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     

Construction, characterization and FISH mapping of a bacterial artificial chromosome library of Chinese pangolin (Manis pentadactyla)

Chinese pangolins as a representative species in the order Pholidota have highly specified morphological characters and occupy an important place in the mammalian phylogenetic tree. To obtain genomic data for this species, we have constructed a bacterial artificial chromosome (BAC) library of Chinese pangolin. The library contains 208,272 clones with an average insert size of 122.1 kb and represents approximately eight times the Chinese pangolin haploid genome (if we assume that the Chinese pangolins have a genome size similar to human). One hundred and twenty randomly-selected BAC clones were mapped onto Chinese pangolin chromosomes by fluorescence in situ hybridization (FISH), showing a largely unbiased chromosomal distribution. Several clones containing repetitive DNA and ribosomal DNA genes were also found. The BAC library and FISH mapped BAC clones are useful resources for comparative genomics and cytogenetics of mammals and in particular, the ongoing genome sequencing project of Chinese pangolins.     

Construction of a bacterial artificial chromosome (BAC) library for potato molecular cytogenetics research.

Lack of reliable techniques for chromosome identification is the major obstacle for cytogenetics research in plant species with large numbers of small chromosomes. To promote molecular cytogenetics research of potato (Solanum tuberosum, 2n = 4x = 48) we developed a bacterial artificial chromosome (BAC) library of a diploid potato species S. bulbocastanum. The library consists of 23,808 clones with an average insert size of 155 kb, and represents approximately 3.7 equivalents to the potato genome. The majority of the clones in the BAC library generated distinct signals on specific potato chromosomes using fluorescence in situ hybridization (FISH). The hybridization signals provide excellent cytological markers to tag individual potato chromosomes. We also demonstrated that the BAC clones can be mapped to specific positions on meiotic pachytene chromosomes. The excellent resolution of pachytene FISH can be used to construct a physical map of potato by mapping molecular marker-targeted BAC clones on pachytene chromosomes.     

Development of 50 gene-associated microsatellite markers using BAC clones and the construction of a linkage map of swine chromosome 4

The development of informative polymorphic markers is essential for QTL mapping. We developed 50 microsatellite markers from BAC clones containing genes that were predicted to map swine chromosome 4 (SSC4) according to comparative analysis between human and swine chromosomes, and constructed a linkage map that consisted of 37 markers including 24 markers closely linked to genes in BAC clones. Microsatellite markers were developed by direct-sequencing of BAC clones and our results demonstrated that this method was effective for developing microsatellite markers in specific regions on chromosomes. Effective development of microsatellite markers closely linked to genes can further accelerate the comparative studies of chromosomes between different species.     

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.     

The structure of a conserved region of porcine genome, represented in human genome by chromosome 17

Radiation mapping of nine genes (H3F3B, HLR1, MYL4, STAT5B, THRA1, TOP2A, MCP1, NF1, and MPO) to porcine chromosome 12 was carried out. Also, subchromosomal location of the NF1 gene along with the two loci containing the DNA sequences homologous to the DNA of the two human BAC clones was determined. The NF1 position was ascertained via microdissection of chromosome 12 with subsequent PCR amplification of the gene fragment with specific primers. BAC clones were mapped using FISH. Comparative analysis of the gene order in porcine chromosome 12 and in the homologous human chromosome 17 was performed. It was demonstrated that the gene orders in these chromosomes differed relative to the position of the MPO gene.     

Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines.     

Fourteen chromosomal localizations and an update of the cytogenetic map of the rabbit

In order to improve the informativeness of the cytogenetic map of the rabbit genome, fourteen markers were regionally mapped to individual chromosomes. The localizations comprise eleven gene loci (PRLR, GHR, HK1, ACE, TF, 18S+28S rDNA, CYP2C4, PMP2, TCRB, ALOX15 and MT1) and three microsatellite loci (Sat13, Sol33 and D1Utr6). Five of the genes contain known microsatellite sequences. To achieve these localizations, homologous and heterologous small insert clones, and clones from a rabbit Bacterial Artificial Chromosome (BAC) library were used as probes for fluorescence in situ hybridization experiments. Results indicate that especially BAC clones are a valuable tool for cytogenetic mapping. Some of the genes were selected for mapping on the basis of human- rabbit comparative painting data, to achieve localizations on gene-poor rabbit chromosomes. Our data are, in general, in agreement with the human-rabbit comparative painting data. By mapping microsatellite sequences that have also been used in linkage studies, links are provided between the genetic and physical maps of the rabbit genome. Linkage groups I, VI and XI could be assigned to chromosomes 1, 5 and 3 respectively. Moreover, in this paper we give an overview of the current status of the rabbit cytogenetic map. This map now comprises 62 physically mapped genes, which are scattered over all autosomes, except chromosome 2, and the X chromosome.     

Linkage and comparative mapping of the locus controlling susceptibility towards E. COLI F4ab/ac diarrhoea in pigs

In 1995, Edfors-Lilja and coworkers mapped the locus for the E. COLI K88ab (F4ab) and K88ac (F4ac) intestinal receptor to pig chromosome 13 (SSC13). Using the same family material we have refined the map position to a region between the microsatellite markers Sw207 and Sw225. Primers from these markers were used to screen a pig BAC library and the positive clones were used for fluorescent in situ hybridization (FISH) analysis. The results of the FISH analysis helped to propose a candidate gene region in the SSC13q41-->q44 interval. Shotgun sequencing of the FISH-mapped BAC clones revealed that the candidate region contains an evolutionary breakpoint between human and pig. In order to further characterise the rearrangements between SSC13 and human chromosome 3 (HSA3), detailed gene mapping of SSC13 was carried out. Based on this mapping data we have constructed a detailed comparative map between SSC13 and HSA3. Two candidate regions on human chromosome 3 have been identified that are likely to harbour the human homologue of the gene responsible for susceptibility towards E. COLI F4ab/ac diarrhoea in pigs.     

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     

Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5× genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and <5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.     

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.     

Comparative mapping of mink (Mustela vison) chromosome 8p: localization of three human BAC clones

Using fluorescent in situ hybridization (FISH), three human BAC clones, localized in the terminal region of human chromosome 17p (HSA17p13; 1.44--3.68 Mp), were mapped to chromosome 8p of American mink (MVI8p). It was demonstrated that in MVI8p the region, homeologous to HSA17p13, was split into three fragments, which were detected within terminal, pericentric, and probably nucleolus-organizing regions. Using human BAC clones as heterologous markers for mapping of the gene sequences to the chromosomes of American mink, regional localization of eight sequences (PRPF8, SLC43A2, and RILP in MVI8p25; C17orf31 in MVI8p21-22; and CTNS, TAX1BP3, and P2RX5 in MVI8p11) was deduced.     

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.     

Comparative mapping of mink (<Emphasis Type="Italic">Mustela vision</Emphasis>) chromosome 8p: Localization of three human BAC clones

Using fluorescent in situ hybridization (FISH), three human BAC clones, localized in the terminal region of human chromosome 17p (HSA17p13; 1.44–3.68 Mp), were mapped in chromosome 8p of American mink (MVI8p). It was demonstrated that in MVI8p the region, homeologous to HSA17p13, was split into three fragments, which were detected within terminal, pericentric, and probably nucleolus-organizing regions. Using human BAC clones as heterologous markers for mapping of the gene sequences to the chromosomes of American mink, regional localization of eight sequences (PRPF8, SLC43A2, and RILP in MVI8p25; C17orf31 in MVI8p21-22; and CARKL, CTNS, TAX1BP3, and P2RX5 in MVI8p11) was deduced.     

A comparative map of bovine chromosome 19 based on a combination of mapping on a bacterial artificial chromosome scaffold map, a whole genome radiation hybrid panel and the human draft sequence

We have constructed a medium density physical map of bovine chromosome 19 using a combination of mapping loci on both a bovine bacterial artificial chromosome (BAC) scaffold map and a whole genome radiation hybrid (WGRH) panel. The resulting map contains 70 loci spanning the length of bovine chromosome 19. Three contiguous groups of BACs were identified on the basis of multiple loci mapping to individual BAC clones. Bovine chromosome 19 was found in this study to be comprised almost entirely from regions of human chromosome 17, with a small region putatively assigned to human chromosome 10. Fourteen breakpoints between the bovine and human chromosomes were detected, with a possibility of five more based on ordering of the WGRH map.     

Simultaneous painting of three genomes in hexaploid wheat by BAC-FISH.

Fluorescence in situ hybridization (FISH) is widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts, making them amenable for FISH mapping. In our BAC-FISH experiments, we selected 56 restriction fragment length polymorphism (RFLP)-locus-specific BAC clones from the libraries of Triticum monococcum and Aegilops tauschii, which are the A- and D-genome donors of wheat (Triticum aestivum, 2n = 6x = 42), respectively. The BAC clone 676D4 from the T. monococcum library contains a dispersed repeat that preferentially hybridizes to A-genome chromosomes, and two BAC clones, 9I10 and 9M13, from the Ae. tauschii library contain a dispersed repeat that preferentially hybridizes to the D-genome chromosomes. These repeats are useful in simultaneously discriminating the three different genomes in hexaploid wheat, and in identifying intergenomic translocations in wheat or between wheat and alien chromosomes. Sequencing results show that both of these repeats are transposable elements, indicating the importance of transposable elements, especially retrotransposons, in the genome evolution of wheat.     

High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice

Large-scale physical mapping has been a major challenge for plant geneticists due to the lack of techniques that are widely affordable and can be applied to different species. Here we present a physical map of rice chromosome 10 developed by fluorescence in situ hybridization (FISH) mapping of bacterial artificial chromosome (BAC) clones on meiotic pachytene chromosomes. This physical map is fully integrated with a genetic linkage map of rice chromosome 10 because each BAC clone is anchored by a genetically mapped restriction fragment length polymorphism marker. The pachytene chromosome-based FISH mapping shows a superior resolving power compared to the somatic metaphase chromosome-based methods. The telomere-centromere orientation of DNA clones separated by 40 kb can be resolved on early pachytene chromosomes. Genetic recombination is generally evenly distributed along rice chromosome 10. However, the highly heterochromatic short arm shows a lower recombination frequency than the largely euchromatic long arm. Suppression of recombination was found in the centromeric region, but the affected region is far smaller than those reported in wheat and barley. Our FISH mapping effort also revealed the precise genetic position of the centromere on chromosome 10.     

Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n=6 in the female and 7 in the male, the karyotypic evolution of which through extensive tandem fusions and several centric fusions has been well-documented by recent molecular cytogenetic studies. In an attempt to define the fusion orientations of conserved chromosomal segments and the molecular mechanisms underlying the tandem fusions, we have constructed a highly redundant (more than six times of whole genome coverage) bacterial artificial chromosome (BAC) library of Indian muntjac. The BAC library contains 124,800 clones with no chromosome bias and has an average insert DNA size of 120 kb. A total of 223 clones have been mapped by fluorescent in situ hybridization onto the chromosomes of both Indian muntjac and Chinese muntjac and a high-resolution comparative map has been established. Our mapping results demonstrate that all tandem fusions that occurred during the evolution of Indian muntjac karyotype from the acrocentric 2n=70 hypothetical ancestral karyotype are centromere–telomere (head–tail) fusions.     

Physical and cDNA mapping in the DBH region of human chromosome 9q34

Chromosome 9q34 has been extensively studied and mapped due to the presence of known disease genes, principally tuberous sclerosis 1 (TSC1), in this region. During the course of our mapping of this region we constructed a 555-kb contig beginning approximately 50 kb proximal to the dopamine-beta-hydroxylase (DBH) gene and extending, with one small deletion, distal to the D9S114 marker. The contig consists of 11 P1 clones, four PAC clones, one BAC clone and six cosmid clones and contains 27 new nonpolymorphic STSs. We have found the region to be unstable in P1, PAC and BAC cloning vehicles and have identified several deleted genomic clones. In addition, we have isolated and mapped the 3' portions of three putative genes located within or immediately distal to the DBH gene, including one large gene that runs on the opposite strand to DBH and utilizes portions of two DBH exons. The genomic clones of the contig, cDNAs and new STSs will be useful reagents for the further study and mapping of this region.