Comparative FISH mapping of bovid X chromosomes reveals homologies and divergences between the subfamilies bovinae and caprinae

Comparative FISH mapping of river buffalo (Bubalus bubalis, BBU), sheep (Ovis aries, OAR), and cattle (Bos taurus, BTA) X chromosomes revealed homologies and divergences between the X chromosomes in the subfamilies Bovinae and Caprinae. Twenty-four and 17 loci were assigned for the first time to BBU X and OAR X, respectively, noticeably extending the physical map in these two species. Seventeen loci (four of which for the first time) were also FISH mapped to BTA X and used for comparative mapping studies on the three species, which show three morphologically different X chromosomes: an acrocentric (BBU X), an acrocentric with distinct short arms (OAR X), and a submetacentric (BTA X). The same order of loci were found on BTA X and BBU X, suggesting that a centromere transposition, with loss (cattle) or acquisition (river buffalo) of constitutive heterochromatin, differentiated the X chromosomes of these two bovids. Comparison of bovine (cattle and river buffalo) and caprine (sheep) X chromosomes revealed at least five common chromosome segments, suggesting that multiple transpositions, with retention or loss of constitutive heterochromatin, had occurred during their karyotypic evolution.     

Comparative FISH-mapping of six expressed gene loci to river buffalo and sheep chromosomes.

Six expressed gene loci (NF1, CRYB1, CHRNB1, TP53, P4HB and GH1), recently assigned to cattle chromosome 19 by both radiation hybrid analysis and FISH-mapping, were comparatively FISH-mapped to river buffalo chromosome (BBU) 3p and sheep chromosome (OAR) 11, extending the physical map in these two important bovids. The six loci mapped to the same homoeologous chromosome bands of BBU 3p and OAR 11, and their gene order was centromere-NF1-CRYB1-CHNRB1-TP53-(GH1, P4HB).     

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.     

The river buffalo (Bubalus bubalis, 2n = 50) cytogenetic map: assignment of 64 loci by fluorescence in situ hybridization and R-banding

Sixty-four genomic BAC-clones mapping five type I (ADCYAP1, HRH1, IL3, RBP3B and SRY) and 59 type II loci, previously FISH-mapped to goat (63 loci) and cattle (SRY) chromosomes, were fluorescence in situ mapped to river buffalo R-banded chromosomes, noticeably extending the physical map of this species. All mapped loci from 26 bovine syntenic groups were located on homeologous chromosomes and chromosome regions of river buffalo and goat (cattle) chromosomes, confirming the high degree of chromosome homeologies among bovids. Furthermore, an improved cytogenetic map of the river buffalo with 293 loci from all 31 bovine syntenic groups is reported.     

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.     

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.     

Chromosomal localization of sixty autosomal loci in sheep (OVIS ARIES, 2n = 54) by fluorescence in situ hybridization and R-banding

Sixty autosomal loci (5 type I and 55 type II) from 24 bovine syntenic groups, and previously FISH-mapped to goat and river buffalo chromosomes, were localized by fluorescence in situ on sheep (OVIS ARIES, 2n = 54) chromosomes, thereby notably extending the cytogenetic map of this economically important species. Caprine BAC clones were hybridized to R-banded chromosome preparations. FITC-signals and RBPI- banding (R-banding by late BrdU-incorporation and propidium iodide staining) were simultaneously visualized and captured by a colour CCD-camera. All mapped loci were localized on homoeologous chromosomes and chromosome regions (bands) of sheep, goat and river buffalo, further supporting chromosome and genetic (loci) homoeologies among bovids.     

Fluorescence in situ hybridization mapping of six loci containing genes involved in the dioxin metabolism of domestic bovids

Six loci containing genes involved in the dioxin metabolism (ARNT, AHR, CYP1A1, CYP1A2, CYP1B1 and AHRR) were assigned, for the first time, to cattle (Bos taurus, 2n = 60, BTA), river buffalo (Bubalus bubalis, 2n = 50, BBU), sheep (Ovis aries, 2n = 54, OAR) and goat (Capra hircus, 2n = 60, CHI) chromosomes by comparative FISH-mapping and R-banding using bovine BAC-clones. The following chromosome locations were found: ARNT to BTA3q21, BBU6q21, OAR1p21 and CHI3q21, AHR to BTA4q15, BBU8q15, OAR4q15 and CHI4q15; CYP1A1 and CYP1A2 to BTA21q17, BBU20q17, OAR18q17 and CHI21q17; CYP1B1 to BTA11q16, BBU12q22, OAR3p16 and CHI11q16, AHRR to BTA20q24, BBU19q24, OAR16q24 and CHI20q24. All loci were mapped at the same homoeologous chromosomes and chromosome bands of the four bovid species. Comparisons with corresponding human locations were also reported.     

Comparative FISH-mapping of the survival of motor neuron gene (SMN) in domestic bovids

A comparative fluorescence in situ mapping of the SMN gene was performed on R-banded chromosome preparations of cattle (Bos taurus, BTA, 2n = 60), river buffalo (Bubalus bubalis, BBU, 2n = 50), sheep (Ovis aries, OAR, 2n = 54) and goat (Capra hircus, CHI, 2n = 60), as well as on those of a calf from Piedmont breed affected by arthrogryposis. SMN was located on BTA20q13.1, OAR16q13.1, CHI20q13.1 and BBU19q13. These chromosomes and chromosome bands are believed to be homeologous, confirming the high degree of chromosome homeologies among bovids. The position of SMN was refined in cattle, compared to the two previous localizations, while it is a new gene assignment in the other three bovids. A comparative fiber-FISH performed on extended chromatin of both normal cattle and calf affected by arthrogryposis revealed more extended FITC signals in the calf, compared to the normal cattle (control), suggesting a possible duplication of the SMN gene in the calf affected by arthrogryposis. .     

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.     

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) is a source of milk and meat, and also serves as a draft animal. In this study, a 5000-rad whole-genome radiation hybrid (RH) panel for river buffalo was constructed and used to build preliminary RH maps for BBU3 and BBU10 chromosomes. The preliminary maps contain 66 markers, including coding genes, cattle expressed sequence tags (ESTs) and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci that will allow detailed comparative maps between buffalo, cattle and other species whose genomes may be mapped in the future. A large quantity of DNA has been prepared from the cell lines forming the river buffalo RH panel and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.     

Chromosome localization of the 31 type I Texas bovine markers in sheep and goat chromosomes by comparative FISH-mapping and R-banding

Bovine BAC clones containing the 31 genes, referred to as the Texas markers used earlier to definitively assign the 31 bovine syntenic groups (U) to cattle chromosomes, were mapped by fluorescent in situ hybridization to sheep and goat R-banded chromosomes according to ISCNDB2000. All 31 markers were localized on homoeologous chromosomes and chromosome bands of the two species in agreement with previous localizations obtained both in cattle and river buffalo, definitively confirming chromosome homoeologies between Caprinae and Bovinae. In addition, we have extended physical maps of sheep and goat as 11 genes (HSD3B1, INHBA, CSN10, IGF2R, PIGR, MAP1B, DSC1, ELN, TNFRSF6, CGN1, IGF2) and 14 genes (SOD1, HSD3B1, CSN10, IGF2R, RB1, TG, PIGR, MAP1B, IGH@, LTF, DSC1, TNFRSF6, CGN1, IGF2) were assigned for the first time to goat and sheep chromosomes, respectively.     

An advanced sheep (Ovis aries, 2n = 54) cytogenetic map and assignment of 88 new autosomal loci by fluorescence in situ hybridization and R-banding

Presented herein is an updated sheep cytogenetic map that contains 452 loci (291 type I and 161 type II) assigned to specific chromosome bands or regions on standard R-banded ideograms. This map, which significantly extends our knowledge of the physical organization of the ovine genome, includes new assignments for 88 autosomal loci, including 74 type I loci (known genes) and 14 type II loci (SSRs/microsatellite marker/STSs), by FISH-mapping and R-banding. Comparison of the ovine map to the cattle and goat cytogenetic maps showed that common loci were located within homologous chromosomes and chromosome bands, confirming the high level of conservation of autosomes among ruminant species. Eleven loci that were FISH-mapped in sheep (B3GAT2, ASCC3, RARSL, BRD2, POLR1C, PPP2R5D, TNRC5, BAT2, BAT4, CDC5L and OLA-DRA) are unassigned in cattle and goat. Eleven other loci (D3S32, D1S86, BMS2621, SFXN5, D5S3, D5S68, CSKB1, D7S49, D9S15, D9S55 and D29S35) were assigned to specific ovine chromosome (OAR) bands but have only been assigned to chromosomes in cattle and goat.     

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 7 and comparative mapping to the cattle and human genomes

A preliminary radiation hybrid (RH) map containing 50 loci on chromosome 7 of the domestic river buffalo Bubalus bubalis (BBU; 2n = 50) was constructed based on a comparative mapping approach. The RH map of BBU7 includes thirty-seven gene markers and thirteen microsatellites. All loci have been previously assigned to Bos taurus (BTA) chromosome BTA6, which is known for its association with several economically important milk production traits in cattle. The map consists of two linkage groups spanning a total length of 627.9 cR(5,000). Comparative analysis of the BBU7 RH(5,000) map with BTA6 in cattle gave new evidence for strong similarity between the two chromosomes over their entire length and exposed minor differences in locus order. Comparison of the BBU7 RH(5,000) map with the Homo sapiens (HSA) genome revealed similarity with a large chromosome segment of HSA4. Comparative analysis of loci in both species revealed more variability than previously known in gene order and several chromosome rearrangements including centromere relocation. The data obtained in our study define the evolutionarily conserved segment on BBU7 and HSA4 to be between 3.5 megabases (Mb) and 115.8 Mb in the HSA4 (genome build 36) DNA sequence.     

Preliminary radiation hybrid map for river buffalo chromosome 6 and comparison to bovine chromosome 3

We present the first radiation hybrid (RH) map of river buffalo (Bubalus bubalis) chromosome 6 (BBU6) developed with a recently constructed river buffalo whole-genome RH panel (BBURH(5000)). The preliminary map contains 33 cattle-derived markers, including 12 microsatellites, 19 coding genes and two ESTs, distributed across two linkage groups. Retention frequencies for markers ranged from 14.4% to 40.0%. Most of the marker orders within the linkage groups on BBU6 were consistent with the cattle genome sequence and RH maps. This preliminary RH map is the starting point for comparing gene order between river buffalo and cattle, presenting an opportunity for the examination of micro-rearrangements of these chromosomes. Also, resources for positional candidate cloning in river buffalo are enhanced.     

Sexing river buffalo (Bubalus bubalis L.), sheep (Ovis aries L.), goat (Capra hircus L.), and cattle spermatozoa by double color FISH using bovine (Bos taurus L.) X- and Y-painting probes

River buffalo, sheep, and goat spermatozoa were cross-hybridized using double color fluorescence in situ hybridization (FISH) with bovine Xcen- and Y-chromosome painting probes, prepared by DOP-PCR of laser-microdissected-catapulted chromosomes, to investigate the possibility of using bovine probes for sexing sperm of other members of the family Bovidae. Before sperm analysis, the probes were hybridized on metaphase chromosomes of each species, as control. Frozen-thawed spermatozoa of cattle, river buffalo, sheep, and goat were decondensed in suspension with 5 mM DTT. Sperm samples obtained from three individuals of each species were investigated, more than 1,000 spermatozoa were scored in each animal. FISH analysis of more than 12,000 sperm revealed high level of sperm with X- or Y-signals in all of the species investigated, indicating FISH efficiency over 99%. Significant interspecific differences were detected in the frequency of aberrant spermatozoa (aneuploid and diploid) between goat (0.393%) and sheep (0.033%) (P < 0.01), goat and cattle (0.096%) (P < 0.5), as well as between river buffalo (0.224%) and sheep (P < 0.5). There was no significant difference between river buffalo and cattle. The present study demonstrated that it is possible to use bovine X-Y painting probes for sexing and analyzing sperm of other species of the family, thus facilitating future studies on the incidence of chromosome abnormalities in sperm as well as on sex predetermination of embryos for the livestock industry. Mol. Reprod. Dev. 67: 108-115, 2004.     

Comparison of the human with the sheep genomes by use of human chromosome-specific painting probes

Human chromosome specific painting probes were hybridized on sheep (Ovis aries, 2n = 54) chromosomes by FISH. The painting results on sequentially stained RBA-banded preparations demonstrated high degree of conserved regions between human and sheep genomes. A total of 48 human chromosome segments were detected in sheep chromosomes. Comparisons with sheep gene mapping data available and previous Zoo-FISH data obtained in sheep, cattle, and river buffalo were performed. Received: 13 October 1998 / Accepted: 18 February 1999     

Comparative mapping of twenty-eight bovine loci in sheep (Ovis aries, 2n = 54) and river buffalo (Bubalus bubalis, 2n = 50) by FISH

Peripheral blood cell cultures were treated for late incorporation of both BrdU and Hoechst-33258 to obtain R-banding pattern preparations. Twenty-eight bovine cosmids from 19 bovine syntenic groups (U), three of which contain type I loci and 25 which contain microsatellite loci and have previously been assigned to cattle chromosomes, were comparatively FISH-mapped to sheep and river buffalo chromosomes according to the standard karyotypes (13 loci for the first time in the latter species). The results enrich the physical maps of both species with information relative to the following loci and to the corresponding syntenic groups: IDVGA35 and IDVGA53 (U6), IDVGA61 and IDVGA84 (U13), JAB10 (U5), IDVGA41 and IDVGA57 (U27), IDVGA87 (U11), IDVGA32 and IDVGA10 (U19), IDVGA49, IDVGA66 and IDVGA68 (U1), ZNF164 (U23), IDVGA74 and IDVGA70 (U9), IDVGA47, IDVGA46 and IDVGA58 (U21), MAP1B (U14), IDVGA79 (U4), CATHL (U12), IDVGA71 (U8), IDVGA59 (U26), IDVGA29 (U29), IDVGA7 (U7), IDVGA82 (X), IDVGA50 (Y). All mapped loci were localized on homoeologous chromosomes and chromosome regions of the two species, confirming the high degree of chromosome homoeologies between the subfamilies Bovinae and Caprinae.     

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 1 (BBU1)

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR(5000). The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH(5000)) panel. The retention frequency of individual markers across the panel ranged from 17.8 to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs.