Extended cytogenetic maps of sheep chromosome 1 and their cattle and river buffalo homoeologues: comparison with the OAR1 RH map and human chromosomes 2, 3, 21 and 1q

Cytogenetic maps are useful tools for several applications, such as the physical anchoring of linkage and RH maps or genome sequence contigs to specific chromosome regions or the analysis of chromosome rearrangements. Recently, a detailed RH map was reported in OAR1. In the present study, we selected 38 markers equally distributed in this RH map for identification of ovine genomic DNA clones within the ovine BAC library CHORI-243 using the virtual sheep genome browser and performed FISH mapping for both comparison of OAR1 and homoeologous chromosomes BBU1q-BBU6 and BTA1-BTA3 and considerably extending the cytogenetic maps of the involved species-specific chromosomes. Comparison of the resulting maps with human-identified homology with HSA2q, HSA3, HSA21 and HSA1q reveals complex chromosome rearrangements differentiating human and bovid chromosomes. In addition, we identified 2 new small human segments from HSA2q and HSA3q conserved in the telomeric regions of OAR1p and homoeologous chromosome regions of BTA3 and BBU6, and OAR1q, respectively. Evaluation of the present OAR1 cytogenetic map and the OAR1 RH map supports previous RH assignments with 2 main exceptions. The 2 loci BMS4011 and CL638002 occupy inverted positions in these 2 maps.     

A radiation hybrid map of sheep chromosome 23 based on ovine BAC-end sequences

More than 375,000 BAC-end sequences (BES) of the CHORI-243 ovine BAC library have been deposited in public databases. blastn searches with these BES against HSA18 revealed 1806 unique and significant hits. We used blastn-anchored BES for an in silico prediction of gene content and chromosome assignment of comparatively mapped ovine BAC clones. Ovine BES were selected at approximately 1.3-Mb intervals of HSA18 and incorporated into a human-sheep comparative map. An ovine 5000-rad whole-genome radiation hybrid panel (USUoRH5000) was typed with 70 markers, all of which mapped to OAR23. The resulting OAR23 RH map included 43 markers derived from BES with high and unique BLAST hits to the sequence of the orthologous HSA18, nine EST-derived markers, 16 microsatellite markers taken from the ovine linkage map and two bovine microsatellite markers. Six new microsatellite markers derived from the 43 mapped BES and the two bovine microsatellite markers were linkage-mapped using the International Mapping Flock (IMF). Thirteen additional microsatellite markers were derived from other ovine BES with high and unique BLAST hits to the sequence of the orthologous HSA18 and also positioned on the ovine linkage map but not incorporated into the OAR23 RH map. This resulted in 24 markers in common and in the same order between the RH and linkage maps. Eight of the BES-derived markers were mapped using fluorescent in situ hybridization (FISH), to thereby align the RH and cytogenetic maps. Comparison of the ovine chromosome 23 RH map with the HSA18 map identified and localized three major breakpoints between HSA18 and OAR23. The positions of these breakpoints were equivalent to those previously shown for syntenic BTA24 and HSA18. This study presents evidence for the usefulness of ovine BES when constructing a high-resolution comprehensive map for a single sheep chromosome. The comparative analysis confirms and refines knowledge about chromosomal conservation and rearrangements between sheep, cattle and human. The constructed RH map demonstrates the resolution and utility of the newly constructed ovine RH panel.     

A comparative radiation hybrid map of sheep chromosome 10

Comparative radiation hybrid (RH) maps of individual ovine chromosomes are essential to identify genes governing traits of economic importance in sheep, a livestock species for which whole genome sequence data are not yet available. The USUoRH5000 radiation hybrid panel was used to generate a RH map of sheep chromosome 10 (OAR10) with 59 markers that span 1,422 cR over an estimated 92 Mb of the chromosome, thus providing markers every 2 Mb (equivalent to every 24 cR). The markers were derived from 46 BAC end sequences (BESs), a single EST, and 12 microsatellites. Comparative analysis showed that OAR10 shares remarkable conservation of gene order along the entire length of cattle chromosome 12 and that OAR10 contains four major homologous synteny blocks, each related to segments of the homologous human chromosome 13. Extending the comparison to the horse, dog, mouse, and chicken genome showed that these blocks share conserved synteny across species.     

A radiation hybrid comparative map of ovine chromosome 1 aligned to the virtual sheep genome

Ovis aries chromosome one (OAR1) is the largest submetacentric chromosome in the sheep genome and is homologous to regions on human chromosomes 1, 2, 3 and 21. Using the USUoRH5000 ovine whole-genome radiation hybrid (RH) panel, we have constructed a RH map of OAR1 comprising 102 framework and 75 placed/binned markers across five linkage groups spanning 3759.43 cR5000, with an average marker density of 21.2 cR5000/marker. The alignment of our OAR1 RH map shows good concordance with the recently developed virtual sheep genome, with fewer than 1.86% discrepancies. A comparative map of OAR1 was constructed by examining the location of RH-mapped orthologues in sheep within the genomes of cow, human, horse and dog. Analysis of the comparative map indicates that conserved syntenies within the five ovine RH linkage groups underwent internal chromosomal rearrangements which, in general, reflect the evolutionary distances between sheep and each of these four species. The ovine RH map presented here integrates all available mapping data and includes new genomic information for ovine chromosome 1.     

Twelve loci from HSA10, HSA11 and HSA20 were comparatively FISH-mapped on river buffalo and sheep chromosomes

Ten type I loci from HSA10 (IL2RA and VIM), HSA11 (HBB and FSHB) and HSA20 (THBD, AVP/OXT, GNAS1, HCK and TOP1) and two domestic cattle type II loci (CSSM30 and BL42) were FISH mapped to R-banded river buffalo (BBU) and sheep (OAR) chromosomes. IL2RA (HSA10) maps on BBU14q13 and OAR13q13, VIM (HSA10) maps on BBU14q15 and OAR13q15, HBB (HSA11) maps on BBU16q25 and OAR15q23, FSHB (HSA11) maps on BBU16q28 and OAR15q26, THBD (HSA20) maps on BBU14q15 and OAR13q15 while AVP/OXT, GNAS1, HCK, and TOP1 (HSA20) as well as CSSM30 and BL42 map on the same large band of BBU14q22 and OAR13q22. All loci were mapped on the same homologous chromosomes and chromosome bands of the two species, and these results agree with those earlier reported in cattle homologous chromosomes 15 and 13, respectively, confirming the high degree of both banding and physical map similarities among the bovid species. Indirect comparisons between physical maps achieved on bovid chromosomes and those reported on HSA10, HSA11 and HSA20 were performed.     

Comparative FISH mapping of mucin 1, transmembrane (MUC1) among cattle, river buffalo, sheep and goat chromosomes: comparison between bovine chromosome 3 and human chromosome 1

Four bovine BAC clones (0494F01, 0069D07, 0060B06, and 0306A12) containing MUC1, as confirmed by mapping MUC1 on a RH3000 radiation hybrid panel, were hybridised on R-banded chromosomes of cattle (BTA), river buffalo (BBU), sheep (OAR) and goat (CHI). MUC1 was FISH-mapped on BTA3q13, BBU6q13, OAR1p13 and CHI3q13 and both chromosomes and chromosome bands were homoeologous confirming the high degree of chromosome homoeologies among bovids and adding more information on the pericentromeric regions of these species' chromosomes. Indeed, MUC1 was more precisely assigned to BTA3 and assigned for the first time to BBU6, OAR1p and CHI3. Moreover, detailed and improved cytogenetic maps of BTA3, CHI3, OAR1p and BBU6 are shown and compared with HSA1.     

Comparative FISH-mapping of twelve loci in river buffalo and sheep chromosomes: comparison with HSA8p and HSA4q

Twelve loci (11 of type I and 1 of type II) previously FISH-mapped in cattle were comparatively FISH-mapped in both river buffalo chromosome 1p (BBU1p) and homologous chromosome 26 of sheep (OAR26), extending the cytogenetic maps in both chromosome species and providing a more precise localization of these loci in single chromosome bands than previous locations on BTA27. Bovine BAC clones containing DCTD, C4orf20, CASP3, TLR3, MSR1, FAT, LONRF1, DLC1, C8orf41, CSSM036, LSM1 and EIF4EBP1 were used for FISH on RBPI-banded chromosomes. All loci were located on the same homologous chromosome bands (R-band positive) of both species further confirming the high degree of banding and gene (order of loci) homologies among bovids. Detailed cytogenetic maps of OAR26 and BBU1p were performed and compared with that of BTA27 as well as with those of both HSA8p and HSA4q, revealing complex chromosome rearrangements differentiating OAR26/BBU1p/BTA27 from human chromosomes.     

Comparative mapping of sheep chromosome 2q

Sheep chromosome 2q (OAR2q), which is homologous with human chromosome 2q (HSA2q), and cattle chromosome 2 (BTA2), is known to contain several loci contributing to carcass traits. However, the chromosomal rearrangements differentiating these chromosomes among the three species have not yet been determined and thus precise correspondences between the locations of sheep and human genes are not known. Twenty-six genes from HSA2q (2q21.1-->2q36) have been assigned to OAR2q by genetic linkage mapping to refine this area of the sheep genome. Seventy-six genes were initially selected from HSA2q. Sixty-eight percent of the PCR primer sets designed for these genes amplified successfully in sheep, and 34% amplified polymorphic products. Part of the proximal arm of OAR2q was found to be inverted compared with HSA2q. The breakpoint has been localised near the growth differentiation factor 8 gene (GDF8), spanning 380 kb between the positions of the hypothetical protein (FLJ20160) (HSA2:191008944-191075046) and glutaminase (GLS) (HSA2:191453847-191538510) (Build36.1).     

Thirteen type I loci from HSA4q, HSA6p, HSA7q and HSA12q were comparatively FISH-mapped in four river buffalo and sheep chromosomes

Thirteen goat BAC clones containing coding sequences from HSA7, HSA12q, HSA4 and HSA6p were fluorescence in situ mapped to river buffalo (Bubalus bubalis, BBU) and sheep (Ovis aries, OAR) R-banded chromosomes. The following type I loci were mapped: BCP to BBU8q32 and OAR4q32, CLCN1 to BBU8q34 and OAR4q34, IGFBP3 to BBU8q24 and OAR4q27, KRT to BBU4q21 and OAR 3q21, IFNG to BBU4q23 and OAR3q23, IGF1 to BBU4q31 and OAR3q31, GNRHR to BBU7q32 and OAR6q32, MTP to BBU7q21 and OAR6q15, PDE6B to BBU7q36 and OAR6q36, BF to BBU2p22 and OAR20q22, EDN1 to BBU2p24 and OAR20q24, GSTA1 to BBU2p22 and OAR20q22, OLADRB (MHC) to BBU2p22 and OAR20q22. All mapped loci appeared to be located on homologous chromosomes and chromosome bands in both bovids. Comparison between gene orders in bovid (BBU and OAR) and human (HSA) chromosomes revealed complex rearrangements, especially between BBU7/OAR6 and HSA4, as well as between BBU2p/OAR20 and HSA6p.     

A comparative map of interstitial bovine chromosome 5 with human chromosomes 12 and 22

We have constructed a high-density comparative radiation hybrid map of the interstitial region of bovine chromosome 5 (BTA5) using a recently constructed 12,000-rad, whole-genome, cattle-hamster radiation hybrid (WGRH) panel. Sixty-two bovine EST markers were selected which have orthologous sequences on human chromosomes 12 and 22 (HSA12 and HSA22). Sixty markers were included in the multi-point framework map at LOD 3.0. Our comprehensive RH map contains more than twice as many markers (88) than previous generation maps. Because of a higher marker density and increased resolution of the RH(12,000) panel, all markers were placed into a single linkage group based on two-point analysis at a LOD score 6.0. As a result, this new comparative map reveals new blocks of synteny and extensive gene order alterations between species. Breakpoints of synteny are located with high accuracy. Overall, this work reveals widespread chromosomal rearrangements between bovine, human and mouse genomes.     

Evolutionary breakpoints through a high-resolution comparative map between porcine chromosomes 2 and 16 and human chromosomes

This study reports a high-resolution comparative map between human chromosomes and porcine chromosomes 2 (SSC2) and 16 (SSC16), pointing out new homologies and evolutionary breakpoints. SSC2 is of particular interest because of the presence of several important QTLs. Among 226 porcine ESTs selected according to their expected localization, 151 were RH mapped and ordered on SSC2. This study confirmed the extensive conservation between SSC2 and HSA11 and HSA19 and refined the homology with HSA5 (three blocks defined). Furthermore the SSC2q pericentromeric region was shown to be homologous to another human chromosome (HSA1). A complex organization of these syntenies was demonstrated on SSC2q. Our strategy led us to improve also the SSC16 RH map by adding 45 markers. Two-color fluorescence in situ hybridization of markers representative of each synteny confirmed block order. Finally, 29 breakpoints were identified in both species, and porcine BACs containing two breakpoints were isolated.     

A new 4016-marker radiation hybrid map for porcine-human genome analysis

We constructed a 5000-rad comprehensive radiation hybrid (RH) map of the porcine (Sus scrofa) genome and compared the results with the human genome. Of 4475 typed markers, 4016 (89.7%) had LOD >5 compared with the markers used in our previous RH map by means of two-point analysis and were grouped onto the 19 porcine chromosomes (SSCs). All mapped markers had LOD >3 as determined by RHMAPPER analysis. The current map comprised 430 microsatellite (MS) framework markers, 914 other MS markers, and 2672 expressed sequence tags (ESTs). The whole-genome map was 8822.1 cR in length, giving an average marker density of 0.342 Mb/cR. The average retention frequency was 35.8%. Using BLAST searches of porcine ESTs against the RefSeq human nucleotide and amino acid sequences (release 22), we constructed high-resolution comparative maps of each SSC and each human chromosome (HSA). The average distance between ESTs in the human genome was 1.38 Mb. SSC contained 50 human chromosomal syntenic groups, and SSC11, SSC12, and SSC16 were only derived from the HSA13q, HSA17, and HSA5 regions, respectively. Among 38 porcine terminal regions, we found that at least 20 regions have been conserved between the porcine and human genomes; we also found four paralogous regions for the major histocompatibility complex (MHC) on SSC7, SSC2, SSC4, and SSC1. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chromosome painting between human and lorisiform prosimians: evidence for the HSA 7/16 synteny in the primate ancestral karyotype

Multidirectional chromosome painting with probes derived from flow-sorted chromosomes of humans (Homo sapiens, HSA, 2n = 46) and galagos (Galago moholi, GMO, 2n = 38) allowed us to map evolutionarily conserved chromosomal segments among humans, galagos, and slow lorises (Nycticebus coucang, NCO, 2n = 50). In total, the 22 human autosomal painting probes detected 40 homologous chromosomal segments in the slow loris genome. The genome of the slow loris contains 16 sytenic associations of human homologues. The ancient syntenic associations of human chromosomes such as HSA 3/21, 7/16, 12/22 (twice), and 14/15, reported in most mammalian species, were also present in the slow loris genome. Six associations (HSA 1a/19a, 2a/12a, 6a/14b, 7a/12c, 9/15b, and 10a/19b) were shared by the slow loris and galago. Five associations (HSA 1b/6b, 4a/5a, 11b/15a, 12b/19b, and 15b/16b) were unique to the slow loris. In contrast, 30 homologous chromosome segments were identified in the slow loris genome when using galago chromosome painting probes. The data showed that the karyotypic differences between these two species were mainly due to Robertsonian translocations. Reverse painting, using galago painting probes onto human chromosomes, confirmed most of the chromosome homologies between humans and galagos established previously, and documented the HSA 7/16 association in galagos, which was not reported previously. The presence of the HSA 7/16 association in the slow loris and galago suggests that the 7/16 association is an ancestral synteny for primates. Based on our results and the published homology maps between humans and other primate species, we propose an ancestral karyotype (2n = 60) for lorisiform primates.     

A 12 000-rad whole-genome radiation hybrid panel in sheep: application to the study of the ovine chromosome 18 region containing a QTL for scrapie susceptibility

Whole-genome radiation hybrid (RH) panels have been constructed for several species, including cattle. RH panels have proven to be an extremely powerful tool to construct high-density maps, which is an essential step in the identification of genes controlling important traits, and they can be used to establish high-resolution comparative maps. Although bovine RH panels can be used with ovine markers to construct sheep RH maps based on bovine genome organization, only some (c. 50%) of the markers available in sheep can be successfully mapped in the bovine genome. So, with the development of genomics and genome sequencing projects, there is a need for a high-resolution RH panel in sheep to map ovine markers. Consequently, we have constructed a 12 000-rad ovine whole-genome RH panel. Two hundred and eight hybrid clones were produced, of which 90 were selected based on their retention frequency. The final panel had an average marker retention frequency of 31.8%. The resolution of this 12 000-rad panel (SheepRH) was estimated by constructing an RH framework map for a 23-Mb region of sheep chromosome 18 (OAR18) that contains a QTL for scrapie susceptibility.     

Use of flow-sorted canine chromosomes in the assignment of canine linkage, radiation hybrid, and syntenic groups to chromosomes: refinement and verification of the comparative chromosome map for dog and human

The mapping of the canine genome has recently been accelerated by the availability of chromosome-specific reagents and publication of radiation hybrid (RH), genetic linkage, and dog/human comparative maps, but the assignment of mapping groups to chromosomes is incomplete. To assign published radiation hybrid, linkage, and "syntenic" groups to chromosomes, individual markers found within each group have been amplified from canine and vulpine flow-sorted, chromosome-specific DNAs as templates. Here a further 102 type I genetic markers (previously mapped in human) and 21 further type II markers are assigned to canine chromosomes using marker-specific PCR. We have assigned all linkage, RH, and syntenic groups in the two most recently published canine genome maps to chromosomes. This demonstrates directly that there is at least one published mapping group for each of the 38 canine autosomes and thus that the coverage of the canine chromosome map is approaching completion. The dog/human comparative map is one of the most complex so far described, with 90 separate segments of chromosomal homology previously seen in dog-on-human cross-species chromosome-painting studies. The total of 142 type I markers now placed on canine chromosomes using this method of marker mapping has allowed us to confirm the placement of the great majority (83) of the 90 homologous segments. The positions of the remaining homologous segments were confirmed in new cross-species chromosome-painting experiments (dog-on-human, fox-on-human).     

A high-resolution comparative RH map of the telomeric end of bovine chromosome 2 with human chromosomes 1 and 2

High density livestock to human comparative maps are necessary for the implementation of comparative positional candidate gene cloning. We have constructed a high-density comparative radiation hybrid (RH) map of the telomeric end of bovine chromosome 2 (BTA2) using a 12,000-rad whole genome cattle-hamster radiation hybrid (WGRH) panel. Eighteen bovine EST markers with orthologues on human chromosomes 1 and 2 (HSA1 and HSA2), together with nine microsatellite markers, were typed against the 180 cell lines of the WGRH panel. Twenty-one markers were included in the multi-point framework map at LOD =3.0. The comparative analysis reveals a new segment of highly conserved synteny between HSA2 and BTA2.     

High-resolution comprehensive radiation hybrid maps of the porcine chromosomes 2p and 9p compared with the human chromosome 11

We are constructing high-resolution, chromosomal 'test' maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH2(12,000-rad))to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH2(12,000-rad) panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH(7,000-rad) panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH(12,000-rad) FW maps, respectively, with an accumulated map distance of 4046.5 cR(12,000 )and 1355.2 cR(7,000 )for SSC2p, and 4244.1 cR(12,000) and 1802.5 cR(7,000) for SSC9p. The kb/cR ratio in the IMNpRH2(12,000-rad) FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH(7,000-rad) FW maps was 47.1 and 36.3, respectively, or an approximately 3.0-fold increase in map resolution in the IMNpRH(12,000-rad) panel over the IMpRH(7,000-rad) panel. The integrated IMNpRH(12,000-rad) andIMpRH(7,000-rad) maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.     

Evolutionary aspects of the genomic organization of rat chromosome 10

Using FISH and RH mapping a chromosomal map of rat chromosome 10 (RNO10) was constructed. Our mapping data were complemented by other published data and the final map was compared to maps of mouse and human chromosomes. RNO10 contained segments homologous to mouse chromosomes (MMU) 11, 16 and 17, with evolutionary breakpoints between the three segments situated in the proximal part of RNO10. Near one of these breakpoints (between MMU17 and 11) we found evidence for an inversion ancestral to the mouse that was not ancestral to the condition in the rat. Within each of the chromosome segments identified, the gene order appeared to be largely conserved. This conservation was particularly clear in the long MMU11-homologous segment. RNO10 also contained segments homologous to three human chromosomes (HSA5, 16, 17). However, within each segment of conserved synteny were signs of more extensive rearrangements. At least 13 different evolutionary breakpoints were indicated in the rat-human comparison. In contrast to what was found between rat and mouse, the rat-human evolutionary breaks were distributed along the entire length of RNO10.