Refinement of bovine chromosome 2 linkage map near the mh locus reveals rearrangements between the bovine and human genomes

The locus responsible for the appearance of muscular hypertrophy (mh) in double muscled cattle breeds has recently been shown to encode a secreted growth factor designated myostatin (MSTN). This conclusion was based in part on the placement of MSTN in the interval to which mh had been mapped on bovine chromosome 2 (BTA2). During the mapping phase of the study, numerous yeast artificial chromosome (YAC) clones were isolated that contained genetic markers closely linked to mh. Other YACs and cosmids were identified that contained genes selected from human chromosome 2q (HSA2q), with the goal of defining the position of breakpoints in conserved synteny between the bovine and human comparative maps, thereby permitting accurate selection of positional candidate genes. An efficient subcloning procedure was developed to obtain microsatellites (ms) from YAC clones, to increase the number of informative meioses in herds segregating for mh. The same procedure was used to place the human orthologues of engrailed-1 (EN1), interleukin 1 beta (IL1B), and paired-box-containing 8 (PAX8) genes on the cattle map to further define the positions of breakpoints in conserved synteny and gene order. Twenty-three of 28 ms identified from YAC subclone libraries were informative in the mapping families. Seven mapped to the centromeric end of BTA2, which contains the mh locus, improving marker density and informativeness. The two MSTN and four EN1 gene-associated ms markers developed from YACs, map to positions 1·5 and 61·6 cm in the BTA2 linkage group, respectively. In addition, ms markers developed from cosmids containing either IL1B or PAX8, map to positions 56·6 and 56·9 cm in the BTA11 linkage group, respectively. These linkage data confirm the location and orientation of orthologous segments of HSA2q that were previously indistinguishable on the bovine map, and demonstrates the presence of microrearrangements of gene order (segments <10 cm ) and conserved synteny between the human and bovine genomes.     

A genetic and physical map of bovine Chromosome 11

A genetic map of bovine Chromosome (Chr) 11 (BTA11, synteny group U16) has been constructed from 330 animals belonging to 21 families, which constitute the international bovine reference panel (IBRP). This map is based on 13 polymorphic microsatellite markers, two of which were chosen in previously published maps. Three markers have been isolated from cosmids. Two of the three cosmids have been physically localized by fluorescence in situ hybridization (FISH), to anchor the genetic map on the chromosome. In addition, a biallelic polymorphism in the -lactoglobulin gene (LGB) has been genetically positioned relative to the microsatellite markers. The most probable order of the markers is: cen-INRA044-BM716-INRA177-(TGLA 327, INRA198, INRA131)-INRA111-INRABERN169-(INRA115, INRA032)-INRA108-INRABERN162-INRA195-LGB. T The total linkage group spans 126 cM, which probably corresponds to most of the chromosome length. The average intermarker distance is about 10.5 cM, allowing the potential detection of a genetic linkage with any Economic Trait Loci (ETL) of this chromosome.     

Radiation hybrid and genetic linkage mapping of two genes related to fat metabolism in cattle: fatty acid synthase (FASN) and glycerol-3-phosphate acyltransferase mitochondrial (GPAM)

Fatness traits, such as fat deposition, carcass composition, fat content, and the percentage of fat in milk, are economically relevant to cattle production. Fatty acid synthase (FASN) and glycerol-3-phosphate acyltransferase mitochondrial (GPAM) are two enzymes that play a central role in de novo lipogenesis. Both could be putative candidate genes for quantitative trait loci (QTL). Several clones containing the fatty acid synthase (FASN) and glycerol-3-phosphate acyltransferase mitochondrial (GPAM) genes were isolated after screening the INRA bovine bacterial artificial chromosome (BAC) library using PCR. Five microsatellite loci were derived from the BAC clones containing the genes of interest with heterozygosity values ranging from 27 to 78%, using DNA samples from the International Bovine Reference Panel (IBRP). The newly developed markers were genotyped on the IBRP animals and on a radiation hybrid panel to compare the obtained linkage and RH maps. Radiation hybrid maps were developed for chromosome BTA19 and BTA26 regions containing FASN and GPAM genes, respectively. The two genes and their associated microsatellite markers were located on the genetic or RH maps or on both. These microsatellite markers could be useful to study the QTL effect on fat synthesis in reference population.     

A Genetic Linkage Map of the Male Goat Genome

This paper presents a first genetic linkage map of the goat genome. Primers derived from the flanking sequences of 612 bovine, ovine and goat microsatellite markers were gathered and tested for amplification with goat DNA under standardized PCR conditions. This screen made it possible to choose a set of 55 polymorphic markers that can be used in the three species and to define a panel of 223 microsatellites suitable for the goat. Twelve half-sib paternal goat families were then used to build a linkage map of the goat genome. The linkage analysis made it possible to construct a meiotic map covering 2300 cM, i.e., >80% of the total estimated length of the goat genome. Moreover, eight cosmids containing microsatellites were mapped by fluorescence in situ hybridization in goat and sheep. Together with 11 microsatellite-containing cosmids previously mapped in cattle (and supposing conservation of the banding pattern between this species and the goat) and data from the sheep map, these results made the orientation of 15 linkage groups possible. Furthermore, 12 coding sequences were mapped either genetically or physically, providing useful data for comparative mapping.     

High-resolution genetic map of bovine chromosome 29 through focused marker development

Chromosome-specific libraries aid in the development of genetic maps and focus marker development in areas of the genome with identified quantitative trait loci (QTL). A small-insert BTA29 library constructed by microdissection of a 1:29 Rb-fusion cell line, was screened for dinucleotide repeats (CA)(15) and/or (GA)(15) with the goal of generating new genetic markers for this, the smallest bovine autosome. A total of 90 primer pairs were designed and 82 of these successfully amplified bovine genomic DNA by PCR. In addition to these 82 loci, primer pairs were developed for nine putative genes identified from the sequenced clones by BLAST searches of GenBank. A somatic cell panel was used to test for synteny of the new loci with two previously mapped BTA29 markers located on the MARC bovine linkage map. A total of 75 of the 82 microsatellite (ms) loci were integrated into the MARC bovine linkage map. Linkage analysis placed 69 ms markers on BTA29, five on BTAX and one on BTA1. Combined results of the somatic cell and linkage analyses place 79 new markers (ms and gene-related) on BTA29, six loci on BTAX and two loci on BTA1. The results of this effort significantly increase the marker density on BTA29, expanding the ability to fine map QTL associated with this chromosome.     

Linkage mapping of the locus responsible for congenital multiple ocular defects in cattle on bovine Chromosome 18

Congenital multiple ocular defects (MOD) in Japanese black cattle is a hereditary ocular disorder with an autosomal recessive manner of inheritance, showing developmental defects of the lens, retina, and iris, persistent embryonic eye vascularization, and microphthalmia. In the present study, we mapped the locus responsible for the disorder by linkage analysis using 240 microsatellite markers covering the entire bovine genome and an inbred pedigree obtained from commercial herds. The linkage analysis demonstrated a significant linkage between the disorder locus and markers on the proximal region of bovine Chromosome (BTA) 18 with the maximum LOD score of 5.1. Homozygosity mapping using the haplotype of the linked markers further refined the critical region. The results revealed the localization of the locus responsible for MOD in an approximately 6.6-cM region of BTA18. Comparison of published linkage and radiation hybrid (RH) maps of BTA18 with its evolutionary ortholog, human Chromosome (HSA) 16, revealed several potential candidate genes for the disorder including the MAF and FOXC2 genes.     

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.     

Comparative mapping of the ovine clpg locus

We used a comparative mapping approach to identify segments of conserved synteny between human Chromosome 14 (HSA14), bovine Chromosome 21 (BTA21), and the portion of ovine Chromosome 18 (OAR18) that contains the clpg locus. A bovine radiation hybrid map of the region was constructed with available Type II genetic markers and seven candidate genes to establish the comparative interval between BTA21 and HSA14. We developed polymorphic microsatellite and SNP markers associated with five candidate genes and placed them on the ovine and/or bovine genetic maps by multipoint linkage analysis. Three additional genes were mapped by virtue of their physical linkage to genetically mapped makers. Development of integrated linkage and physical maps facilitates the selection of positional candidate genes from the gene rich human map. The physically linked candidate genes PREF-1 and MEG3 map to the interval containing the clpg locus. Comparative biology suggests imprinting of MEG3 and/or the influences of PREF-1 on cellular differentiation, should be examined for their role in the parent-of-origin dependent influence of mutant clpg alleles on sheep muscle characteristics. Received: 3 February 2000 / Accepted: 19 April 2000     

Single nucleotide polymorphism (SNP) discovery and linkage mapping of bovine cytokine genes

Polymorphic markers at bovine gene loci facilitate the integration of cattle genetic maps with those of humans and mice. To this end, 31 single nucleotide polymorphism (SNP) markers were developed for seven bovine chemokine genes. Loci were amplified from bovine genomic DNA by the polymerase chain reaction, and candidate amplicons were sequenced to determine their identity. Amplified loci from 24 founding parents and select progeny from a beef cattle reference population were sequenced and analyzed for SNPs. SNP haplotype alleles were determined by examining segregation patterns and used to establish the locus position on the bovine linkage map. Loci for growth-related proteins (GRO3, GRO1, and GROX) were clustered with the related CXC chemokine genes, interleukin (IL) 8, and epithelial cell inflammatory protein 1, at 84 cM from the centromeric end of the bovine chromosome (BTA) 6 linkage group. Bovine loci for a cluster of IL8 receptors, a stromal cell-derived factor 1, interferon-γ, and tumor necrosis factor-α were mapped at 90, 55, 59, and 34 cM, respectively, from the centromeric ends of the BTA 2, 28, 5, and 23 linkage groups. The positions of these bovine loci were compared with those of orthologous loci on the human map to refine the boundaries of conserved synteny. These seven loci provide examples of SNP development in which the efficiency was largely dependent on the availability of bovine genomic or cDNA sequence. The polymorphic nature of these SNP haplotype markers suggests that they will be useful for mapping complex traits in cattle, such as resistance to infectious disease. Received: 30 April 1999 / Accepted: 12 July 1999     

A high-resolution radiation hybrid map of bovine chromosome 5q1.3-q2.5 compared with human chromosome 12q

In this study we present a comprehensive 3000-rad radiation hybrid map on bovine chromosome 5 (BTA5) of a region between 12.8 and 74.0 cM according to the linkage map, which contains a quantitative trait loci for ovulation rate. We mapped 28 gene-associated sequence tagged site markers derived from sequences of bovine BAC clones and 10 microsatellite markers to the BTA5 region. In comparison with HSA12q, four blocks of conserved synteny were apparent showing three chromosomal breakpoints and two inversions in this segment of BTA5. Therefore, we have improved breakpoint resolution in the human-bovine comparative map, which enhances the determination of candidate genes underlying traits of interest mapped to BTA5.     

PCR MULTIPLEXED MICROSATELLITE PANELS TO EXPEDITE CANINE GENETIC DISEASE LINKAGE ANALYSIS

ABSTRACT

Modern dog breeds possess large numbers of genetic diseases for which there are currently few candidate genes or diagnostic tests. Linkage of a microsatellite marker to a disease phenotype is often the only available tool to aid in the development of screening tests for disease carriers. Detection of linkage to a specific disease phenotype requires screening of large numbers of markers across known affected and unaffected animals. To establish high throughput genome scanning this study placed 100 canine microsatellite markers, arranged by fragment size and fluorescent dye label, into 12 PCR multiplexed panels. The highest degree of multiplexing was 11 markers per panel while the lowest was five markers per panel; each panel was run in one gel lane on automated DNA sequencers. Selection of the markers was based upon chromosomal or linkage group locations, degree of polymorphism, PCR multiplex compatibility and ease of interpretation. The marker set has an average spacing of 22.25?centiMorgan (cM). Marker polymorphism was evaluated across 28 American Kennel Club (AKC) recognized breeds. The utility of buccal swab vs. blood samples was also validated in this study as all template DNA was derived from swabs obtained and submitted by participating dog breeders and owners. The PCR multiplexed microsatellite panels and sampling method described in this report will provide investigators with a cost effective and expedient means of pursuing linkage studies of specific canine genetic diseases.     

Comparative mapping of human Chromosome 2 identifies segments of conserved synteny near the bovine mh locus

The ``double-muscling' (mh) locus has been localized to an interval between the centromere and the microsatellite marker TGLA44 on bovine Chromosome (Chr) 2 (BTA2). We identified segments of conserved synteny that correspond to this region of BTA2 by assigning large genomic clones containing bovine homologs of seven genes from the long arm of human Chr 2 (HSA2q). Polymorphic markers developed from these clones integrated the physical and linkage maps of BTA2 from 2q12 to 2q44 and extended genetic coverage towards the centromere. This comparative analysis suggests the mh locus resides on HSA2q near both the protein C and collagen type III alpha-1 genes. Overall, our data reveal a complex rearrangement of gene order between BTA2q12-44 and HSA2q14-37 that underscores the need to establish boundaries of conserved synteny when applying comparative mapping information to identify genes or traits of interest. Received: 3 March 1997 / Accepted: 12 May 1997     

A second-generation linkage map of the sheep genome

A genetic map of Ovis aries (haploid n = 27) was developed with 519 markers (504 microsatellites) spanning ∼3063 cM in 26 autosomal linkage groups and 127 cM (female specific) of the X Chromosome (Chr). Genotypic data were merged from the IMF flock (Crawford et al., Genetics 140, 703, 1995) and the USDA mapping flock. Seventy-three percent (370/504) of the microsatellite markers on the map are common to the USDA-ARS MARC cattle linkage map, with 27 of the common markers derived from sheep. The number of common markers per homologous linkage group ranges from 5 to 22 and spans a total of 2866 cM (sex average) in sheep and 2817 cM in cattle. Marker order within a linkage group was consistent between the two species with limited exceptions. The reported translocation between the telomeric end of bovine Chr 9 (BTA 9) and BTA 14 to form ovine Chr 9 is represented by a 15-cM region containing 5 common markers. The significant genomic conservation of marker order will allow use of linkage maps in both species to facilitate the search for quantitative trait loci (QTLs) in cattle and sheep. Received: 20 September 1992 / Accepted: 18 November 1997     

Isolation of a Cosmid Sublibrary for a Region of Chromosome 12 Frequently Amplified in Human Cancers Using a Complex Chromosome Microdissection Probe

Chromosome-specific cosmid libraries are an extremely useful resource for positional cloning projects. Once a particular region of interest has been identified, it would be of value to have an approach for isolating chromosome band-specific cosmids that could be assembled into a sublibrary for rapid screening. We constructed a region-specific sublibrary of 700 cosmids by screening a chromosome 12-specific cosmid library with a complex probe generated by degenerate oligonucleotide-primed PCR of a microdissected homogeneously staining region containing sequences amplified from chromosome 12q13–q15. Based on fluorescencein situhybridization, approximately 60% of the cosmids in the sublibrary were derived from the microdissected region. To demonstrate further the utility of this sublibrary, a 150-kb contig containing the SAS and CDK4 genes was constructed, as well as several additional contigs between CDK4 and MDM2. This study demonstrates the possibility of utilizing probes generated by microdissection for assembling band-specific sublibraries that are amenable to rapid screening with multiple markers.     

Physically mapped,cosmid-derived microsatellite markers as anchor loci on bovine chromosomes

To identify physical and genetic anchor loci on bovine chromosomes, 13 cosmids, obtained after the screening of partial bovine cosmid libraries with the (CA)_n microsatellite motif, were mapped by fluorescence in situ hybridization (FISH). Eleven cosmid probes yielded a specific signal on one of the bovine chromosomes and identified the following loci: D5S2, D5S3, D6S3, D8S1, D11S5, D13S1, D16S5, D17S2, D19S2, D19S3, D21S8. Two cosmids produced centromeric signals on many chromosomes. The microsatellite-containing regions were subcloned and sequenced. The sequence information revealed that the two centromeric cosmids were derived from bovine satellites 1.723 and 1.709, respectively. A cosmid located in the subtelomeric region of Chromosome (Chr) 17 (D17S2) had features of a chromosome-specific satellite. Primers were designed for eight of the nonsatellite cosmids, and seven of these microsatellites were polymorphic with between three and eight alleles on a set of outbred reference families. The polymorphic and chromosomally mapped loci can now be used to physically anchor other bovine polymorphic markers by linkage analysis. The microsatellite primers were also applied to DNA samples of a previously characterized panel of somatic hybrid cell lines, allowing the assignment of seven microsatellite loci to defined syntenic groups. These assignments confirmed earlier mapping results, revealed a probable case of false synteny, and placed two formerly unassigned syntenic groups on specific chromosomes.     

A microsatellite-based analysis for the detection of selection on BTA1 and BTA20 in northern Eurasian cattle (Bos taurus) populations

Background

Microsatellites surrounding functionally important candidate genes or quantitative trait loci have received attention as proxy measures of polymorphism level at the candidate loci themselves. In cattle, selection for economically important traits is a long-term strategy and it has been reported that microsatellites are linked to these important loci.

Methods

We have investigated the variation of seven microsatellites on BTA1 (Bos taurus autosome 1) and 16 on BTA20, using bovine populations of typical production types and horn status in northern Eurasia. Genetic variability of these loci and linkage disequilibrium among these loci were compared with those of 28 microsatellites on other bovine chromosomes. Four different tests were applied to detect molecular signatures of selection.

Results

No marked difference in locus variability was found between microsatellites on BTA1, BTA20 and the other chromosomes in terms of different diversity indices. Average D'' values of pairwise syntenic markers (0.32 and 0.28 across BTA 1 and BTA20 respectively) were significantly (P < 0.05) higher than for non-syntenic markers (0.15). The Ewens-Watterson test, the Beaumont and Nichol''s modified frequentist test and the Bayesian F_ST-test indicated elevated or decreased genetic differentiation, at SOD1 and AGLA17 markers respectively, deviating significantly (P < 0.05) from neutral expectations. Furthermore, lnRV, lnRH and lnRθ'' statistics were used for the pairwise population comparison tests and were significantly less variable in one population relative to the other, providing additional evidence of selection signatures for two of the 51 loci. Moreover, the three Finnish native populations showed evidence of subpopulation divergence at SOD1 and AGLA17. Our data also indicate significant intergenic linkage disequilibrium around the candidate loci and suggest that hitchhiking selection has played a role in shaping the pattern of observed linkage disequilibrium.

Conclusion

Hitchhiking due to tight linkage with alleles at candidate genes, e.g. the POLL gene, is a possible explanation for this pattern. The potential impact of selective breeding by man on cattle populations is discussed in the context of selection effects. Our results also suggest that a practical approach to detect loci under selection is to simultaneously apply multiple neutrality tests based on different assumptions and estimations.     

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.     

Development and assignment of bovine-specific PCR systems for the Texas nomenclature marker genes and isolation of homologous BAC probes

In 1996, Popescu et al. published the Texas standard nomenclature of the bovine karyotype in which 31 marker genes, already mapped in man, were chosen to permit unambiguous identification and numbering of each bovine chromosome. However, specific PCR systems were not available for each marker gene thus preventing the assignment of part of these markers by somatic cell hybrid analysis. In addition, some difficulties remained with the nomenclature of BTA25, BTA27 and BTA29. In this work, specific PCR systems were developed for each of the marker genes except VIL1 (see results), from either existing bovine or human sequences, and a bovine BAC library was screened to obtain the corresponding BAC clones. These PCR systems were used successfully to confirm the assignment of each marker gene (except for LDHA, see results) by analysis on the INRA hamster-bovine somatic cell hybrid panel. The difficulties observed for LDHA and VIL1 are probably due to the fact that these genes belong to large gene families and therefore suggest that they may not be the most appropriate markers for a standardisation effort. This panel of BACs is available to the scientific community and has served as a basis for the establishment of a revised standard nomenclature of bovine chromosomes.     

A genetic map of nine loci on bovine Chromosome 2

A male-specific genetic linkage map of nine loci on bovine Chromosome (Chr) 2 (BTA2) was constructed from 306 offspring belonging to six paternal halfsib families. Loci studied were the structural genes for liver/bone/kidney alkaline phosphatase (ALPL), Gardner-Rasheed feline sarcoma (v-fgr) oncogene homolog (FGR), alpha-L-fucosidase 1 (FUCA1), and fibronectin 1 (FN1), and the microsatellite loci ARO28, DU17S2, DU17S3, DU17S4, and DU17S5. Genotyping was performed by restriction fragment length polymorphism (RFLP) for structural genes and polymerase chain reaction (PCR) for the microsatellites. Two genetically independent linkage groups were identified. The order of genes in the first linkage group, L31, is (ARO28-FN1)-FGR-FUCA1-ALPL, covering a map distance of 34.1 cM between terminal markers. The second linkage group, L32, consists of DU17S2-DU17S5-DU17S4-DU17S3 and is 41.3 cM in length. Genetic linkage between FN1 and FGR confirms previous physical assignment of these genes to the same synteny group. Currently, the genetic linkage of FN1 and FGR is unique to cattle and thus localizes a site of chromosomal evolution to a 22-cM interval between the two loci.     

Consensus and comprehensive linkage maps of bovine chromosome 24

This study describes development of a consensus genetic linkage map of bovine chromosome 24 (BTA24). Eight participating laboratories contributed data for 58 unique markers including a total of 25 409 meioses. Eighteen markers, which were typed in more than one reference population, were used as potential anchors to generate a consensus framework map. The framework map contained 16 loci ordered with odds greater than 1000:1 and spanned 79.3 cM. Remaining markers were included in a comprehensive map relative to these anchors. The resulting BTA24 comprehensive map was 98.3 cM in length. Average marker intervals were 6.1 and 2.5 cM for framework and comprehensive maps, respectively. Marker order was generally consistent with previously reported BTA24 linkage maps. Only one discrepancy was found when comparing the comprehensive map with the published USDA-MARC linkage map. Integration of genetic information from different maps provides a high-resolution BTA24 linkage map.