Cytogenetic localization of 136 genes in the horse: comparative mapping with the human genome

The aim of this study was to increase the number of type I markers on the horse cytogenetic map and to improve comparison with maps of other species, thus facilitating positional candidate cloning studies. BAC clones from two different sources were FISH mapped: homologous horse BAC clones selected from our newly extended BAC library using consensus primer sequences and heterologous goat BAC clones. We report the localization of 136 genes on the horse cytogenetic map, almost doubling the number of cytogenetically mapped genes with 48 localizations from horse BAC clones and 88 from goat BAC clones. For the first time, genes were mapped to ECA13p, ECA29, and probably ECA30. A total of 284 genes are now FISH mapped on the horse chromosomes. Comparison with the human map defines 113 conserved segments that include new homologous segments not identified by Zoo-FISH on ECA7 and ECA13p.     

Development and applications of a set of chromosome-specific cytogenetic DNA markers in potato

Reliable and easy to use techniques for chromosome identification are critical for many aspects of cytogenetic research. Unfortunately, such techniques are not available in many plant species, especially those with a large number of small chromosomes. Here we demonstrate that fluorescence in situ hybridization (FISH) signals derived from bacterial artificial chromosomes (BACs) can be used as chromosome-specific cytogenetic DNA markers for chromosome identification in potato. We screened a potato BAC library using genetically mapped restriction fragment length polymorphism markers as probes. The identified BAC clones were then labeled as probes for FISH analysis. A set of 12 chromosome-specific BAC clones were isolated and the FISH signals derived from these BAC clones serve as convenient and reliable cytological markers for potato chromosome identification. We mapped the 5S rRNA genes, the 45S rRNA genes, and a potato late blight resistance gene to three specific potato chromosomes using the chromosome-specific BAC clones. Received: 19 January 2000 / Accepted: 27 March 2000     

Chromosome localization of the 31 type I Texas bovine markers in sheep and goat chromosomes by comparative FISH-mapping and R-banding

Bovine BAC clones containing the 31 genes, referred to as the Texas markers used earlier to definitively assign the 31 bovine syntenic groups (U) to cattle chromosomes, were mapped by fluorescent in situ hybridization to sheep and goat R-banded chromosomes according to ISCNDB2000. All 31 markers were localized on homoeologous chromosomes and chromosome bands of the two species in agreement with previous localizations obtained both in cattle and river buffalo, definitively confirming chromosome homoeologies between Caprinae and Bovinae. In addition, we have extended physical maps of sheep and goat as 11 genes (HSD3B1, INHBA, CSN10, IGF2R, PIGR, MAP1B, DSC1, ELN, TNFRSF6, CGN1, IGF2) and 14 genes (SOD1, HSD3B1, CSN10, IGF2R, RB1, TG, PIGR, MAP1B, IGH@, LTF, DSC1, TNFRSF6, CGN1, IGF2) were assigned for the first time to goat and sheep chromosomes, respectively.     

Construction of a horse BAC library and cytogenetical assignment of 20 type I and type II markers

A horse BAC library was constructed with about 40, 000 clones and mean insert size of 110 kb representing a 1.5 genome equivalent coverage and a probability of finding a single sequence of 0.75. It was characterized by PCR screening of about 130 sequences of horse microsatellites and exonic gene sequences retrieved from databases. BACs containing 8 microsatellites and 12 genes were subsequently localized by fluorescent in situ hybridization (FISH) on chromosomes. Two linkage groups were newly assigned to chromosomes: LG2 to ECA3 and LG5 to ECA24, and five linkage groups were also oriented—LG3, LG4, LG5, LG8, and LG12—leaving only three groups unassigned. This work showed how this library makes an integrated map a realistic objective for the near future and how it can make comparative mapping more efficient in a search for candidate genes of interest. Received: 9 January 1998 / Accepted: 13 April 1998     

Comprehensive DNA copy number profile and BAC library construction of an Indian individual

Bacterial artificial chromosomes (BACs) are used in genomic variation studies due to their capacity to carry a large insert, their high clonal stability, low rate of chimerism and ease of manipulation. In the present study, an attempt was made to create the first genomic BAC library of an anonymous Indian male (IMBL4) consisting of 100,224 clones covering the human genome more than three times. Restriction mapping of 255 BAC clones by pulse field gel electrophoresis confirmed an average insert size of 120 kb. The library was screened by PCR using SHANK3 (SH3 and multiple ankyrin repeat domains 3) and OLFM3 (olfactomedin 3) specific primers. A selection of clones was analyzed by fluorescent in situ hybridization (FISH) and sequencing. Fine mapping of copy number variable regions by array based comparative genomic hybridization identified 467 CNVRs in the IMBL4 genome. The IMBL4 BAC library represents the first cataloged Indian genome resource for applications in basic and clinical research.     

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.     

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 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.     

FISH mapping and sequence analysis of 87 porcine BAC clones

Ninety-one bacterial artificial chromosomes (BAC) clones, selected effectively at random from our library, were used as probes for fluorescence in situ hybridization. Of these, 87 clones gave a specific signal in one, two or three different pair(s) of swine metaphase chromosomes. The ends of 35 BAC clones were sequenced in order to obtain information for comparative mapping. Fifteen of them gave useful comparative mapping information.     

Isolation, characterization and FISH assignments of horse BAC clones containing type I and II markers

In order to increase the number of markers on the horse cytogenetic map and expand the integration with the linkage map, an equine BAC library was screened for genes and for microsatellites. Eighty-nine intra-exon primers were designed from consensus gene sequences in documented species. After PCR screening, 38 clones containing identified genes were isolated and FISH mapped. These data allowed us to refine the available Zoo-FISH results, to define ten new conserved cytogenetic segments and expand two others, thus leading to the identification of a total of 26 conserved segments between horse and human. Interestingly, a new homeology segment was detected between ECA6p and HSA2q. Screening BAC clones for dinucleotide repeats led to the isolation of 33 microsatellites. Ten of the clones each contained at least a polymorphic microsatellite and one specific gene. The success of the approach in the production of integrative anchor loci and their potential use in localization and analysis of traits of interest by the candidate gene and positional cloning approach, are discussed.     

Conserved synteny of genes between chromosome 15 of Bombyx mori and a chromosome of Manduca sexta shown by five-color BAC-FISH.

The successful assignment of the existing genetic linkage groups (LGs) to individual chromosomes and the second-generation linkage map obtained by mapping a large number of bacterial artificial chromosome (BAC) contigs in the silkworm, Bombyx mori, together with public nucleotide sequence databases, offer a powerful tool for the study of synteny between karyotypes of B. mori and other lepidopteran species. Conserved synteny of genes between particular chromosomes can be identified by comparatively mapping orthologous genes of the corresponding linkage groups with the help of BAC-FISH (fluorescent in situ hybridization). This technique was established in B. mori for 2 differently labeled BAC probes simultaneously hybridized to pachytene bivalents. To achieve higher-throughput comparative mapping using BAC-FISH in Lepidoptera, we developed a protocol for five-color BAC-FISH, which allowed us to map simultaneously 6 different BAC probes to chromosome 15 in B. mori. We identified orthologs of 6 B. mori LG15 genes (RpP0, RpS8, eIF3, RpL7A, RpS23, and Hsc70) for the tobacco hornworm, Manduca sexta, and selected the ortholog-containing BAC clones from an M. sexta BAC library. All 6 M. sexta BAC clones hybridized to a single M. sexta bivalent in pachytene spermatocytes. Thus, we have confirmed the conserved synteny between the B. mori chromosome 15 and the corresponding M. sexta chromosome (hence provisionally termed chromosome 15).     

BAC FISH analysis in Allium cepa.

Onion (Allium cepa L.; 1C=15,000 Mb) is an agriculturally important plant. The genome of onion has been extensively studied at the conventional cytogenetic level, but molecular analyses have lagged behind due to its large genome size. To overcome this bottleneck, a partial bacterial artificial chromosome (BAC) library of onion was constructed. The average insert size of the BAC library was about 100 kb. A total of 48,000 clones, corresponding to 0.32 genome equivalent, were obtained. Fluorescent in situ hybridization (FISH) screening resulted in identification of BAC clones localized on centromeric, telomeric, or several limited interstitial chromosomal regions, although most of the clones hybridized with entire chromosomes. The partial BAC library proved to be a useful resource for molecular cytogenetic studies of onion, and should be useful for further mapping and sequencing studies of important genes of this plant. BAC FISH screening is a powerful method for identification of molecular cytogenetic markers in large-genome plants.     

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.     

Construction of a medium-density horse gene map

A medium-density map of the horse genome (Equus caballus) was constructed using genes evenly distributed over the human genome. Three hundred and twenty-three exonic primer pairs were used to screen the INRA and the CHORI-241 equine BAC libraries by polymerase chain reaction and by filter hybridization respectively. Two hundred and thirty-seven BACs containing equine gene orthologues, confirmed by sequencing, were isolated. The BACs were localized to horse chromosomes by fluorescent in situ hybridization (FISH). Overall, 165 genes were assigned to the equine genomic map by radiation hybrid (RH) (using an equine RH(5000) panel) and/or by FISH mapping. A comparison of localizations of 713 genes mapped on the horse genome and on the human genome revealed 59 homologous segments and 131 conserved segments. Two of these homologies (ECA27/HSA8 and ECA12p/HSA11p) had not been previously identified. An enhanced resolution of conserved and rearranged chromosomal segments presented in this study provides clarification of chromosome evolution history.     

Conservation of avian Z chromosomes as revealed by comparative mapping of the Z-linked aldolase B gene

A chicken Z-linked BAC probe containing the aldolase B gene was used for fluorescence in-situ hybridization (FISH) mapping in four different avian species. The biotinylated BAC clone showed distinct unique hybridization sites on the structurally different Z chromosomes. This result, together with previous data, lends credence to the notion that, despite undergoing structural rearrangements, the gene content of the avian Z chromosome remained conserved during evolution. Our study also demonstrates the feasibility of using large genomic clones for comparative mapping of Z-linked genes in birds.     

Adhesion of ectomycorrhizal bacteria to plant cells: an in vitro evidence

Defensins are a family of host defence peptides that play an important role in the innate immunity of mammalian and avian species. In humans, four beta-defensins have been isolated so far, corresponding to the products of the genes DEFB1 (h-BD1, GenBank accession number NM_005218); DEFB4 (h-Bd2, NM_004942.2), DEFB103 (h-BD3, NM_018661); and DEFB104 (hBD4, NM_080389) mapping on chromosome 8p23.22. We have localized beta-defensin genes on metaphasic chromosomes of great apes and several non-human primate species to determine their physical mapping. Using fluorescent in situ hybridization and BAC probes containing the four beta-defensin genes, we have mapped the homologous regions to the beta-defensin genes on chromosome 8p23-p.22 in non-human primates, while no signals were detected on prosimians chromosomes.     

Assignment of zebrafish genetic linkage groups to chromosomes

In this report the zebrafish genetic linkage groups are assigned to specific chromosomes using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group (LG). Chromosomes were identified using a combination of relative size and arm ratios. The largest genetic maps generally corresponded to the largest chromosomes, but genetic recombination tended to be elevated in the smaller chromosomes and near telomeres. Large insert clones containing genes near telomeres often hybridized to telomeres of multiple chromosome pairs, suggesting the presence of shared subtelomeric repetitive DNAs near telomeres. Evidence from comparative gene mapping in medaka, zebrafish, pufferfish, and humans suggests that the linkage groups of these species have the content of duplicate proto-chromosomes. However, these duplicate linkage groups are not associated with chromosomes of similar size or morphology. This suggests that considerable chromosome restructuring occurred subsequent to the genome duplication in teleosts.     

Assignment of rainbow trout linkage groups to specific chromosomes

The rainbow trout genetic linkage groups have been assigned to specific chromosomes in the OSU (2N=60) strain using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group. There was a rough correlation between chromosome size and size of the genetic linkage map in centimorgans for the genetic maps based on recombination from the female parent. Chromosome size and structure have a major impact on the female:male recombination ratio, which is much higher (up to 10:1 near the centromeres) on the larger metacentric chromosomes compared to smaller acrocentric chromosomes. Eighty percent of the BAC clones containing duplicate genes mapped to a single chromosomal location, suggesting that diploidization resulted in substantial divergence of intergenic regions. The BAC clones that hybridized to both duplicate loci were usually located in the distal portion of the chromosome. Duplicate genes were almost always found at a similar location on the chromosome arm of two different chromosome pairs, suggesting that most of the chromosome rearrangements following tetraploidization were centric fusions and did not involve homeologous chromosomes. The set of BACs compiled for this research will be especially useful in construction of genome maps and identification of QTL for important traits in other salmonid fishes.     

Fluorescent in situ hybridization to ascidian chromosomes

The draft genome of the ascidian Ciona intestinalis has been sequenced. Mapping of the genome sequence to the Ciona 14 haploid chromosomes is essential for future studies of the genome-wide control of gene expression in this basal chordate. Here we describe an efficient protocol for fluorescent in situ hybridization for mapping genes to the Ciona chromosomes. We demonstrate how the locations of two BAC clones can be mapped relative to each other. We also show that this method is efficient for coupling two so-far independent scaffolds into one longer scaffold when two BAC clones represent sequences located at either end of the two scaffolds.     

Equine FISH mapping of 36 genes known to locate on human chromosome ends

The INRA and the CHORI-241 horse BAC libraries were screened by hybridization with DNA probes and/or directly by PCR with primers designed in consensus sequences of genes localized at the end of each human chromosome. BAC clones were retrieved and 36 could be FISH mapped after the expected gene was confirmed in each BAC by sequencing. Our results show that 16 BACs can be considered to be at telomeric or centromeric positions in the horse and 15 were found at the boundary of actually defined conserved segments even-though often located within conserved syntenic fragments between horse and human. There is no straightforward relation between the end position of a marker in human and its end position in the horse. A gene was first anchored to ECA27 by FISH mapping. The localization of these markers expands the cytogenetic map of the horse and will serve as anchors for the integrated and future physical maps. It should also help to better understand the different chromosomal rearrangements that occurred during evolution of genomes derived from a common ancestral karyotype.