Comparative analysis of the cattle and human genomes: detection of ZOO-FISH and gene mapping-based chromosomal homologies

Comparative chromosome painting with individual human chromosome-specific libraries (CSLs) on cattle metaphase chromosomes delineated 46 homologous chromosomal segments between the two species. Continuous arrangement of these segments on individual cattle chromosomes demonstrates a nearly complete coverage of the bovine karyotype and shows physical boundaries of bovine chromosomal segments homologous to individual human chromosomes. Alignment of the available comparative gene mapping data with the homologous segments strongly supports the detected gross homologies between the karyotypes of the two species. In addition to cattle, four human CSLs were hybridized to sheep metaphase chromosomes also, to further verify the known karyotype homology within the Bovidae. Besides its application to karyotype evolution research, the comparative knowledge provides for rapid expansion of the much needed Type I locus-based bovine gene map. Received: 9 September 1995 / Accepted: 4 December 1995     

Comparative mapping of human chromosome 10 to pig chromosomes 10 and 14

Identification of predictive markers in QTL regions that impact production traits in commercial populations of swine is dependent on construction of dense comparative maps with human and mouse genomes. Chromosomal painting in swine suggests that large genomic blocks are conserved between pig and human, while mapping of individual genes reveals that gene order can be quite divergent. High-resolution comparative maps in regions affecting traits of interest are necessary for selection of positional candidate genes to evaluate nucleotide variation causing phenotypic differences. The objective of this study was to construct an ordered comparative map of human chromosome 10 and pig chromosomes 10 and 14. As a large portion of both pig chromosomes are represented by HSA10, genes at regularly spaced intervals along this chromosome were targeted for placement in the porcine genome. A total of 29 genes from human chromosome 10 were mapped to porcine chromosomes 10 (SSC10) and 14 (SSC14) averaging about 5 Mb distance of human DNA per marker. Eighteen genes were assigned by linkage in the MARC mapping population, five genes were physically assigned with the IMpRH mapping panel and seven genes were assigned on both maps. Seventeen genes from human 10p mapped to SSC10, and 12 genes from human 10q mapped to SSC14. Comparative maps of mammalian species indicate that chromosomal segments are conserved across several species and represent syntenic blocks with distinct breakpoints. Development of comparative maps containing several species should reveal conserved syntenic blocks that will allow us to better define QTL regions in livestock.     

Comparative chromosome painting defines the high rate of karyotype changes between pigs and bovids

Human and sheep chromosome-specific probes were used to construct comparative painting maps between the pig (Suiformes), cattle and sheep (Bovidae), and humans. Various yet unknown translocations were observed that would assist in a more complete reconstruction of homology maps of these species. The number of homologous segments that can be identified with sheep probes in the pig karyotype exceeds that described previously by chromosome painting between two non-primate mammals belonging to the same order. Sheep probes painted 62 segments on pig autosomes and delineated not only translocations, but also 9 inversions. All inversions were paracentric and indicate that these rearrangements may be characteristic for chromosomal changes in suiforms. Hybridizations of all sheep painting probes to cattle chromosomes confirmed the chromosome conservation in bovids. In addition, we observed a small translocation that was previously postulated from linkage mapping data, but was not yet described by physical mapping. The chromosome painting data are complemented with a map of available comparative gene mapping data between pig and sheep genomes. A detailed table listing the comparative gene mapping data between pig and cattle genomes is provided. The reanalysis of the pig karyotype with a new generation of human paint probes provides an update of the human/pig comparative genome map and demonstrates two new chromosome homologies. Seven conserved segments not yet identified by chromosome painting are also reported. Received: 2 October 2000 / Accepted: 15 January 2001     

A First Generation Comparative Chromosome Map between Guinea Pig (Cavia porcellus) and Humans

The domesticated guinea pig, Cavia porcellus (Hystricomorpha, Rodentia), is an important laboratory species and a model for a number of human diseases. Nevertheless, genomic tools for this species are lacking; even its karyotype is poorly characterized. The guinea pig belongs to Hystricomorpha, a widespread and important group of rodents; so far the chromosomes of guinea pigs have not been compared with that of other hystricomorph species or with any other mammals. We generated full sets of chromosome-specific painting probes for the guinea pig by flow sorting and microdissection, and for the first time, mapped the chromosomal homologies between guinea pig and human by reciprocal chromosome painting. Our data demonstrate that the guinea pig karyotype has undergone extensive rearrangements: 78 synteny-conserved human autosomal segments were delimited in the guinea pig genome. The high rate of genome evolution in the guinea pig may explain why the HSA7/16 and HSA16/19 associations presumed ancestral for eutherians and the three syntenic associations (HSA1/10, 3/19, and 9/11) considered ancestral for rodents were not found in C. porcellus. The comparative chromosome map presented here is a starting point for further development of physical and genetic maps of the guinea pig as well as an aid for genome assembly assignment to specific chromosomes. Furthermore, the comparative mapping will allow a transfer of gene map data from other species. The probes developed here provide a genomic toolkit, which will make the guinea pig a key species to unravel the evolutionary biology of the Hystricomorph rodents.     

Radiation hybrid map assignments of 11 ESTs obtained from a 28-day-old swine embryo cDNA library to the IMpRH map

In order to improve the map resolution and to locate more genes on the porcine radiation hybrid map, expressed sequence tags (ESTs) were isolated from a 28-day-old normal pig embryo cDNA library. The ESTs were sequenced from the 5'-end and similarities were checked with sequences registered in the NCBI DNA database (http://www.ncbi.nlm.nih.gov/blast/). The ESTs sequences which have high identity scores (>80%) against human genes or ESTs were further sequenced from the 3' untranslated region. The ESTs which were sequenced successfully were used to design primers for PCR analysis of the radiation hybrid panel. Eleven ESTs were physically mapped to porcine chromosomes 2, 4, 8, 10, 13, 14 and X. The localizations are in agreement with the comparative mapping data between human and pig. The results will provide unique information to the comparative map of human and pig.     

Generation of a 5.5-Mb BAC/PAC contig of pig chromosome 6q1.2 and its integration with existing RH, genetic and comparative maps

We generated a sequence-ready BAC/PAC contig spanning approximately 5.5 Mb on porcine chromosome 6q1.2, which represents a very gene-rich genome region. STS content mapping was used as the main strategy for the assembly of the contig and a total of 6 microsatellite markers, 53 gene-related STS and 116 STS corresponding to BAC and PAC end sequences were analyzed. The contig comprises 316 BAC and PAC clones covering the region between the genes GPI and LIPE. The correct contig assembly was verified by RH-mapping of STS markers and comparative mapping of BAC/PAC end sequences using BLAST searches. The use of microsatellite primer pairs allowed the integration of the physical maps with the genetic map of this region. Comparative mapping of the porcine BAC/PAC contig with respect to the gene-rich region on the human chromosome 19q13.1 map revealed a completely conserved gene order of this segment, however, physical distances differ somewhat between HSA19q13.1 and SSC6q1.2. Three major differences in DNA content between human and pig are found in two large intergenic regions and in one region of a clustered gene family, respectively. While there is a complete conservation of gene order between pig and human, the comparative analysis with respect to the rodent species mouse and rat shows one breakpoint where a genome segment is inverted.     

Linkage and physical mapping of prolactin to porcine chromosome 7

Comparative mapping studies between human and pig have shown that there is conserved synteny between human chromosome 6 and pig chromosomes 1 and 7, but some gene locations are not well established. Prolactin ( PRL ), an anterior pituitary hormone, has been mapped to human chromosome 6, and has tentatively mapped to pig chromosome 7 using Southern-RFLP analysis with a limited number of meioses. To confirm the assignment of prolactin to porcine chromosome 7 by physical and linkage analysis, pig cDNA and human genomic DNA sequences were used to design pig-specific PCR primers. The primers amplified a fragment of ≈2·8 kb. Two polymorphic restriction sites were identified within this fragment with the restriction endonuclease Bst UI. Prolactin was significantly linked to six markers on the published PiGMaP map of pig chromosome 7. Prolactin was physically mapped using a pig × rodent somatic cell hybrid panel. An analysis of these data placed PRL on pig 7p1·1–p1·2 with 100% concordance and was in complete agreement with the linkage data. Both mapping techniques placed PRL in a conserved order with the loci in the syntenic region of human chromosome 6.     

A first-generation EST RH comparative map of the porcine and human genome

We have constructed a first-generation EST radiation hybrid comparative map of the porcine genome by assigning 1058 markers to the IMpRH7000 panel. Chromosomal localization was determined with a 2pt LOD of 4.8 for 984 markers, using the IMpRH mapping tool. Annotated ESTs represent 46.2% or 489 of the markers. Marker distribution was not stochastic and ranged from 0.41 for SSC8 to 1.77 for SSC12, respectively. Two hundred fifty-one markers assigned to the physical map of the pig did not find a homologous sequence in V22 of the human genome assembly, indicative of gaps in the assembled human genome sequence. The comparative porcine/human map covers 3290 MB, or 98.3% of the presumed size of the human genome. However, 60 breakpoints were identified between chromosomes, as well as 90 micro-rearrangements within synteny groups. Six porcine chromosomes—SSC2, 5, 6, 7, 12, and 14—correspond to the three gene-richest human chromosomes, HSA17, 19, and 22, and show above average marker density. Porcine Chrs 1, 8, 11, and X display a low DNA/marker ratio and correspond to the 'genome deserts' on HSA 18, 4, 13, and X.     

Improving the comparative map of porcine chromosome 10 with respect to human chromosomes 1, 9 and 10

ZOO-FISH mapping shows human chromosomes 1, 9 and 10 share regions of homology with pig chromosome 10 (SSC10). A more refined comparative map of SSC10 has been developed to help identify positional candidate genes for QTL on SSC10 from human genome sequence. Genes from relevant chromosomal regions of the public human genome sequence were used to BLAST porcine EST databases. Primers were designed from the matching porcine ESTs to assign them to porcine chromosomes using the INRA somatic cell hybrid panel (INRA-SCHP) and the INRA-University of Minnesota Radiation Hybrid Panel (IMpRH). Twenty-eight genes from HSA1, 9 and 10 were physically mapped: fifteen to SSC10 (ACO1, ATP5C1, BMI1, CYB5R1, DCTN3, DNAJA1, EPHX1, GALT, GDI2, HSPC177, OPRS1, NUDT2, PHYH, RGS2, VIM), eleven to SSC1 (ADFP, ALDHIB1, CLTA, CMG1, HARC, PLAA, STOML2, RRP40, TESK1, VCP and VLDLR) and two to SSC4 (ALDH9A1 and TNRC4). Two anonymous markers were also physically mapped to SSC10 (SWR1849 and S0070) to better connect the physical and linkage maps. These assignments have further refined the comparative map between SSC1, 4 and 10 and HSA1, 9 and 10.     

Mapping of 443 porcine EST improves the comparative maps for SSC1 and SSC7 with the human genome

Numerous mapping studies of complex traits in the pig have resulted in quantitative trait loci (QTL) intervals of 10-20 cM. To improve the chances to identify the genes located in such intervals, increased expressed sequence tags (EST)-based marker density, coupled with comparative mapping with species whose genomes have been sequenced such as human and mouse, is the most efficient tool. In this study, we mapped 443 porcine EST with a radiation hybrid (RH) panel (384 had LOD > 6.0) and a somatic cell hybrid panel. Requiring no discrepancy between two-point and multipoint RH data allowed robust assignment of 309 EST, of which most were located on porcine chromosomes (SSC) 1, 4, 7, 8 and X. Moreover, we built framework maps for two chromosomes, SSC1 and SSC7, with mapped QTL in regions with known rearrangement between pig and human genomes. Using the Blast tool, we found orthologies between 407 of the 443 pig cDNA sequences and human genes, or to existing pig genes. Our porcine/human comparative mapping results reveal possible new homologies for SSC1, SSC3, SSC5, SSC6, SSC12 and SSC14 and add markers in synteny breakpoints for chromosome 7.     

A new contribution to the integration of human and porcine genome maps: 623 new points of homology

In this study we examined homologies between 1,735 porcine microsatellites and human sequence. For 1,710 microsatellites we directly used the sequence flanking the repeat available in GenBank. For a set of 305 microsatellites, a BAC library was screened and end-sequencing provided 461 additional sequences. Altogether 2,171 porcine sequences were tentatively aligned with the sequence of the human genome using the fasta program. Human homologies were observed for 652 microsatellite loci and porcine chromosome assignments available for 623 microsatellites provide useful links in the human and pig comparative map. Moreover for 92 STS, a significant sequence similarity was detected using at least two sequences and in all cases corresponding human locations were consistent. The present study allowed the integration of anonymous markers and the porcine linkage map into the framework of the comparative data between human and porcine genomes (http://w3.toulouse.inra.fr/lgc/pig/msat/). Moreover all conserved syntenic segments were defined on human chromosomes.     

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).     

Zoo-FISH with microdissected arm specific paints for HSA2, 5, 6, 16, and 19 refines known homology with pig and horse chromosomes

Microdissected arm specific paints (ASPs) for human (HSA) chromosomes (Chrs) 2, 5, 6, 16, and 19 were used as probes on pig (SSC) and horse (ECA) metaphase chromosomes. Regions homologous to individual human arms were delineated in the two species studied. Of the ten ASPs used, HSA6 and 16 ASPs showed complete synteny conservation of individual arms as single blocks/arms both in pig and horse. A similar trend was, in general, also observed for HSA19 ASPs. However, contrary to these observations, synteny conservation of individual arms of HSA2 and HSA5 was not observed in pig and horse. The arm specific painting data, coupled with the available gene mapping data, showed that, although HSA2 corresponded to two arms/chromosomes each in pig and horse, the breakpoint of this synteny in humans was not located at the centromere, but at HSA2q13 band. Similarly, arm specific paints for HSA5 showed that of the two blocks/chromosomes painted in pig and horse, one corresponded to HSA5q13-pter, the other to HSA5q13-qter. The findings suggest that 5q13 band may also be an evolutionary break point, similar to the one detected on HSA2q13. The microdissected human arm specific painting probes used in the present work provide more accurate and refined comparative information on pig and horse chromosomes than that available through the use of human whole chromosome specific paints. Received: 1 June 1997 / Accepted: 5 September 1997     

A high-resolution comparative RH map of porcine Chromosome (SSC) 2

A high-resolution comparative map was constructed for porcine Chromosome (SSC) 2, where a QTL for back fat thickness (BFT) is located. A radiation hybrid (RH) map containing 33 genes and 25 microsatellite markers was constructed for this chromosome with a 3000-rad porcine RH panel. In total, 16 genes from human Chromosome (HSA) 11p, HSA19p, and HSA5q were newly assigned to SSC2. One linkage group was observed at LOD 3.0, and five linkage groups at LOD 4.0. Comparison of the porcine RH map with homologous human gene orders identified four conserved segments between SSC2 and HSA11, HSA19, and HSA5. Concerning HSA11, a rearrangement of gene order is observed. The segment HSA11p15.4-q13 is inverted on SSC2 when compared with the distal tip of SSC2p, which is homologous to HSA11p15.5. The boundaries of the conserved segments between human and pig were defined more precisely. This high-resolution comparative map will be a valuable tool for further fine mapping of the QTL area. Received: 10 November 2000 / Accepted: 23 January 2001     

Complete homology maps of the rabbit (Oryctolagus cuniculus) and human by reciprocal chromosome painting

Fluorescence in situ hybridization (FISH) was used to construct a homology map to analyse the extent of evolutionary conservation of chromosome segments between human and rabbit (Oryctolagus cuniculus, 2n = 44). Chromosome-specific probes were established by bivariate fluorescence activated flow sorting followed by degenerate oligonucleotide-primed PCR (DOP-PCR). Painting of rabbit probes to human chromosomes and vice versa allowed a detailed analysis of the homology between these species. All rabbit chromosome paints, except for the Y paint, hybridized to human chromosomes. All human chromosome paints, except for the Y paint, hybridized to rabbit chromosomes. The results obtained revealed extensive genome conservation between the two species. Rabbit chromosomes 12, 19 and X were found to be completely homologous to human chromosomes 6, 17 and X, respectively. All other human chromosomes were homologous to two or sometimes three rabbit chromosomes. Many conserved chromosome segments found previously in other mammals (e.g. cat, pig, cattle, Indian muntjac) were also found to be conserved in rabbit chromosomes.     

Comparative mapping between humans and pigs: localization of 58 anchorage markers (TOASTs) by use of porcine somatic cell and radiation hybrid panels

To increase the number of Type I markers that are directly informative for comparative mapping, 58 anchorage markers, TOASTs (Traced Orthologous Amplified Sequence Tags), were mapped in pig. With specific consensus primers, 76 TOASTs were tested in pig: 50 were regionally localized in pig on a somatic cell hybrid panel (SCHP), and 51 were mapped on the whole genome, INRA/University of Minnesota porcine Radiation Hybrid panel (IMpRH). Comparison of marker positions on RH and cytogenetic maps indicated general concordance except for two chromosomal regions. For RH mapping, all markers, apart from one, were significantly linked (LOD > 4.8) to a marker of the first-generation radiation hybrid map. Localization of new markers on the initial map is necessary for drawing a framework map as shown for Chromosome Sscr 14. The addition of four TOASTs has enabled us to propose an improved map, using a threshold likelihood ratio of 1000/1. At the whole-genome level, this work significantly increased (by 50%) the number of precisely mapped genes on the porcine RH map and confirmed that the IMpRH panel is a valuable tool for high-resolution gene mapping in pig. Porcine PCR products were sequenced and compared with human sequences to verify their identity. Most of the localizations made it possible to either confirm or refine the previous comparative data between humans and pigs obtained through heterologous chromosomal painting or gene mapping. Moreover, the use of TOASTs in mapping studies appears to be a complement to other strategies using CATS, human ESTs, or heterologous FISH with BACs which had already been applied to improve the gene density of comparative genomic maps for mammals. Received: 15 March 2000 / Accepted: 27 July 2000     

Multi-species comparative mapping in silico using the COMPASS strategy

MOTIVATION: The completion of human and mouse genome sequences provides a valuable resource for decoding other mammalian genomes. The comparative mapping by annotation and sequence similarity (COMPASS) strategy takes advantage of the resource and has been used in several genome-mapping projects. It uses existing comparative genome maps based on conserved regions to predict map locations of a sequence. An automated multiple-species COMPASS tool can facilitate in the genome sequencing effort and comparative genomics study of other mammalian species. RESULTS: The prerequisite of COMPASS is a comparative map table between the reference genome and the predicting genome. We have built and collected comparative maps among five species including human, cattle, pig, mouse and rat. Cattle-human and pig-human comparative maps were built based on the positions of orthologous markers and the conserved synteny groups between human and cattle and human and pig genomes, respectively. Mouse-human and rat-human comparative maps were based on the conserved sequence segments between the two genomes. With a match to human genome sequences, the approximate location of a query sequence can be predicted in cattle, pig, mouse and rat genomes based on the position of the match relatively to the orthologous markers or the conserved segments. AVAILABILITY: The COMPASS-tool and databases are available at http://titan.biotec.uiuc.edu/COMPASS/     

Regional localisations of VIM, HSD3b, ACTA1 and PGM1 in pigs

The comparative map between human and pig has progressed rapidly over the past 2 years. Nevertheless, some points still need to be clarified, particularly the correspondences between human chromosome 10 (HSA10) and porcine chromosome 10 (SSC10) and between human chromosome 1 (HSA1) and porcine chromosomes. The gene codings for vimentin (VIM) carried by HSA10 and three genes carried by HSA1 (hydroxy delta 5 steroid dehydrogenase 3 beta: HSD3B; alpha actin 1: ACTA1; and phosphoglucomutase 1: PGM1) were selected and the regional localisations on pig chromosomes were determined using a well-characterised somatic cell hybrid panel.