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.     

Physical mapping of the major histocompatibility complex class II and class III regions of the rat

A contig of overlapping bacterial and P1-derived artificial chromosome (BAC, PAC) clones derived from the inbred rat strain BN was constructed that encompasses the class II and the class III regions of the rat MHC (RT1 complex). The genomic structure of the rat, human, and mouse class II and class III regions is highly similar. However, different from human and mouse, a copy of the C4, Cyp21, and Stk19 genes is found that maps to the class II region in the rat. Gene trees constructed from human, rat, and mouse C4, Cyp21, and Stk19 sequences show species-specific clustering of the duplicated genes. The class II/III contig reported here links two previously published PAC contigs of the BN rat that contain the centromeric and the telomeric class I regions, RT1-A and RT1-C/E/M, respectively. Thus, the MHC of the rat is now completely mapped in a single contig of BAC/PAC clones derived from a single RT1 haplotype and encompasses about 3.7 Mb.     

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.     

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.     

The feline major histocompatibility complex is rearranged by an inversion with a breakpoint in the distal class I region

In order to determine the genomic organization of the major histocompatibility complex (MHC) of the domestic cat (Felis catus), DNA probes for 61 markers were designed from human MHC reference sequences and used to construct feline MHC BAC contig map spanning ARE1 in the class II region to the olfactory receptor complex in the extended class I region. Selected BAC clones were then used to identify feline-specific probes for the three regions of the mammalian MHC (class II–class III–class I) for radiation hybrid mapping and fluorescent in situ hybridization to refine the organization of the domestic cat MHC. The results not only confirmed that the p-arm of domestic cat B2 is inverted relative to human Chromosome 6, but also demonstrated that one inversion breakpoint localized to the distal segment of the MHC class I between TRIM39 and TRIM26. The inversion thus disjoined the ~2.85 Mb of MHC containing class II–class III–class I (proximal region) from the ~0.50 Mb of MHC class I/extended class I region, such that TRIM39 is adjacent to the Chromosome B2 centromere and TRIM26 is adjacent to the B2 telomere in the domestic cat.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Closing a gap in the physical map of the ovine major histocompatibility complex

A 184 kb gap in an ovine MHC physical map was successfully closed by identification of two overlapping clones (304C7 and 222G18) from a Chinese fine wool merino sheep BAC library. The location and tiling path of the two clones were confirmed by BAC‐end sequencing and PCR amplification of loci in overlapping regions. Full‐length sequencing of the clones identified 13 novel ovine genes in the gap between loci Notch4 and Btnl2, and eight of them belonging to the Butyrophilin‐like (Btn‐like or Btnl) gene family. The scattered distribution of the Btnl gene cluster at the gap provided a clue to explain the difficulties previously experienced in closing the gap. Completed BAC contigs of the ovine MHC will facilitate sequencing of the entire ovine leukocyte antigen (OLA) region, providing detailed information for comparative studies of MHC evolution.     

A BAC-based physical map of Zhikong scallop (Chlamys farreri Jones et Preston)

Zhikong scallop (Chlamys farreri) is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (～5.8× genome coverage) fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs) and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization), contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs.     

Homology-driven assembly of a sequence-ready mouse BAC contig map spanning regions related to the 46-Mb gene-rich euchromatic segments of human chromosome 19

Draft sequence derived from the 46-Mb gene-rich euchromatic portion of human chromosome 19 (HSA19) was utilized to generate a sequence-ready physical map spanning homologous regions of mouse chromosomes. Sequence similarity searches with the human sequence identified more than 1000 individual orthologous mouse genes from which 382 overgo probes were developed for hybridization. Using human gene order and spacing as a model, these probes were used to isolate and assemble bacterial artificial chromosome (BAC) clone contigs spanning homologous mouse regions. Each contig was verified, extended, and joined to neighboring contigs by restriction enzyme fingerprinting analysis. Approximately 3000 mouse BACs were analyzed and assembled into 44 contigs with a combined length of 41.4 Mb. These BAC contigs, covering 90% of HSA19-related mouse DNA, are distributed throughout 15 homology segments derived from different regions of mouse chromosomes 7, 8, 9, 10, and 17. The alignment of the HSA19 map with the ordered mouse BAC contigs revealed a number of structural differences in several overtly conserved homologous regions and more precisely defined the borders of the known regions of HSA19-syntenic homology. Our results demonstrate that given a human draft sequence, BAC contig maps can be constructed quickly for comparative sequencing without the need for preestablished mouse-specific genetic or physical markers and indicate that similar strategies can be applied with equal success to genomes of other vertebrate species.     

Physical and genetic mapping of the rainbow trout major histocompatibility regions: evidence for duplication of the class I region

One of the most unexpected discoveries in MHC genetics came from studies dealing with the teleost MHC. Initially discovered in zebrafish, the MHC class I and II regions of all bony fish are not linked. Previous segregation analysis in trout suggested that the class I and II regions reside on completely different chromosomes. To learn more about MHC genomics in trout, we have isolated BAC clones harboring class Ia and Ib loci, a single BAC clone containing an MH class II gene ( DAB), as well as BAC clones containing the ABCB2 gene. Upon PCR and sequence confirmation, BAC clones were labeled and used as probes for in situ hybridization on rainbow trout metaphase chromosomes for determination of the physical locations of the trout MH regions. Finally, SNPs, RFLPs, and microsatellites found within the BAC clones allowed for these regions to be assigned to specific linkage groups on the OSU x Hotcreek (HC) and OSU x Arlee (ARL) genetic linkage maps. Our data demonstrate that the trout MH regions are located on at least four different chromosomes and the corresponding linkage groups, while also providing direct evidence for the partial duplication of the MH class I region in trout.     

Physical localization and order of genes in the class I region of the bovine MHC

Fluorescence in situ hybridization (FISH) analyses were used to order 16 bacterial artificial chromosomes (BAC) clones containing loci from the bovine lymphocyte antigen (BoLA) class I and III regions of bovine chromosome 23 (BTA23). Fourteen of these BACs were assigned to chromosomal band locations of mitotic and pachytene chromosomes by single- and dual-colour FISH. Dual-colour FISH confirmed that class II DYA is proximal to and separated from BoLA class I genes by approximately three chromosome bands. The FISH results showed that tumour necrosis factor alpha (TNFA), heat shock protein 70 (HSP70.1) and 21 steroid dehydrogenase (CYP21) are closely linked in the region of BTA23 band 22 along with BoLA class I genes, and that male enhanced antigen (MEA) mapped between DYA and the CYP21/TNFA/HSP70.1 gene region. All BAC clones containing BoLA class I genes mapped distal to CYP21/TNFA/HSP70.1 and centromeric to prolactin (PRL). Myelin oligodendrocyte glycoprotein (MOG) was shown to be imbedded within the BoLA class I gene cluster. The cytogenetic data confirmed that the disrupted distribution of BoLA genes is most likely the result of a single large chromosomal inversion. Similar FISH results were obtained when BoLA DYA and class I BAC clones were mapped to discrete chromosomal locations on the BTA homologue in white-tailed deer, suggesting that this chromosomal inversion predates divergence of the advanced ruminant families from a common ancestor.     

Evidence that the separation of Mhc class II from class I loci in the zebrafish,Danio rerio,occurred by translocation

In the zebrafish, Danio rerio, and other teleosts, the class I and class II loci of the major histocompatibility complex ( Mhc) reside on different chromosomes. To shed light on the events that might have generated this difference from tetrapods, in which these two types of loci are clustered in a single chromosomal region, the organization of the class II loci in linkage group 8 of the zebrafish was determined by the characterization of contigs of PAC clones. Three contigs were defined: DAB, DCB, and DBB. The 350-kb-long DAB contig contained only four genes: DDB, DAB, SLC7A4, and DAA. The 150-kb-long DCB contig contained the DCB, DCA, and fz10 genes at an undetermined distance from the DAB contig. And the 120-kb-long DBB contig comprised the DBB gene presumably in another linkage group. The low gene density of the linkage group 8 contigs, contrasting with the high gene density of the zebrafish class I region, and the close association with genes [ SLC7A4 coding for an amino acid transporter, and fz10 (frizzled 10) coding for a receptor of the WNT glycoprotein] that are not linked with the tetrapod Mhc, is interpreted to mean that the separation of the class II from class I loci in teleosts occurred by translocation rather than by genomic or chromosomal duplication.     

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.     

Characterization of the turkey MHC chromosome through genetic and physical mapping

Previous studies in the chicken have identified a single microchromosome (GGA16) containing the ribosomal DNA (rDNA) and two genetically unlinked MHC regions, MHC-B and MHC-Y. Chicken DNA sequence from these loci was used to develop PCR primers for amplification of homologous fragments from the turkey (Meleagris gallopavo). PCR products were sequenced and overgo probes were designed to screen the CHORI 260 turkey BAC library. BAC clones corresponding to the turkey rDNA, MHC-B and MHC-Y were identified. BAC end and subclone sequencing confirmed identity and homology of the turkey BAC clones to the respective chicken loci. Based on subclone sequences, single-nucleotide polymorphisms (SNPs) segregating within the UMN/NTBF mapping population were identified and genotyped. Analysis of SNP genotypes found the B and Y to be genetically unlinked in the turkey. Silver staining of metaphase chromosomes identified a single pair of microchromosomes with nucleolar organizer regions (NORs). Physical locations of the rDNA and MHC loci were determined by fluorescence in situ hybridization (FISH) of the BAC clones to metaphase chromosomes. FISH clearly positioned the rDNA distal to the Y locus on the q-arm of the MHC chromosome and the MHC-B on the p-arm. An internal telomere array on the MHC chromosome separates the B and Y loci.     

A bacterial artificial chromosome based physical map of the Ustilago maydis genome.

Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and co-digested with HaeIII to reduce fragments to 50-750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P < or = 1 x 10(-6) to 1 x 10(-24)). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P < or = 1 x 10(-7) to 1 x 10 (-12). The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P < or = 1 x 10(-7). The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18-20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.     

FISH physical mapping with barley BAC clones

Fluorescence in situ hybridization (FISH) is a useful technique for physical mapping of genes, markers, and other single- or low-copy sequences. Since clones containing less than 10 kb of single-copy DNA do not reliably produce detectable signals with current FISH techniques in plants, a bacterial artificial chromosome (BAC) partial library of barley was constructed and a FISH protocol for detecting unique sequences in barley BAC clones was developed. The library has a 95 kb average barley insert, representing about 20% of a barley genome. Two BAC clones containing hordein gene sequences were identified and partially characterized. FISH using these two BAC clones as probes showed specific hybridization signals near the end of the short arm of one pair of chromosomes. Restriction digests of these two BAC clones were compared with restriction patterns of genomic DNA; all fragments contained in the BAC clones corresponded to bands present in the genomic DNA, and the two BAC clones were not identical. The barley inserts contained in these two BAC clones were faithful copies of the genomic DNA. FISH with four BAC clones with inserts varying from 20 to 150 kb, showed distinct signals on paired chromatids. Physical mapping of single- or low-copy sequences in BAC clones by FISH will help to correlate the genetic and physical maps. FISH with BAC clones also provide an additional approach for saturating regions of interest with markers and for constructing contigs spanning those regions.     

A physical map of the polytenized region (101EF-102F) of chromosome 4 in Drosophila melanogaster

Chromosome 4, the smallest autosome ( approximately 5 Mb in length) in Drosophila melanogaster contains two major regions. The centromeric domain ( approximately 4 Mb) is heterochromatic and consists primarily of short, satellite repeats. The remaining approximately 1.2 Mb, which constitutes the banded region (101E-102F) on salivary gland polytene chromosomes and contains the identified genes, is the region mapped in this study. Chromosome walking was hindered by the abundance of moderately repeated sequences dispersed along the chromosome, so we used many entry points to recover overlapping cosmid and BAC clones. In situ hybridization of probes from the two ends of the map to polytene chromosomes confirmed that the cloned region had spanned the 101E-102F interval. Our BAC clones comprised three contigs; one gap was positioned distally in 102EF and the other was located proximally at 102B. Twenty-three genes, representing about half of our revised estimate of the total number of genes on chromosome 4, were positioned on the BAC contigs. A minimal tiling set of the clones we have mapped will facilitate both the assembly of the DNA sequence of the chromosome and a functional analysis of its genes.     

Cytogenetic localization of 44 new coding sequences in the horse

The purpose of this study was to increase the number of genes assigned by in situ hybridization to equine chromosomes and thus the number of links for comparative mapping with other species. Forty-four new sequences were added to the horse cytogenetic map by FISH mapping of BAC clones containing genes (35) or ESTs (9). Three approaches were developed: use of horse BAC clones screened with (i) horse EST primers, (ii) interspecific consensus intraexonic primers, and (iii) use of goat BAC containing genes previously localized on goat chromosomes. Present data suggest that the second approach is the most promising. A total of 46 segments containing one or several genes could be compared, among which 40 loci could be included in 16 synteny groups between human and horse, displaying one ordered segment and several breaking points along chromosomes. All single BAC localizations confirm the most recent mapping data. Twenty-six out of 31 chromosomes now contain a gene mapped by in situ hybridization, and 14 new arm-to-arm segment homologies were revealed. Received: 2 May 2000 / Accepted: 27 July 2000     

The complete map of the Ig heavy chain constant gene region reveals evidence for seven IgG isotypes and for IgD in the horse

This report contains the first map of the complete Ig H chain constant (IGHC) gene region of the horse (Equus caballus), represented by 34 overlapping clones from a new bacterial artificial chromosome library. The different bacterial artificial chromosome inserts containing IGHC genes were identified and arranged by hybridization using overgo probes specific for individual equine IGHC genes. The analysis of these IGHC clones identified two previously undetected IGHC genes of the horse. The newly found IGHG7 gene, which has a high homology to the equine IGHG4 gene, is located between the IGHG3 and IGHG4 genes. The high degree of conservation shared between the nucleotide sequences of the IGHG7 and IGHG4 genes is unusual for the IGHG genes of the horse and suggests that these two genes duplicated most recently during evolution of the equine IGHG genes. Second, we present the genomic nucleotide sequence of the equine IGHD gene, which is located downstream of the IGHM gene. Both the IGHG7 and IGHD genes were found to be expressed at the mRNA level. The order of the 11 IGHC genes in the IGH-locus of the horse was determined to be 5'-M-D-G1-G2-G3-G7-G4-G6-G5-E-A-3', confirming previous studies using lambda phage clones, with the exception that the IGHG5 gene was found to be the most downstream-located IGHG gene. Fluorescence in situ hybridization was used to localize the IGHC region to Equus caballus (ECA) 24qter, the horse chromosome corresponding to human chromosome 14, where the human IGH locus is found.     

A segment of the apospory-specific genomic region is highly microsyntenic not only between the apomicts Pennisetum squamulatum and buffelgrass, but also with a rice chromosome 11 centromeric-proximal genomic region

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.