Physical and linkage mapping of the bovine genome with cosmids

We have initiated a mapping strategy using cosmid clones to chromosomally anchor a high-resolution bovine genetic linkage map. Ten cosmids containing microsatellites were assigned to bovine chromosomes by fluorescence in situ suppression hybridization (FISH). Four cosmid clones, three of which contain an informative microsatellite, were assigned to autosomes 5, 13, 24, and 28. The assignment to autosome 13 anchors bovine syntenic group U11. Two additional cosmid clones, each containing informative microsatellites, are assigned to autosomes 9 and 29, auchoring bovine linkage groups U2 and U8, respectively. Four cosmid clones, three of which contain informative microsatellites, also provide the first assignment to autosome 25, anchoring bovine syntenic group U7 and orienting the corresponding linkage group relative to the centromere.     

Mapping of 50 cosmid clones isolated from a flow-sorted human X chromosome library by fluorescence in situ hybridization.

Fifty cosmids have been mapped to metaphase chromosomes by fluorescence in situ hybridization under conditions that suppress signals from repetitive DNA sequences. The cosmid clones were isolated from a flow-sorted human X chromosome library. Thirty-eight of the clones were localized to chromosome X and 12 to autosomes such as chromosomes 3, 7, 8, 14, and 17. Although most of the cosmids mapped to the X chromosome appeared to be scattered along both the short and long arms, 10 cosmids were localized to the centromeric region of the chromosome. Southern blot analysis revealed that only two of these clones hybridized to probe pXBR-1, which detects the DXZ1 locus. In addition, 4 out of 5 cosmids mapped on chromosome 8 also localized on the centromeric region. While localization of X-specific cosmids will facilitate the physical mapping of the human X chromosome, cosmids mapped to the centromeric regions of chromosomes X and 8 should be especially useful for studying the structure and organization of these regions.     

A cosmid clone for the 5HT1A receptor (HTR1A) reveals a TaqI RFLP that shows tight linkage to dna loci D5S6, D5S39, and D5S76.

A human neuroreceptor clone (G21), which was isolated by cross-hybridization with the human clone for the beta 2-adrenergic receptor, has recently been shown to encode the gene for the 5HT1A receptor (HTR1A) subtype. In situ hybridization to human metaphase chromosomes mapped the G21 sequence to chromosome 5 at bands 5q11.2-q13. The clone G21 recognizes a SacI RFLP with low heterozygosity (0.13). To increase the informativeness of the HTR1A locus we have isolated two new cosmid clones containing the receptor gene. No polymorphic microsatellites were present in the cosmids. However, one cosmid revealed a new TaqI RFLP that showed tight linkage to new highly polymorphic microsatellites for the loci D5S76, D5S39, and D5S6 in seven British and Icelandic reference pedigrees (maximum LOD of 13.2 with D5S76).     

Contribution to the physically anchored linkage map of the pig

Thirty-three microsatellites have been mapped on the PiGMaP porcine genetic map. By comparison with the previously published PiGMaP maps, the maps of chromosome 2 (140 cM/70 cM) and chromosome 3 (180 cM/110 cM) were extended and new markers were mapped on the p-arm extremity of chromosome 7 and on the centromeric extremity of chromosome 15. New orders are proposed for markers on chromosomes 3 and 17. Six microsatellites isolated from cosmids were also localized on the cytogenetic map by fluorescent in situ hybridization. We tested the subcloning ligation mixture–polymerase chain reaction (SLiM-PCR) method for isolating microsatellites from cosmids. Subcloning is more effective when the cosmid harbours several microsatellites whereas SLiM-PCR is more straightforward when the cosmid contains a single microsatellite. Fifteen anonymous microsatellites were regionally assigned by using a hybrid cell panel. For map integration, the determination of a regional assignment of anonymous microsatellites by using a hybrid cell panel offers an alternative to microsatellite isolation from cosmids and their localizations by in situ hybridization.     

Chromosomal localization and molecular characterization of 53 cosmid-derived bovine microsatellites

Gene mapping in cattle has progressed rapidly in recent years largely owing to the introduction of powerful genetic markers, such as the microsatellites, and through advances in physical mapping techniques such as synteny mapping and fluorescence in situ hybridization (FISH). Microsatellite markers are often not physically mapped because they are generally isolated from small insert plasmid libraries, which makes their chromosomal localization inefficient. In this report we describe the FISH mapping of a large group of cosmid-derived bovine microsatellite markers, as our contribution to the European mapping initiative, BovMap. One objective of BovMap is to develop a set of anchored loci for the cattle genome map.Two cosmid libraries were screened with probes corresponding to the (AC) _n microsatellite motif. Positive clones were mapped by FISH, and then a subset was further analyzed by sequencing the region flanking the microsatellite repeat. In total, 58 clones were hybridized with chromosomes and identified loci on 22 of the 31 different bovine chromosomes. Three clones contained satellite DNA. Two or more markers were placed on 12 chromosomes. Sequencing of the microsatellites and flanking regions was performed directly from 43 cosmids, as previously reported (Ferretti et al. Anim. Genet. 25, 209–214, 1994). Primers were developed for 39 markers and used to describe the polymorphism associated with the corresponding loci.     

Ordering of markers in the pericentromeric region of chromosome 10

Seven polymorphic cosmids previously assigned to 10cen-q11.2 were mapped between D10S34 and RBP3, and ordered by interphase in situ hybridization and yeast artificial chromosome analysis. Some of the presumed unique sequences from the centromeric region have homologies either within the same region or within the centromeric region of other chromosomes.     

Integrating the porcine physical and linkage map using cosmid-derived markers

An essential part in the development of informative linkage maps is to include genetic markers that have been anchored by physical mapping. Here a set of 18 porcine cosmid-derived genetic markers are reported that have been mapped by linkge analysis, and that also have been physically localized by fluorescence in situ hybridization (FISH). Three different strategies were used to establish polymorphic markers from the cosmid clones. Firstly, dinucleotide microsatellite loci were derived by sequencing cosmid subclones containing (CA), repeats. Secondly, variable SINE 3′ poly(A) tracts (SINEVA) were identified by direct SINE-PCR amplification of cosmid clones. Thirdly, the cosmids were used in Southern blot hybridization to detect restriction fragment length polymorphisms (RFLPs). Compared with the most recent consensus compilation of the porcine gene map, the present assignment of markers to chromosomes Zp, 3, 4, 10, 12q, and 16 represents the first loci mapped to these chromosomes, for which linkage as well as in situ data are now available.     

The cosmid CSSM25 assigns syntenic group U2 to bovine Chromosome 9 and is localized to ovine Chromosome 8

The cosmid-derived microsatellite CSSM 25 has previously been shown to map to bovine syntenic group U2 by link-age and hybrid somatic cell analysis. We have mapped the cosmid by fluorescent in situ hybridization to bovine Chromosome (Chr) 9q17-21 and ovine Chr 8q17-21 and hence assign U2 to Chr 9 in cattle. Bovine Chr 9 and ovine Chr 8 show strong banding pattern homology, and the localization of CSSM 25 to the same region confirms the strong conservation of gene locations on these chromosomes.     

Characterization of Two Chromosome 12 Cosmid Libraries and Development of STSs from Cosmids Mapped by FISH

We have constructed and characterized two related human chromosome 12-specific cosmid libraries. DNA from flow-sorted chromosomes from a somatic cell hybrid was cloned into a cosmid vector. Approximately 61% of the cosmids in the nearly 26,200 member arrayed libraries (LLt2NC01 and LLt2NC02) contain human DNA inserts, and 31% of the cosmids derived from human DNA contain CA repeats. One hundred and fifty-two cosmids isolated from the libraries have been mapped by fluorescence in situ hybridization (FISH). Cosmids containing human DNA inserts were localized by FISH exclusively to chromosome 12, confirming the chromosomal specificity of the libraries. The cosmids have been localized to all parts of this chromosome, although some regions are more highly represented than others. Partial sequence information was obtained from 44 mapped cosmids, and oligonucleotide primer pairs were synthesized that define unique sequence tagged sites (STSs). These mapped cosmids, and unique STSs derived from them, provide a set of useful clones and primer pairs for screening YAC libraries and developing contigs centered on regions of interest within chromosome 12. In addition, 120 of the mapped cosmids contain CA repeats, and thus they also provide a useful resource for defining highly polymorphic simple tandem repeat elements that serve as genetic markers for linkage analysis and disease gene localization.     

Identification,characterisation and clinical applications of cosmids from the telomeric and centromeric regions of the long arm of chromosome 22

Using human telomeric repeats and centromeric alpha repeats, we have identified adjacent single copy cosmid clones from human chromosome 22 cosmid libraries. These single copy cosmids were mapped to chromosome 22 by fluorescence in situ hybridisation (FISH). Based on these cosmids, we established contigs that included part of the telomeric and subtelomeric regions, and part of the centromeric and pericentromeric regions of the long arm of human chromosome 22. Each of the two cosmid contigs consisted of five consecutive steps and spanned approximately 100–150 kb at both extreme ends of 22q. Moreover, highly informative polymorphic markers were identified in the telomeric region. Our results suggest that the telomere specific repeat (TTAGGG) _n encompasses a region that is larger than 40 kb. The cosmid contigs and restriction fragment length polymorphism markers described here are useful tools for physical and genetic mapping of chromosome 22, and constitute the basis of further studies of the structure of the subtelomeric and pericentromeric regions of 22q. We also demonstrate the use of these clones in clinical diagnosis of different chromosome 22 aberrations by FISH.     

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.     

Synteny mapping in the bovine: genes from human chromosome 4.

Genes homologous to those located on human chromosome 4 (HSA4) were mapped in the bovine to determine regions of syntenic conservation among humans, mice, and cattle. Previous studies have shown that two homologs of genes on HSA4, PGM2 and PEPS, are located in bovine syntenic group U15 (chromosome 6). The homologous mouse genes, Pgm-1 and Pep-7, are on MMU5. Using a panel of bovine x hamster hybrid somatic cells, we have assigned homologs of 11 additional HSA4 loci to their respective bovine syntenic groups. D4S43, D4S10, QDPR, IGJ, ADH2, KIT, and IF were assigned to syntenic group U15. This syntenic arrangement is not conserved in the mouse, where D4s43, D4s10, Qdpr, and Igj are on MMU5 while Adh-2 is on MMU3. IL-2, FGB, FGG, and F11, which also reside on MMU3, were assigned to bovine syntenic group U23. These data suggest that breaks and/or fusions of ancestral chromosomes carrying these genes occurred at different places during the evolution of humans, cattle, and mice.     

Rapid mapping of cosmid clones on pig chromosomes by fluorescence in situ hybridization

Nineteen cosmids have been mapped to pig chromosomes by fluorescence in situ hybridization. Two kinds of cosmid clones were isolated as potential physical and genetic markers for the pig genome. Anonymous cosmids were obtained by screening a commercial cosmid library and were localized to Chromosomes (Chrs) 1, 2, 6, 7, 8, 10, 11, 12, 13, and 14. Some of these cosmids were found to reveal RFLP type DNA polymorphism. Microsatellite-containing cosmid clones were isolated by screening a pig cosmid library with a (CA)₁₀ probe and were regionally mapped to Chrs 2, 6, 7, 13, and 14. Ten of the 19 chromosomes in the pig were labeled with these probes. Two-color fluorescence in situ hybridization was used to increase the efficiency of the cosmid localizations.     

Development and genetic mapping of 127 new microsatellite markers in barley

To enhance the marker density of existing genetic maps of barley (Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.Communicated by G. Wenzel     

A Physical Map of 15 Loci on Human Chromosome 5q23-q33 by Two-Color Fluorescence in Situ Hybridization

The q23-q33 region of human chromosome 5 encodes a large number of growth factors, growth factor receptors, and hormone/neurotransmitter receptors. This is also the general region into which several disease genes have been mapped, including diastrophic dysplasia, Treacher Collins syndrome, hereditary startle disease, the myeloid disorders that are associated with the 5q-syndrome, autosomal-dominant forms of hereditary deafness, and limb girdle muscular dystrophy. We have developed a framework physical map of this region using cosmid clones isolated from the Los Alamos arrayed chromosome 5-specific library. Entry points into this library included 14 probes to genes within this interval and one anonymous polymorphic marker locus. A physical map has been constructed using fluorescence in situ hybridization of these cosmids on metaphase and interphase chromosomes, and this is in good agreement with the radiation hybrid map of the region. The derived order of loci across the region is cen-IL4-IL5-IRF1-IL3-IL9-EGR1-CD14-FGFA-GRL-D5S207-ADRB2-SPARC-RPS14-CSF1R-ADRA1, and the total distance spanned by these loci is approximately 15 Mb. The framework map, genomic clones, and contig expansion within 5q23-q33 should provide valuable resources for the eventual isolation of the clinically relevant loci that reside in this region.     

Microsatellite mapping of the induced sphaerococcoid mutation genes in Triticum aestivum

The S1, S2 and S3 genes of the induced sphaerococcoid mutation in common wheat (Triticum aestivum) were mapped using three different F₂ populations consisting of 71–96 individual plants. Twenty-four microsatellite markers from homeologous group 3 of T. aestivum were used to map the S1, S2 and S3 genes on chromosomes 3D, 3B and 3A, respectively. The S1 locus was found to be closely linked to the centromeric marker Xgwm456 of the long arm (2.9 cM) and mapped not far (8.0 cM) from the Xgdm72 marker of the short arm of chromosome 3D. The S2 gene was tightly linked to 2 centromeric markers (Xgwm566, Xgwm845) of chromosome 3B. S3 was located between Xgwm2 (5.1 cM), the marker of the short arm, and Xgwm720 (6.6 cM), the marker of the long arm, both of chromosome 3A. Mapping the S1, S2 and S3 loci of the induced sphaerococcoid mutation near the centromeric regions supports the hypothesis that the sphaerococcum type may be due to gene duplication resulting from DNA recombination in the centromeric region. Received: 20 June 1999 / Accepted: 29 July 1999     

Isolation of coding sequences from bovine cosmids by means of exon trapping

Exon trapping was employed to identify coding sequences from a collection of 46 bovine cosmids, previously characterized for the presence of microsatellite markers and physically mapped to chromosomes by FISH. The sequence analysis of 104 clones revealed 18 putative exons, 10 of which showed near identity to known sequences. Among these were the human (cytosine-5)-methyltransferase (DNMT), ATP-citrate lyase (ACLY), the mouse Lbcl1 oncogene, the bovine mitochondrial aconitase (ACO2) and β-arrestin 1 (ARR1). The chromosomal localization of the cloned exons was inferred from the localization of the parent cosmids. DNMT and ACLY were not previously known in cattle, but the physical localization of the cloned bovine exons is in agreement with the published comparative human and bovine maps. The trapping of exons for bovine ACO2 and ARR1 confirms the available mapping information based on synteny and provides a physical assignment for the genes. Received: 23 November 1996 / Accepted: 3 March 1997     

Syntenic Assignment of Human Chromosome 1 Homologous Loci in the Bovine

Three mouse chromosomes (MMU 1, 3, and 4) carry homologs of human chromosome 1 (HSA 1) genes. A similar situation is found in the bovine, where five bovine chromosomes (BTA 2, 3, 5, 16, and unassigued syntenic group U25) contain homologs of HSA 1 loci. To evaluate further the syntenic relationship of HSA 1 homologs in cattle, 10 loci have been physically mapped through segregation analysis in bovine-rodent hybrid somatic cells. These loci, chosen for their location on HSA 1, are antithrombin 3 (AT3), renin (REN), complement component receptor 2 (CR2), phosphofructokinase muscle type (PFKM), Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog (FGR), α fucosidase (FUCA1), G-protein β1 subunit (GNB1), α 1A amylase, (AMY1), the neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), and α skeletal actin (ACTA1). AT3, REN, CR2, and GNB1 mapped to BTA 16, PFKM to BTA 5, AMY1A and NRAS to BTA 3, FGR and FUCA1 to BTA 2, and ACTA1 to BTA 28.     

Synteny mapping in the bovine: genes from human chromosome 5.

In an effort to generate a more complete bovine syntenic map of Type I comparative anchor loci, seven homologs to genes found on HSA5 were mapped using a panel of bovine x rodent hybrid somatic cells. Five HSA5 genes, CSF2, RPS14, PDGFRB, FGFA, and CSF1R, were assigned to bovine syntenic group U22 (chromosome 7), while two others, C9 and HGMCR, mapped to U10 and U5, respectively. Previous studies had assigned the HSA5 marker SPARC to bovine syntenic group U22. The mapping of genes spanning the length of HSA5 in cattle and also in mouse permits syntenic comparisons between prototypic genomes of three mammalian orders, providing insight into the evolutionary history of this region of the ancestral mammalian genome.     

Genetic mapping of 66 new microsatellite (SSR) loci in bread wheat

In hexaploid bread wheat ( Triticum aestivum L. em. Thell), ten members of the IWMMN ( International Wheat Microsatellites Mapping Network) collaborated in extending the microsatellite (SSR = simple sequence repeat) genetic map. Among a much larger number of microsatellite primer pairs developed as a part of the WMC ( Wheat Microsatellite Consortium), 58 out of 176 primer pairs tested were found to be polymorphic between the parents of the ITMI ( International Triticeae Mapping Initiative) mapping population W7984 x Opata 85 (ITMI pop). This population was used earlier for the construction of RFLP ( Restriction Fragment Length Polymorphism) maps in bread wheat (ITMI map). Using the ITMI pop and a framework map (having 266 anchor markers) prepared for this purpose, a total of 66 microsatellite loci were mapped, which were distributed on 20 of the 21 chromosomes (no marker on chromosome 6D). These 66 mapped microsatellite (SSR) loci add to the existing 384 microsatellite loci earlier mapped in bread wheat.