig> of these bac   greatly facilitated the establishment of this complete contig.
our work completes the studies begun by barbosa et al  <cit> , who presented  <dig> small contigs.
the analysis of porcine bes anchoring on the human genome demonstrates that the fragment  <dig> containing porcine marker sw <dig> corresponds to a fragment framing the centromere of hsa <dig> .
taking into account the average distance between two hits of porcine bes on human genome, we can say that if additional fragments from other human chromosomes are located in this rearranged region of the porcine genome, they should measure < <dig> kb.
analysis of the anchoring of porcine bes on the human genome highlighted also additional results: 1) it was indeed possible to define the position of the  <dig> synteny breakpoints .
on the human genome, these synteny breakpoints  are situated between genes ring1-vps <dig>  elovl5-gclc and bmp5-col21a <dig>  each of these  <dig> synteny breakpoints are localized in a interval of ~100– <dig> kb, between the human positions  <dig>  mb- <dig>  mb,  <dig>  mb- <dig>  mb and  <dig>  mb- <dig>  mb.
2) the fourth synteny breakpoint  was identified thanks to the anchoring of the bes in the human genome: the two fragments framing hsa <dig> centromere were found in a different order on the pig genome without the presence of a centromere .
3) for several bes, alignment on the human genome showed  <dig> possible positions with similar likelihood.
this result suggested the existence of duplicated sequences close to the human centromere, which was confirmed by a dot plot on itself of the sequence of this human region.
phylogeny studies of human chromosome  <dig> demonstrated the repositioning of this centromere among primate species in a region equivalent to 6p <dig>  in eulemur macaco, callithrix jacchus and lagothrix lagothrica, to 6q <dig> in macaca fascicularis and presbytis cristata, and to 6p <dig> in the great apes  <cit> .
the repeated sequences we highlighted might then correspond to sequences of an ancestral centromere positioned in hsa6p <dig>   <cit> .
4) lastly, in silico analysis of the bes alignments on the human genome provided evidence of the absence from the pig genome of a fragment of ~400– <dig> kb found in human genome.
one can argue that the  <dig> bes of bac bi0688h <dig> match on the human genome with sequences distant of  <dig> kb .
the absence of anchoring of other bes between  <dig>  mb and  <dig>  mb, whereas one hit is found for each  <dig> kb in the surrounding region, confirmed that this fragment is absent from the pig genome.
this region appears to be heavily rearranged between the porcine and human genomes.
we wanted to determine if these rearrangements are specific of suines or cetartiodactyles, or occur in many other species.
to identify gene order in other species, we used the genomic sequence when available.
for gallus gallus, the assembly of the genome has not been totally completed; this is why we enriched information obtained from the sequence by rh mapping data when it was necessary .
the order of genes seems to be conserved between the chicken and human genomes except for the rxrb and ring <dig> genes .
these  <dig> genes which may not exist in the chicken genome are located in other species in the extended class ii region of mhc.
the chicken mhc, named the b locus, is a  <dig> kb dna sequence containing  <dig> genes  <cit> .
it defines a minimal essential set of mhc genes conserved over  <dig> million years of divergence between birds and mammals  <cit> .
as the available assembly of the cow genome consists of many independent sequence scaffolds, we determined the gene order by rh mapping  <cit>  .
our results reveal that genes from gclc to khdrbs <dig>  are similarly ordered on the human and bovine genomes in this region, which suggests that the internal rearrangements of these  <dig> fragments, comparing the human and porcine genomes, are specific to the evolution of the suine family.
everts-van der wind et al.
<cit>  reported that  <dig> cattle chromosomes are entirely similar to a complete human chromosome arm, and in particular, that p and q arms of hsa <dig> are homologs of bta <dig> and bta <dig>  our study modifies slightly this report, in that khdrbs <dig> , the first gene localized on the long arm of human chromosome  <dig>  is probably located on bovine chromosome  <dig>  as the number of genes in this region, as in other species, is very limited; it is difficult to develop and map markers in this region.
the end of bta <dig> might thus signal the location of the centromere in the ancestors of cetartiodactyles, in which the fission between the  <dig> chromosomal arms occurred.
in this region, a further comparison of the bovine genomic sequence with the human sequence will be of interest.
we compared the gene order in the pig  and cattle  with the situation in the genomic sequence of mus musculus, canis familiaris, gallus gallus and homo sapiens .
we observed at first that on the pig genome, a large fragment  of ~ <dig>  mb is shifted between ring <dig> and vps <dig>   <dig> genes belonging to the extended class ii region of the mhc.
if mhc is one of the most dynamic regions of the genome  <cit> , a high level of conservation of genome organization provides evidence in this region of extended class ii genes  <cit> .
fourteen genes occur in this region between ring <dig> and the vps <dig> in dog, cat, human and mouse genomes, which only differs in the presence of processed pseudogenes  <cit> .
this small fragment of  <dig> kb containing ring <dig> and vps <dig> is thus dynamically involved in chromosomal evolution.
it is interesting to note that the translocation of the  <dig>  mb fragment occurs in this region.
in this region, the genomic organization of the fragment and its flanking regions is similar in mouse, human, dog and chicken, whereas translocation is observed in pig and cattle.
this suggests that this genomic reorganization occurred after the divergence of the common ancestor of cetartiodactyles from the ferungulates ancestor between  <dig> to  <dig> million years ago  <cit> .
the gene order observed in pig for the  <dig>  mb fragment is different from the one identified in other species, indicating that these internal rearrangements occurred after ruminant-suine speciation.
we identified a situation really different from the map presented by everts-van der wind et al.
for the proximal part of bta <dig> close to the centromere  <cit> .
further study of the rearrangements that occurred in ruminants in this region of extended class ii of the mhc complex is clearly needed.
we used grimm to compare the different maps available and to present a possible evolution of this region between the various species  <cit> .
the unrooted tree we obtained  presents a very different view from a classical phylogenic tree  <cit> , highlighting a high rate of rearrangements in this region that have occurred recently in cetartiodactyles.
the study of local rearrangements also provides a view different from the recent multispecies comparison of this region  <cit> .
in order to filter out noise induced by mapping errors, this analysis from murphy et al only took into account fragments > <dig> mb, defined by three genes.
among other results, they documented the reuse of evolutionary breakpoint regions identified between conserved homologous synteny blocks.
our study indicates that such a study should be redone on a more detailed scale, when the accuracy of mapping data is adequate.
CONCLUSIONS
a qtl affecting fattening and growth had been mapped in the porcine region ssc 7q <dig> .
as we had shown that a small fragment is translocated between porcine and human genome, it was important to establish a final comparative map of this region.
the rh map and the bac contig we developed in this region allowed us to attest that the translocated fragment is approximately  <dig>  mb long.
it consists in three blocks, found in hsa <dig> between  <dig>  and  <dig>  mb, one of these blocks containing a few genes, framing the centromere on the human chromosome.
the multispecies study allowed us to confirm that the translocation of this  <dig>  mb fragment occurred in the common ancestor of pig and ruminants, whereas internal rearrangements are found only in the pig genome.
the exact knowledge of the genomic content of this region permits us now to establish a list of all possible candidate genes that may affect the growth and fattening of pigs.

 chromosome contains a region of the porcine genome in which the highest number of quantitative trait loci have been mapped, affecting most notably growth, fat deposition and carcass composition. among all qtl detected on this chromosome, many have been mapped in an interval spanning the sla complex in position 7p12-q development of a high resolution porcine gene map and comparison with the human map is a major step towards the identification of the genes responsible for qtl effects. the development of two radiation hybrid panels imprh and imnprh constructed after irradiation of porcine cells at and rads provides tools of choice for such comparative mapping studies at complementary resolutions: 1) one breakage could be observed on at least one hybrid of the panel for markers at an average distance of > and kb, 2) the order of a set of markers can be unambiguously determined, provided that the distance between adjacent markers range from to mb on imprh panel and from to kb on imnprh panel. at first glance, the chromosomal region in which qtl have been mapped on ssc seemed identical to the p arm of hsa. a more detailed study showed that a fragment of ~ mb found at a pericentromeric location on hsa is situated at mb from the expected location on the porcine genome, precisely in the region of interest. this fragment was defined by three genes located close to the centromere at mb on hsa6p -6p and mapped on ssc7q close to rab2l and daxx genes localized at mb on hsa6p. a preliminary physical map of this region was proposed by barbosa, but limited bac contigs were built in this rearranged region. however, it failed to establish a complete comparative map of this region and to ensure that no additional fragments found elsewhere on the human genome are located in the porcine qtl region. we now report the building of a complete bac map covering the whole region. this was achieved by taking advantage of the fingerprinting of a × bac library, undertaken to establish primary bac contigs. we monitored the assembly of the complete contig by mapping genes and markers on the high resolution imnprh panel, as previously done in the halothane region. sequence comparison of bac ends from this contig with the human sequence allowed us to identify bac containing the four synteny breaking points detected in this region. a local bovine rh map was also established in this region to determine if the rearrangements observed on the pig genome are specific of suines or if they occurred in an ancestral species. a multispecies comparison of this region was finally performed.  development of a preliminary porcine gene map to study the complete rearrangements that occurred in this region of an estimated mb and containing the genes lano, bmp and bag, it was necessary to: 1) define the chromosomal blocks arranged differently between hsa pericentromeric region and ssc7; 2) determine if additional human fragments moved in this porcine region. in the first approach, we increased the density of genes mapped in this region of the imnprh panel. using the iccare browser, we identified genes for which porcine est were available. we chose genes located on hsa6p and genes located on hsa6p -6p: col11a, hsd17b, ring, vps, rps, gclc, tinag, hcrtr, col21a and dst. no gene was selected below prim2a since no porcine sequence was available for the few genes located in this region very close to the centromere of hsa when possible, primers were developed in the 3' utr sequence of selected genes. for gclc only, we had to choose primers in two consecutive exons framing an intron. markers developed for these genes, as well as markers previously developed for rxrb, elovl and prim2a, were analyzed on the clones of imnprh panel. in addition to markers produced in the frame of this project, we included in the analysis markers already mapped in this region on imprh and imnprh panels, in particular hke, rab2l, lano,bmp and bag. an average retention frequency of 40% was observed, ranging from 27% for prim2a to 55% for rab2l and hke a draft 1000: framework map was then built with carthagene software, using a stepwise locus adding strategy. the robustness of the map was tested by comparison of the likelihood of the reference map to the likelihood of maps derived from the reference map by all possible permutations in a window of markers and by inversion of parts of the map using the simulated annealing algorithm. the additional genes were mapped at their most likely location relatively to markers of this preliminary framework map, considering the distance between framework markers as fixed. to compare the order of genes between ssc and hsa we determined the location of these genes on the ncbi build assembly of the human genome available at ucsc. we confirmed the insertion in pig of a large fragment between ring and vps genes, which are only kb apart on the human genome. the fragment found in pig between these two genes is located on human genome close to the centromere on the hsa6p arm. the mapping of these genes on the porcine rh map allowed us to distinguish two main conserved fragments: one fragment containing bmp – hctr – sw – tinag – lano – gclc, and a second fragment containing dst, bag and prim2a. the order of genes is conserved inside each fragment; however, the blocks are oppositely oriented in the porcine and human genomes. a large distance is observed between gclc and bag suggesting that another fragment is inserted between them, possibly a fragment called "6" in the human genome. this additional porcine fragment is particularly interesting because it contains sw a marker mapped at the most likely location of the qtl of interest. at that step, it was not possible to determine whether the rearranged fragment includes a short fragment located on the q arm of hsa the first gene found on hsa6q arm also mapped on the porcine genome, being flj located on ssc. markers in bold belong to the framework rh map, whereas markers in italic are positioned at the most likely location. other markers have been developed in the frame of the contig building. the arrows on the side represent the fragments conserved between the pig and human genomes. primer sequences, retention fraction, rh map location on porcine map and human location are indicated. bac contig construction in order to confirm the rearrangements previously observed between hsa and ssc, we built a complete bac contig of this region. as in previous studies, an almost complete bac contig of the sla region has been developed, but a gap remained to be filled precisely in this region that was very different between the human and pig genomes. we screened by pcr the inra bac library using primers defined for microsatellites sw and sw and for the following genes ring hsd17b bmp hcrtr tinag, lano, gclc, prim2a, bag and dst. for each of these genes or markers, we identified one positive bac. to accelerate the establishment of a complete bac contig, we took advantage of the availability of preliminary bac contigs built after the fingerprinting of bac of the inra library. it appeared that of these bac were mapped in different inra contigs, containing from to bac. in the frame of the inra physical mapping project, inra bac regularly dispersed on the inra contigs had been selected. the sequence of their extremities were determined at cns and compared with the human sequence. analysis of porcine bes anchored on the human region between and mb, or between and mb, allowed us to identify bac located on the pig genome in the chosen region. among these bac, belonged to one of the inra contigs already identified. additional bac allowed us to identify others inra contigs located in this same region, and the last clones correspond to bac that had not been fingerprinted. to fill several gaps, the inra bac library was screened with markers derived from bes, and additional inra bac were recruited. for bac of interest whose bes had not been sequenced by the cns, we sequenced the extremities in order to get on with the construction of the contig; these sequences were submitted to embl nucleotide sequence database. to fill the remaining gaps, we selected additional bac on the international bac map. the building of the complete bac contig was also monitored regularly by the mapping of bac end- markers on the imnprh rh panel, verifying that the order of the markers and the distances between them were consistent with the bac contig results. in particular, markers found at the extremities of the inra contigs were mapped on the rh panel. for these markers, an average retention frequency of 35% was observed. a 1000: framework map was then established, including previously mapped genes and markers derived from bes. the others bac ends were mapped at their most likely location relatively to markers of the framework. this rh map covering cr is in total agreement with the bac contig covering the whole region. analysis of bac contig the availability of bes from bac located between prim2a and gclc permitted characterization of the intermediate fragment in which no genes were mapped. all the significant homologies detected with these bes show that this fragment corresponds to fragment located on hsa in the interval – mb framing the centromere. among bes anchored at this human region, bi0666e01f is highly homologous to an intron of khdrbs demonstrating the presence of the gene in this porcine region. in addition, the comparison of porcine bes produced from bac bi0222c bi0223c and bi0819c revealed that these sequences share significant homology with human fragments at a distance of kb, suggesting the presence of duplication in the human genome. a dot-plot of the human region mb- mb on itself was performed with dotter, using its default settings. the dot-plot showed that this region contains many large repeated sequences. a fragment of kb located at mb- mb is duplicated in position mb- mb, and a kb fragment from mb to mb is duplicated and turned over in position mb- mb on hsa. in the pig, this fragment covers ~ cr, which corresponds to ~ kb suggesting that a single copy is present in the pig genome. finally, for bac bi0688h and bi0046e mapped in the same region, hits obtained for the two bes of both clones were kb apart, which is not compatible with the size of a bac. analysis of this human region on ensembl database indicates that no porcine bes matches the human genome between and mb, whereas bes were anchored in the surrounding region on average each kb. altogether these results suggest that a kb region is missing in the pig genome. from this analysis, an estimate can be made of the size of the rearranged region on the pig genome – considering that the size of this region on the porcine genome is similar to the size of human region also found on the porcine genome. the sizes of the three rearranged fragments are: ~ mb for fragment mb for fragment and mb for fragment the whole rearranged fragment covers thus ~ mb of the porcine genome. to confirm the reality of the rearrangements identified during the building of the bac contig, we tried to identify a porcine bac spanning each of the breakpoints, with bes anchored on the expected locations on human genome. in order to increase the probability of identifying these bac, we also selected from the international map bac overlapping inra bac close to synteny breaking points. for bac with no significant homology, we used fasta to detect any potential homology, using less stringent parameters. in pig, one bac anchored on the expected human locations was identified, for the breaking points a-b, c-d, e-f and g-h, bt0141e, bi0222c and rpci44_0428o ). analysis of the anchorage on the human map of all bes available in these regions, allowed us to estimate more precisely the location of the breakpoints on the human genome: a/h breakpoint is located at – mb, i/c at – mb, b/g at –, f/d after mb and before mb, e point after mb. multispecies comparison of the organization of the studied region to compare the order of genes in this region between various species, we identified the location on the genome of sequenced species of several genes representing each chromosomal fragment described previously: fragment, fragment, fragment, fragment, fragment, fragment and fragment. their locations were determined by blasting the porcine sequences against current genome assemblies: mouse sequence build assembly from ncbi, dog whole genome shotgun assembly v, and chicken galgal assembly. to enrich the set of data available for the chicken genome, we developed markers for vps and rab2l which were not identified on the sequence of chicken genome with the ests available for these genes. we mapped these markers on chickrh panel and analyzed the results on chickrh server. it was also important to include a species phylogenetically close to the pig in the comparison. we selected cattle, a species for which both detailed comparative mapping studies have been performed and a partial genomic sequence is available. however, in the region of interest, the assembly of the bovine genome is by no means complete and available rh maps are insufficiently detailed. this led us to develop a bovine rh map in this region. we used the iccare browser, to select bovine est available for genes of this region: rxrb, ring hke hsd17b vps rab2l, elovl tinag, lano, col21a dst, bag prim2a, khdrbs phf and ptp4a. we developed pairs of primers for each gene and we mapped them on the rh bovine panel. an average retention frequency of 36% was observed, ranging from 26% for glo to 50% for ptp4a vectors obtained for the new genes were analyzed together with markers previously available for bta. a 1000: framework map including genes, was built with carthagene software in the region from prim2a to vps. in addition gene khdrbs is linked with a lod score at the top of this map. four additional genes hsd17b rxrb, ring and hke were found totally linked and mapped at their most likely location relatively to markers of the framework. elovl was found linked to markers previously mapped in a distal position. phf and ptp4a were found significantly linked together, but totally unlinked to other genes. in the dog genome, all the selected genes are grouped into two large fragments located on chromosome. in the mouse genome, the same region is similarly split into conserved fragments: fragments and are located on mmu1; fragments and on mmu9; and fragments and on mmu in chicken, a conserved fragment containing fragments and is located on gga two other genes, vps and rab2l, are mapped in a linkage group that also contains markers derived from chicken mhc region located on gga. no information is available for rxrb and ring. if the order of genes is thus globally conserved in these species, a very different situation is observed on the bovine genome: of the selected genes are localized on bta but in an order very different from the generally conserved order, and others genes are located on chromosome the analysis of these results shows that the whole fragment inserted on the pig genome between fragments and seems very well conserved in chicken, dog, mouse and human. in cattle and pig, belonging both to the clade of cetartiodactyles, fragments and are found at a different location. however, in this mb large fragment, a total conservation of gene order was observed between human and cattle genomes. a global analysis of the chromosomal rearrangements that occurred during evolution was carried out using grimm.. the results obtained are summarized on figure markers in bold belong to the framework rh map whereas markers in italic are positionned with the best likelihood. primer sequences, retention fraction, rh map location on bovine map and human location are indicated. discussion the study of porcine chromosome is of particular interest due to the presence of many qtl affecting traits of economic importance and of mhc. one approach to study this chromosome is to identify all the genes located on this chromosome and to compare their order relatively to that in the human genome. previously, a strong conservation of gene order was found between a large fragment of porcine ssc and human hsa p arm. we had highlighted the existence of a fragment estimated to be less than mb rearranged between ssc and hsa. this fragment defined by genes located on hsa6p -6p is found in the pig genome between genes located on hsa6p. the building of a bac contig in the chromosomal region surrounding sla complex provided additional information, even if the contig remained incomplete. this study describes a situation that is even more complex, suggesting the presence of vps within the rearranged region. in the pig genome, this rearranged chromosomal region is included in the region where the qtl of interest is mapped, between markers sw and nfy. in order to identify all candidate genes that may be responsible for the qtl effects, it was particularly important to establish a detailed comparative mapping analysis of this region. we first built a dense porcine rh map using the high resolution imnprh212000rad panel, allowing us to order unambiguously genes at distances of to kb. our data confirmed the shift of the rearranged fragment and its precise location in the pig genome between ring and vps genes. in contrast to barbosa et al, we show that vps is located close to rab2l in both species. we also determined more precisely the extent of the fragment rearranged between the human and pig genomes. the upper frontier of this fragment is located between elovl and gclc, located respectively at and mb on hsa the lower boundary of the fragment is defined by the first gene located on hsa6q also mapped on ssc this first gene was previously designated as flj and was located at mb on hsa. we showed that gluld located at mb on hsa also mapped on ssc and constitutes the limit currently identified in this fragment. the gene-based rh map that we built allowed us to define sub-fragments of this region arranged differently on the pig and human genomes. two of these sub-fragments are delimited by genes: bmp and gclc for the first one, and prim2a and col21a for the second. no genes were identified in the third fragment, which contains the anonymous microsatellite sw this map greatly refines the internal rearrangements suggested by our previous work. the sum of the sizes of fragments and is ~ mb and is similar to the orthologous human region. fragment covers mb in the human map, whereas the size of this fragment in the porcine rh map suggests a size of approximately kb which would indicate that a fragment of kb is absent in the porcine genome. the fragment containing sw on the pig genome might thus be orthologous to part of the region flanking the centromere of hsa but at that step it remained to be proved. to confirm the order of genes suggested by rh mapping and study this region in more detail, we built a contig of bac covering the entire region. the availability of elementary contigs resulting from the fingerprinting of clones from the inra bac library and sequences of extremities of of these bac greatly facilitated the establishment of this complete contig. our work completes the studies begun by barbosa et al, who presented small contigs. the analysis of porcine bes anchoring on the human genome demonstrates that the fragment containing porcine marker sw corresponds to a fragment framing the centromere of hsa. taking into account the average distance between two hits of porcine bes on human genome, we can say that if additional fragments from other human chromosomes are located in this rearranged region of the porcine genome, they should measure < kb. analysis of the anchoring of porcine bes on the human genome highlighted also additional results: 1) it was indeed possible to define the position of the synteny breakpoints. on the human genome, these synteny breakpoints are situated between genes ring1-vps elovl5-gclc and bmp5-col21a each of these synteny breakpoints are localized in a interval of ~100– kb, between the human positions mb- mb, mb- mb and mb- mb. 2) the fourth synteny breakpoint was identified thanks to the anchoring of the bes in the human genome: the two fragments framing hsa centromere were found in a different order on the pig genome without the presence of a centromere. 3) for several bes, alignment on the human genome showed possible positions with similar likelihood. this result suggested the existence of duplicated sequences close to the human centromere, which was confirmed by a dot plot on itself of the sequence of this human region. phylogeny studies of human chromosome demonstrated the repositioning of this centromere among primate species in a region equivalent to 6p in eulemur macaco, callithrix jacchus and lagothrix lagothrica, to 6q in macaca fascicularis and presbytis cristata, and to 6p in the great apes. the repeated sequences we highlighted might then correspond to sequences of an ancestral centromere positioned in hsa6p. 4) lastly, in silico analysis of the bes alignments on the human genome provided evidence of the absence from the pig genome of a fragment of ~400– kb found in human genome. one can argue that the bes of bac bi0688h match on the human genome with sequences distant of kb. the absence of anchoring of other bes between mb and mb, whereas one hit is found for each kb in the surrounding region, confirmed that this fragment is absent from the pig genome. this region appears to be heavily rearranged between the porcine and human genomes. we wanted to determine if these rearrangements are specific of suines or cetartiodactyles, or occur in many other species. to identify gene order in other species, we used the genomic sequence when available. for gallus gallus, the assembly of the genome has not been totally completed; this is why we enriched information obtained from the sequence by rh mapping data when it was necessary. the order of genes seems to be conserved between the chicken and human genomes except for the rxrb and ring genes. these genes which may not exist in the chicken genome are located in other species in the extended class ii region of mhc. the chicken mhc, named the b locus, is a kb dna sequence containing genes. it defines a minimal essential set of mhc genes conserved over million years of divergence between birds and mammals. as the available assembly of the cow genome consists of many independent sequence scaffolds, we determined the gene order by rh mapping. our results reveal that genes from gclc to khdrbs are similarly ordered on the human and bovine genomes in this region, which suggests that the internal rearrangements of these fragments, comparing the human and porcine genomes, are specific to the evolution of the suine family. everts-van der wind et al. reported that cattle chromosomes are entirely similar to a complete human chromosome arm, and in particular, that p and q arms of hsa are homologs of bta and bta our study modifies slightly this report, in that khdrbs, the first gene localized on the long arm of human chromosome is probably located on bovine chromosome as the number of genes in this region, as in other species, is very limited; it is difficult to develop and map markers in this region. the end of bta might thus signal the location of the centromere in the ancestors of cetartiodactyles, in which the fission between the chromosomal arms occurred. in this region, a further comparison of the bovine genomic sequence with the human sequence will be of interest. we compared the gene order in the pig and cattle with the situation in the genomic sequence of mus musculus, canis familiaris, gallus gallus and homo sapiens. we observed at first that on the pig genome, a large fragment of ~ mb is shifted between ring and vps genes belonging to the extended class ii region of the mhc. if mhc is one of the most dynamic regions of the genome, a high level of conservation of genome organization provides evidence in this region of extended class ii genes. fourteen genes occur in this region between ring and the vps in dog, cat, human and mouse genomes, which only differs in the presence of processed pseudogenes. this small fragment of kb containing ring and vps is thus dynamically involved in chromosomal evolution. it is interesting to note that the translocation of the mb fragment occurs in this region. in this region, the genomic organization of the fragment and its flanking regions is similar in mouse, human, dog and chicken, whereas translocation is observed in pig and cattle. this suggests that this genomic reorganization occurred after the divergence of the common ancestor of cetartiodactyles from the ferungulates ancestor between to million years ago. the gene order observed in pig for the mb fragment is different from the one identified in other species, indicating that these internal rearrangements occurred after ruminant-suine speciation. we identified a situation really different from the map presented by everts-van der wind et al. for the proximal part of bta close to the centromere. further study of the rearrangements that occurred in ruminants in this region of extended class ii of the mhc complex is clearly needed. we used grimm to compare the different maps available and to present a possible evolution of this region between the various species. the unrooted tree we obtained presents a very different view from a classical phylogenic tree, highlighting a high rate of rearrangements in this region that have occurred recently in cetartiodactyles. the study of local rearrangements also provides a view different from the recent multispecies comparison of this region. in order to filter out noise induced by mapping errors, this analysis from murphy et al only took into account fragments > mb, defined by three genes. among other results, they documented the reuse of evolutionary breakpoint regions identified between conserved homologous synteny blocks. our study indicates that such a study should be redone on a more detailed scale, when the accuracy of mapping data is adequate.  a qtl affecting fattening and growth had been mapped in the porcine region ssc 7q. as we had shown that a small fragment is translocated between porcine and human genome, it was important to establish a final comparative map of this region. the rh map and the bac contig we developed in this region allowed us to attest that the translocated fragment is approximately mb long. it consists in three blocks, found in hsa between and mb, one of these blocks containing a few genes, framing the centromere on the human chromosome. the multispecies study allowed us to confirm that the translocation of this mb fragment occurred in the common ancestor of pig and ruminants, whereas internal rearrangements are found only in the pig genome. the exact knowledge of the genomic content of this region permits us now to establish a list of all possible candidate genes that may affect the growth and fattening of pigs. 
new micro rearrangements were highlighted between the porcine and human genomes. a 1000: framework map covering cr was established and a complete contig of the region was developed. internal rearrangements of this fragment were suggested, and partial contigs had been built, but further characterization of this region and identification of all human chromosomal fragments orthologous to this porcine fragment had to be carried out. a whole physical map of the region was constructed by integrating radiation hybrid mapping, bac fingerprinting data of the inra bac library and anchoring bac end sequences on the human genome. on porcine chromosome the region surrounding the major histocompatibility complex contains several quantitative trait loci influencing many traits including growth, back fat thickness and carcass composition. previous studies highlighted that a fragment of ~ mb is located within the swine leucocyte antigen complex. the study of this complete bac contig showed that human chromosomal fragments homologs of this heavily rearranged qtl region are all located in the region of hsa that surrounds the centromere. comparison of the organization of this region in pig, cattle, human, mouse, dog and chicken genomes revealed that 1) the translocation of the fragment described previously is observed only on the bovine and porcine genomes and 2) the new internal micro rearrangements are specific of the porcine genome. we estimate that the region contains several rearrangements and covers mb of the porcine genome. a bovine rh map was also developed in this region by mapping genes. this work allows us to define a list of all candidate genes that could explain these qtl effects.
 on porcine chromosome the region surrounding the major histocompatibility complex contains several quantitative trait loci influencing many traits including growth, back fat thickness and carcass composition. previous studies highlighted that a fragment of ~ mb is located within the swine leucocyte antigen complex. internal rearrangements of this fragment were suggested, and partial contigs had been built, but further characterization of this region and identification of all human chromosomal fragments orthologous to this porcine fragment had to be carried out.  a whole physical map of the region was constructed by integrating radiation hybrid mapping, bac fingerprinting data of the inra bac library and anchoring bac end sequences on the human genome. genes and reference microsatellites were ordered on the high resolution imnprh212000rad radiation hybrid panel. a 1000: framework map covering cr was established and a complete contig of the region was developed. new micro rearrangements were highlighted between the porcine and human genomes. a bovine rh map was also developed in this region by mapping genes. comparison of the organization of this region in pig, cattle, human, mouse, dog and chicken genomes revealed that 1) the translocation of the fragment described previously is observed only on the bovine and porcine genomes and 2) the new internal micro rearrangements are specific of the porcine genome.  we estimate that the region contains several rearrangements and covers mb of the porcine genome. the study of this complete bac contig showed that human chromosomal fragments homologs of this heavily rearranged qtl region are all located in the region of hsa that surrounds the centromere. this work allows us to define a list of all candidate genes that could explain these qtl effects. 