Comparative mapping of cattle chromosome 19: cytogenetic localization of 19 BAC clones

Here we present the results of fluorescent in situ hybridization (FISH) mapping of a set of cattle BAC clones preselected for assignment on cattle chromosome 19 (BTA19). The BAC clones were anchored to human chromosome 17 (HSA17) sequences by BLASTn similarity search of cattle BAC-ends against the human genome sequence (NCBI build 33). Five blocks of homologous synteny were defined in the comparative map of BTA19 and HSA17 built with FISH data and the human genome coordinates. The positions for four evolutionary breakpoints in the bovine and human chromosomes were identified. Comparison of the FISH comparative map with previously published comparative RH, physical, and cytogenetic maps of BTA19 did not reveal major conflicts and allowed for the extension of the boundaries of homology between BTA19 and HSA17. Comparative analysis of HSA17, BTA19, and mouse chromosome 11 (MMU11) demonstrates that most likely mice retain the ancestral organization of the synteny group, and both cattle and human chromosomes underwent several major internal rearrangements after the divergence of Primates, Rodentia, and Cetartiodactyla.     

A comparative map of chicken chromosome 24 and human chromosome 11

To improve the physical and comparative map of chicken chromosome 24 (GGA24; former linkage group E49C20W21) bacterial artificial chromosome (BAC) contigs were constructed around loci previously mapped on this chromosome by linkage analysis. The BAC clones were used for both sample sequencing and BAC end sequencing. Sequence tagged site (STS) markers derived from the BAC end sequences were used for chromosome walking. In total 191 BAC clones were isolated, covering almost 30% of GGA24, and 76 STS were developed (65 STS derived from BAC end sequences and 11 STS derived within genes). The partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases, and revealed matches to 19 genes, expressed sequence tags (ESTs) and genomic clones located on human chromosome 11q22-q24 and mouse chromosome 9. Furthermore, 11 chicken orthologues of human genes located on HSA11q22-q24 were directly mapped within BAC contigs of GGA24. These results provide a better alignment of GGA24 with the corresponding regions in human and mouse and identify several intrachromosomal rearrangements between chicken and mammals.     

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.     

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

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

A BAC clone-based physical map of ovine major histocompatibility complex

An ovine bacterial artificial chromosome (BAC) library containing 190,000 BAC clones was constructed and subsequently screened to construct a BAC-based physical map for the ovine major histocompatibility complex (MHC). Two hundred thirty-three BAC clones were selected by 84 overgo probes designed on human, mouse, and swine MHC sequence homologies. Ninety-four clones were ordered by DNA fingerprinting to form contigs I, II, and III that correspond to ovine MHC class I-class III, class IIa, and class IIb. The minimum tiling paths of contigs I, II, and III are 15, 4, and 4 BAC clones, spanning approximately 1900, 400, and 300 kb, respectively. The order and orientation of most BAC clones in each contig were confirmed by BAC-end sequencing. An open gap exists between class IIa and class III. This work helps to provide a foundation for detailed study of ovine MHC genes and of evolution of MHCs in mammals.     

Genetic and physical mapping of a high recombination region on chromosome 7H(1) in barley

Approaches utilizing microlinearity between related species allow for the identification of syntenous regions and orthologous genes. Within the barley Chromosome 7H(1) is a region of high recombination flanked by molecular markers cMWG703 and MWG836. We present the constructed physical contigs linked to molecular markers across this region using bacterial artificial chromosomes (BAC) from the cultivar Morex. Barley expressed sequence tags (EST), identified by homology to rice chromosome 6 between the rice molecular markers C425A and S1434, corresponded to the barley syntenous region of Chromosome 7H(1) Bins 2–5 between molecular markers cMWG703-MWG836. Two hundred and thirteen ESTs were genetically mapped yielding 267 loci of which 101 were within the target high recombination region while 166 loci mapped elsewhere. The 101 loci were joined by 43 other genetic markers resulting in a highly saturated genetic map. In order to develop a physical map of the region, ESTs and all other molecular markers were used to identify Morex BAC clones. Seventy-four BAC contigs were formed containing 2–102 clones each with an average of 19 and a median of 13 BAC clones per contig. Comparison of the BAC contigs, generated here, with the Barley Physical Mapping Database contigs, resulted in additional overlaps and a reduction of the contig number to 56. Within cMWG703-MWG836 are 24 agriculturally important traits including the seedling spot blotch resistance locus, Rcs5. Genetic and physical analysis of this region and comparison to rice indicated an inversion distal of the Rcs5 locus. Three BAC clone contigs spanning the Rcs5 locus were identified. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of comparative physical and sequence mapping to annotate mouse chromosome 16 and human chromosome 21

Distal mouse chromosome 16 (MMU16) shares conserved linkage with human chromosome 21 (HSA21), trisomy for which causes Down syndrome (DS). A 4.5-Mb physical map extending from Cbr1 to Tmprss2 on MMU16 provides a minimal tiling path of P1 artificial chromosomes (PACs) for comparative mapping and genomic sequencing. Thirty-four expressed sequences were positioned on the mouse map, including 19 that were not physically mapped previously. This region of the mouse:human comparative map shows a high degree of evolutionary conservation of gene order and content, which differs only by insertion of one gene (in mouse) and a small inversion involving two adjacent genes. "Low-pass" (2.2x) mouse sequence from a portion of the contig was ordered and oriented along 510 kb of finished HSA21 sequence. In combination with 68 kb of unique PAC end sequence, the comparison provided confirmation of genes predicted by comparative mapping, indicated gene predictions that are likely to be incorrect, and identified three candidate genes in mouse and human that were not observed in the initial HSA21 sequence annotation. This comparative map and sequence derived from it are powerful tools for identifying genes and regulatory regions, information that will in turn provide insights into the genetic mechanisms by which trisomy 21 results in DS.     

A BAC-based physical map of the channel catfish genome

Catfish is the major aquaculture species in the United States. To enhance its genome studies involving genetic linkage and comparative mapping, a bacterial artificial chromosome (BAC) contig-based physical map of the channel catfish (Ictalurus punctatus) genome was generated using four-color fluorescence-based fingerprints. Fingerprints of 34,580 BAC clones (5.6x genome coverage) were generated for the FPC assembly of the BAC contigs. A total of 3307 contigs were assembled using a cutoff value of 1x10(-20). Each contig contains an average of 9.25 clones with an average size of 292 kb. The combined contig size for all contigs was 0.965 Gb, approximately the genome size of the channel catfish. The reliability of the contig assembly was assessed by both hybridization of gene probes to BAC clones contained in the fingerprinted assembly and validation of randomly selected contigs using overgo probes designed from BAC end sequences. The presented physical map should greatly enhance genome research in the catfish, particularly aiding in the identification of genomic regions containing genes underlying important performance traits.     

A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 ± 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa , version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.     

Genome sequence comparison between Chinese hamster ovary (CHO) DG44 cells and mouse using end sequences of CHO BAC clones based on BAC-FISH results

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology as a mammalian host cell platform for expressing genes of interest. Previously, we constructed a detailed physical chromosomal map of the CHO DG44 cell line by fluorescence in situ hybridization (FISH) imaging using 303 bacterial artificial chromosome (BAC) clones as hybridization probes (BAC-FISH). BAC-FISH results revealed that the two longest chromosomes were completely paired. However, other chromosomes featured partial deletions or rearrangements. In this study, we determined the end sequences of 303 BAC clones (BAC end sequences), which were used for BAC-FISH probes. Among 606 BAC-end sequences (BESs) (forward and reverse ends), 558 could be determined. We performed a comparison between all determined BESs and mouse genome sequences using NCBI BLAST. Among these 558 BESs, 465 showed high homology to mouse chromosomal sequences. We analyzed the locations of these BACs in chromosomes of the CHO DG44 cell line using a physical chromosomal map. From the obtained results, we investigated the regional similarities among CHO chromosomes (A–T) and mouse chromosomes (1–19 and sex) about 217 BESs (46.7% of 465 high homologous BESs). Twenty-three specific narrow regions in 13 chromosomes of the CHO DG44 cell line showed high homology to mouse chromosomes, but most of other regions did not show significant correlations with the mouse genome. These results contribute to accurate alignments of chromosomes of Chinese hamster and its genome sequence, analysis of chromosomal instability in CHO cells, and the development of target locations for gene and/or genome editing techniques.     

An ordered BAC contig map of the equine major histocompatibility complex

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.     

High-resolution comparative physical mapping of mouse Chromosome 10 in the region of homology with human Chromosome 21

Comparative mapping of human and mouse chromosomes can be used to predict locations of homologous loci between the species, provides the substrate to examine the process of chromosomal evolution, and facilitates the continuing development of mouse genetic models for human disorders. A YAC contig of the region of mouse Chromosome (Chr) 10 (MMU10) that demonstrates conserved linkage with the distal portion of human Chr 21 (HSA21) has been constructed. The contig contains all known genes mapped in both species, defines the proximal region of homology between MMU10 and HSA22, and contains the evolutionary junction between HSA21 and HSA22 on MMU10. It consists of 23 YACs and 2 PACs, and covers 3.2 Mb of MMU10. The average marker density for this region is 1 marker/69 kb. Nine of 22 expressed sequences are mapped here for the first time in mouse, and two are newly characterized expressed sequences. The contig also contains 12 simple sequence repeats (SSRs) and 16 YAC and PAC endclone markers. YAC fragmentation analysis was used to create a physical map for the proximal 2.2 Mb of the contig. Cloning of the corresponding region of HSA21 has proven difficult, and the mouse contig includes segments absent from previously described sequence ready maps of HSA21. Received: 22 July 1998 / Accepted: 13 November 1998     

Construction of a 1.2-Mb BAC/PAC contig of the porcine gene RYR1 region on SSC 6q1.2 and comparative analysis with HSA 19q13.13

We screened a porcine bacterial artificial chromosome (BAC) and a P1 derived artificial chromosome (PAC) library to construct a sequence-ready approximately 1.2-Mb BAC/PAC contig of the ryanodine receptor-1 gene (RYR1) region on porcine chromosome (SSC) 6q1.2. This genomic segment is of special interest because it harbors the locus for stress susceptibility in pigs and a putative quantitative trait locus for muscle growth. Detailed physical mapping of this gene-rich region allowed us to assign to this contig 17 porcine genes orthologous to known human chromosome 19 genes. Apart from the relatively well-characterized porcine gene RYR1, the other 16 genes represent novel chromosomal assignments and 14 genes have been cloned for the first time in pig. Comparative analysis of the porcine BAC/PAC contig with the human chromosome (HSA) 19q13.13 map revealed a completely conserved gene order of this segment between pig and human. A detailed porcine-human-mouse comparative map of this region was constructed.     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

A physical map of large segments of pig Chromosome 7q11–q14: comparative analysis with human Chromosome 6p21

The aim of this study was to establish a porcine physical map along the chromosome SSC7q by construction of BAC contigs between microsatellites Sw1409 and S0102. The SLA class II contig, located on SSC7q, was lengthened. Four major BAC contigs and 10 short contigs span a region equivalent to 800 cR measured by IMpRH7000 mapping. The BAC contigs were initiated by PCR screening with primers derived from human orthologous segments, extended by chromosome walking, and controlled and oriented by RH mapping with the two available panels, IMpRH7000Rad and IMNpRH12000Rad. The location of 43 genes was revealed by sequenced segments, either from BAC ends or PCR products from BAC clones. The 220 BAC end sequences (BES) were also used to analyze the different marks of evolution. Comparative mapping analysis between pigs and humans demonstrated that the gene organization on HSA6p21 and on SSC7p11 and q11-q14 segments was conserved during evolution, with the exception of long fragments of HSA6p12 which shuffled and spliced the SLA extended class II region. Additional punctual variations (unique gene insertion/deletion) were observed, even within conserved segments, revealing the evolutionary complexity of this region. In addition, 18 new polymorphic microsatellites have been selected in order to cover the entire SSC7p11-q14 region.     

Comparative map between chicken Chromosome 15 and human chromosomal region 12q24 and 22q11-q12

The physical and comparative map of GGA15 was improved by the construction of 9 BAC contigs around loci previously mapped on GGA15 by linkage analysis. In total, 240 BAC clones were isolated, covering 30–35% of GGA15, and 120 STS were developed (104 STS derived from BAC end sequences and 18 STS derived within genes). Seventeen chicken orthologues of human genes located on human Chr 22q11-q12 were directly mapped within BAC contigs of GGA15. Furthermore, the partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases and revealed matches to 26 genes, ESTs, and genomic clones located on HSA22q11-q12 and HSA12q24. These results provide a better alignment of GGA15 with the corresponding regions in human and mouse, and improve our knowledge of the evolution and dynamics of the vertebrate genome. GenBank Accession Numbers: The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers BZ592394-BZ592544.     

A BAC-based physical map of the apple genome

Genome-wide physical mapping is an essential step toward investigating the genetic basis of complex traits as well as pursuing genomics research of virtually all plant and animal species. We have constructed a physical map of the apple genome from a total of 74,281 BAC clones representing approximately 10.5x haploid genome equivalents. The physical map consists of 2702 contigs, and it is estimated to span approximately 927 Mb in physical length. The reliability of contig assembly was evaluated by several methods, including assembling contigs using variable stringencies, assembling contigs using fingerprints from individual libraries, checking consensus maps of contigs, and using DNA markers. Altogether, the results demonstrated that the contigs were properly assembled. The apple genome-wide BAC-based physical map represents the first draft genome sequence not only for any member of the large Rosaceae family, but also for all tree species. This map will play a critical role in advanced genomics research for apple and other tree species, including marker development in targeted chromosome regions, fine-mapping and isolation of genes/QTL, conducting comparative genomics analyses of plant chromosomes, and large-scale genomics sequencing.     

A high resolution physical and RH map of pig chromosome 6q1.2 and comparative analysis with human chromosome 19q13.1

Background

The generation of BAC/PAC contigs in targeted genome regions is a powerful method to establish high-resolution physical maps. In domestic animal species the generation of such contigs is typically initiated with the screening of libraries with probes derived from human genes that are expected to be located in the region of interest by comparative mapping. However, in many instances the available gene-derived probes are too far apart to allow the cloning of BAC/PAC contigs larger than a few hundred kb. High resolution physical mapping allows to estimate the sizes of gaps and to control the orientation of the individual sub-contigs, which helps to avoid errors during the assembly of smaller contigs into final Mb-sized contigs. The recently constructed porcine IMNpRH2 panel allowed us to use this approach for the construction of high-resolution physical maps of SSC 6q1.2.

Results

Two sequence-ready BAC/PAC contigs of the gene-rich region on porcine chromosome 6q1.2 (SSC 6q1.2) containing the RYRl gene were constructed. The two contigs spanned about 1.2 Mb and 2.0 Mb respectively. The construction of these contigs was monitored by the results provided by the mapping of 15 markers on the IMpRH_7000rad and 35 markers on the IMNpRH2_12000rad radiation hybrid panels. Analyses on the IMpRH panel allowed us to globally link and orientate preliminary smaller contigs, whereas analyses on the high resolution IMNpRH2 panel allowed us to finally identify the order of genes and markers.

Conclusions

A framework map of 523 cR₁₂₀₀₀ was established covering the whole studied region. The order of markers on the framework 1000:1 RH map was found totally consistent with the data deduced from the contig map. The kb/cR ratio was very constant in the whole region, with an average value of 6.6 kb/cR. We estimate that the size of the remaining gap between the two contigs is of about 300 kb. The integrated physical and RH map of the investigated region on SSC 6q1.2 was used for a comparative analysis with respect to the syntenic regions on HSA 19q13.1 and MMU 7 and revealed a perfectly conserved gene order across the entire studied interval.

     

SPC-P1: a pathogenicity-associated prophage of Salmonella paratyphi C

Background

The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy.

Results

The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries.

Conclusions

The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.