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.     

High-resolution human/goat comparative map of the goat polled/intersex syndrome (PIS): the human homologue is contained in a human YAC from HSA3q23

The genetic and cytogenetic map around the chromosome 1 region shown to be linked with polledness and intersexuality (PIS) in the domestic goat (Capra hircus) was refined. For this purpose, a goat BAC library was systematically screened with primers from human coding sequences, scraped chromosome 1 DNA, bovine microsatellites from the region, and BAC ends. All the BACs (n = 30) were mapped by fluorescence in situ hybridization (FISH) on goat chromosome 1q41-q45. The genetic mapping of 30 new goat polymorphic markers, isolated from these BACs, made it possible to reduce the PIS interval to a region of less than 1 cM on goat chromosome 1q43. The PIS locus is now located between the two genes ATP1B and COP, which both map to 3q23 in humans. Genetic, cytogenetic, and comparative data suggest that the PIS region is now probably circumscribed to an approximately 1-Mb DNA segment for which construction of a BAC contig is in progress. In addition, a human YAC contig encompassing the blepharophimosis-ptosis-epicanthus-inversus region was mapped by FISH to goat chromosome 1q43. This human disease, mapped to HSA 3q23 and affecting the development and maintenance of ovarian function, could be a potential candidate for goat PIS.     

Refined genetic and comparative physical mapping of the canine copper toxicosis locus

Recently, the copper toxicosis (CT) locus in Bedlington terriers was assigned to canine chromosome region CFA10q26, which is homologous to human chromosome region HSA2p13-21. A comparative map between CFA10q21-26 and HSA2p13-21 was constructed by using genes already localized to HSA2p13-21. A high-resolution radiation map of CFA10q21-26 was constructed to facilitate positional cloning of the CT gene. For this map, seven Type I and eleven Type II markers were mapped. Using homozygosity mapping, the CT locus could be confined to a 42.3 cR₃₀₀₀ region, between the FH2523 and C10.602 markers. On the basis of a partial BAC contig, it was estimated that 1-cR₃₀₀₀ is equivalent to approximately 210 kb, implying that the CT candidate region is therefore estimated to be about 9 Mb. Received: 16 December 1999 / Accepted: 23 February 2000     

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.     

Improvement of the comparative map of chicken chromosome 13

A comparative map was made of chicken chromosome 13 (GGA13) with a part of human chromosome 5 (HSA5). Microsatellite markers specific for GGA13 were used to screen the Wageningen chicken bacterial artificial chromosome (BAC) library. Selected BAC clones were end sequenced and 57 sequence tag site (STS) markers were designed for contig building. In total, 204 BAC clones were identified which resulted in a coverage of about 20% of GGA13. Identification of genes was performed by a bi-directional approach. The first approach starting with sequencing mapped chicken BAC subclones, where sequences were used to identify orthologous genes in human and mouse by a basic local alignment search tool (BLAST) database search. The second approach started with the identification of chicken orthologues of human genes in the HSA5q23-35 region. The chicken orthologous genes were subsequently mapped by fluorescent in situ hybridisation (FISH) and/or single neucleotide polymorphism typing. The total number of genes mapped on GGA13 is increased from 14 to a total of 20 genes. Genes mapped on GGA13 have their orthologues on HSA5q23-5q35 in human and on Mmu11, Mmu13 and Mmu18 in mouse.     

A physical map of the 20q12-q13.1 region associated with type 2 diabetes

Several recent genetic studies have suggested linkage of Type 2 diabetes (non-insulin-dependent diabetes mellitus) susceptibility to a region of chromosome 20q12-q13.1. To facilitate the identification and cloning of a diabetes susceptibility gene(s) in this region, we have constructed correlated radiation hybrid and YAC/BAC contig physical maps of the region. A high-resolution radiation hybrid map encompassing 9.5 Mb between the PLC and the CEBPB genes was constructed using 68 markers: 25 polymorphic markers, 15 known genes, 21 ESTs, and 7 random genomic sequences. The physical order of the polymorphic markers within this radiation hybrid map is consistent with published genetic maps. A YAC/BAC contig that gives continuous coverage between PLC and CEBPB was also constructed. This contig was constructed from 24 YACs, 34 BACs, and 1 P1 phage clone onto which 71 markers were mapped: 23 polymorphic markers, 12 genes, 24 ESTs, and 12 random genomic sequences. The radiation hybrid map and YAC/BAC physical map enable precise mapping of newly identified transcribed sequences and polymorphic markers that will aid in linkage and linkage disequilibrium studies and facilitate identification and cloning of candidate Type 2 diabetes susceptibility genes residing in 20q12-q13.1.     

A fine physical map of the rice chromosome 5

A fine physical map of the rice (Oryza sativa spp. Japonica var. Nipponbare) chromosome 5 with bacterial artificial chromosome (BAC) and PI-derived artificial chromosome (PAC) clones was constructed through integration of 280 sequenced BAC/PAC clones and 232 sequence tagged site/expressed sequence tag markers with the use of fingerprinted contig data of the Nipponbare genome. This map consists of five contigs covering 99% of the estimated chromosome size (30.08 Mb). The four physical gaps were estimated at 30 and 20 kb for gaps 1–3 and gap 4, respectively. We have submitted 42.2-Mb sequences with 29.8 Mb of nonoverlapping sequences to public databases. BAC clones corresponding to telomere and centromere regions were confirmed by BAC-fluorescence in situ hybridization (FISH) on a pachytene chromosome. The genetically centromeric region at 54.6 cM was covered by a minimum tiling path spanning 2.1 Mb with no physical gaps. The precise position of the centromere was revealed by using three overlapping BAC/PACs for ~150 kb. In addition, FISH results revealed uneven chromatin condensation around the centromeric region at the pachytene stage. This map is of use for positional cloning and further characterization of the rice functional genomics. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Chia-Hsiung Cheng and Mei-Chu Chung have equal contributions.     

Construction of a 550 kb BAC contig spanning the genomic region containing the apple scab resistance gene Vf

A positional cloning project was started in apple with the aim of isolating the Vf resistance gene of Malus floribunda 821. Vf confers resistance against apple scab, the most important disease in apple orchards. A chromosome walk starting from two molecular markers (M18-CAPS and AM19-SCAR) flanking Vf was performed, using a bacterial artificial chromosome (BAC) library containing inserts of the cultivar Florina, which is heterozygous for Vf. Thirteen BAC clones spanning the region between the two markers were identified in nine chromosome walking steps. The size of the resulting contig is approximately 550 kb. In order to map the Vf region in more detail, we analyzed over 2000 plants from different populations segregating for Vf with markers produced from BAC end sequences. In this way, we were able to restrict the possible location of the Vf gene to a minimum of five clones spanning an interval of approximately 350 kb. Received: 4 July 1999 / Accepted: 16 September 1999     

A sequence-ready map of the human chromosome 1q telomere

A 260-kb half-YAC clone derived from human chromosome 1q was mapped at high resolution using cosmid subclone fingerprint analysis and was integrated with overlapping clones from the telomeric end of a separately derived 1q44 BAC contig to create a sequence-ready map extending to the molecular telomere of 1q. Analysis of 100 kb of sample sequences from across the 260-kb region encompassed by the half-YAC revealed the presence of EST sequence matches corresponding to 12 separate Unigene clusters and to 12 separate unclustered EST sequences. Low-copy subtelomeric repeats typical of many human telomere regions are present within the distal-most 30 kb of 1q. The previously isolated and radiation hybrid-mapped markers Bda84F03, 1QTEL019, and WI11861 localized at distances approximately 32, 88, and 99 kb, respectively, from the 1q terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 1q and will enable telomeric closure of the human chromosome 1q DNA reference sequence by connecting the molecular 1q telomere to an internal BAC contig.     

A high utility integrated map of the pig genome

Background

The domestic pig is being increasingly exploited as a system for modeling human disease. It also has substantial economic importance for meat-based protein production. Physical clone maps have underpinned large-scale genomic sequencing and enabled focused cloning efforts for many genomes. Comparative genetic maps indicate that there is more structural similarity between pig and human than, for example, mouse and human, and we have used this close relationship between human and pig as a way of facilitating map construction.

Results

Here we report the construction of the most highly continuous bacterial artificial chromosome (BAC) map of any mammalian genome, for the pig (Sus scrofa domestica) genome. The map provides a template for the generation and assembly of high-quality anchored sequence across the genome. The physical map integrates previous landmark maps with restriction fingerprints and BAC end sequences from over 260,000 BACs derived from 4 BAC libraries and takes advantage of alignments to the human genome to improve the continuity and local ordering of the clone contigs. We estimate that over 98% of the euchromatin of the 18 pig autosomes and the X chromosome along with localized coverage on Y is represented in 172 contigs, with chromosome 13 (218 Mb) represented by a single contig. The map is accessible through pre-Ensembl, where links to marker and sequence data can be found.

Conclusion

The map will enable immediate electronic positional cloning of genes, benefiting the pig research community and further facilitating use of the pig as an alternative animal model for human disease. The clone map and BAC end sequence data can also help to support the assembly of maps and genome sequences of other artiodactyls.     

A sequence-ready BAC/PAC contig and partial transcript map of approximately 1.5 Mb in human chromosome 17q25 comprising multiple disease genes

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy mapped to a 4-cM interval on chromosome 17q25 between the short tandem repeat (STR) markers D17S1603 and D17S802. Chromosome 17q25 in general and the 4-cM HNA region in particular are also implicated in the pathogenesis of a number of tumors (tylosis with esophageal cancer, sporadic breast and ovarian tumors) and harbor a psoriasis susceptibility locus. Initial attempts to construct a yeast artificial chromosome contig failed. Therefore, we have now constructed a complete P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) contig of the region flanked by the STR markers D17S1603 and D17S802. The contig contains 22 PAC and 64 BAC clones and covers a physical distance of approximately 1. 5 Mb. A total of 83 sequence-tagged site (STS) markers (10 known STSs and STRs, 56 STSs generated from clone end-fragments, 12 expressed sequence tags, and 5 known genes) were mapped on the contig, resulting in an extremely dense physical map with approximately 1 STS per 20 kb. This sequence-ready PAC and BAC contig will be pivotal for the positional cloning of the HNA gene as well as other disease genes mapping to this region.     

Construction of a BAC Contig for a 3 cM Biologically Significant Region of Mouse Chromosome 1

One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.     

Genetic analysis of jumbled spine and ribs (Jsr) mutation affecting the vertebral development in mice

The jumbled spine and ribs (Jsr) mouse was derived from a spontaneous mutation. As the phenotype, a shortened trunk and kinky tail are characteristic Jsr traits. In this study, on high resolution mapping it was found that Lunatic fringe (Lfng) mapped at the same position as Jsr. Lfng was identified as the candidate gene for Jsr, but sequence analysis of this gene revealed no substitution in the coding region of cDNA. Therefore, we adopted the strategy of positional cloning for Jsr using a mouse bacterial artificial chromosome (BAC) library. A BAC contig was constructed from three BAC clones showing positive signals of Lfng and 11MMHAP75FRD8.seq near the Jsr locus on chromosome 5. Based on the genetic mapping of both T7 and sp6 ends of a clone of BAC382-O-7 (BAC382), the Jsr gene was considered to exist in BAC382 and to be positioned near the sp6 side.     

A clone contig of 12q24.3 encompassing the distal hereditary motor neuropathy type II gene

We previously assigned the disease locus for autosomal dominant hereditary motor neuropathy type II (distal HMN II) within a 13-cM interval at chromosome 12q24.3. We constructed a physical map of the distal HMN II region based on yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) using an STS content mapping approach. The contig contains 26 YAC, 15 PAC, and 60 BAC clones and covers a physical distance of approximately 5 Mb. A total of 99 STS markers, including 25 known STSs and STRs, 49 new STSs generated from clone end-fragments, 20 ESTs, and 5 known genes, were located on the contig. This physical map provides a valuable resource for mapping genes and markers located within the distal HMN II region and facilitates the positional cloning of the distal HMN II gene.