Integration of cytogenetic with recombinational and physical maps of mouse chromosome 16.

To link the cytogenetic map for mouse chromosome 16 (Chr 16) to the more detailed recombinational and physical maps, multiple probes were mapped by fluorescence in situ hybridization (FISH). Sixteen large insert clones (YACs, BACs, and PACs) containing markers that have been assigned to the Chr 16 recombinational map were localized to a cytogenetic band or subband by high-resolution FISH. This linkage of the cytogenetic and recombinational maps provides a useful tool for assigning new probe locations and for defining breakpoints in mice with chromosomal rearrangements. A direct application of these probes is demonstrated by identifying mice trisomic for distal Chr 16 using FISH analysis of interphase nuclei.     

Integration of the cytogenetic and genetic linkage maps of Brassica oleracea

We have assigned all nine linkage groups of a Brassica oleracea genetic map to each of the nine chromosomes of the karyotype derived from mitotic metaphase spreads of the B. oleracea var. alboglabra line A12DHd using FISH. The majority of probes were BACs, with A12DHd DNA inserts, which give clear, reliable FISH signals. We have added nine markers to the existing integrated linkage map, distributed over six linkage groups. BACs were definitively assigned to linkage map positions through development of locus-specific PCR assays. Integration of the cytogenetic and genetic linkage maps was achieved with 22 probes representing 19 loci. Four chromosomes (2, 4, 7, and 9) are in the same orientation as their respective linkage groups (O4, O7, O8, and O6) whereas four chromosomes (1, 3, 5, and 8) and linkage groups (O3, O9, O2, and O1) are in the opposite orientation. The remaining chromosome (6) is probably in the opposite orientation. The cytogenetic map is an important resource for locating probes with unknown genetic map positions and is also being used to analyze the relationships between genetic and cytogenetic maps.     

A high-resolution cytogenetic map of human chromosome 12: Localization of 195 new cosmid markers by direct R-banding fluorescence in situ hybridization

We have constructed a high-resolution cytogenetic map of human chromosome 12 with 195 newly isolated cosmids by direct R-banding flourescence in situ hybridization. The fluorescent signals of 195 clones were evenly distributed throughout chromosome 12, but sublocalized preferentially to R-positive bands. This high-resolution cytogenetic map with an average map distance of 0.73 Mb on bands can, in conjunction with a genetic linkage map, facilitate the analysis of chromosomal and molecular aberrations in genetic diseases and cancers. Moreover, the cytogenetic mapping data provide starting points for establishing contig maps with cosmid clones and yeast artificial chromosomes.     

Cytogenetic localization of genetic markers on porcine chromosome 7q

Four microsatellite markers ( S0078, SWR1210, SW732, and SW304 ) taken from the linkage map of porcine chromosome 7 were assigned to the cytogenetic map of pig chromosome 7 by fluorescence in situ hybridization (FISH) analysis of selected yeast artificial chromosomes (YACs). These four new polymorphic cytogenetic markers provide additional anchor points for integrating the linkage and cytogenetic maps of chromosomal region 7q.     

An Integrated Genetic and Cytogenetic Map for Zhikong Scallop,Chlamys farreri,Based on Microsatellite Markers

The reliability of genome analysis and proficiency of genetic manipulation requires knowledge of the correspondence between the genetic and cytogenetic maps. In the present study, we integrated cytogenetic and microsatellite-based linkage maps for Zhikong scallop, Chlamys farreri. Thirty-eight marker-anchored BAC clones standing for the 19 linkage groups were used to be FISH probes. Of 38 BAC clones, 30 were successfully located on single chromosome by FISH and used to integrate the genetic and cytogenetic map. Among the 19 linkage groups, 12 linkage groups were physically anchored by 2 markers, 6 linkage groups were anchored by 1 marker, and one linkage group was not anchored any makers by FISH. In addition, using two-color FISH, six linkage groups were distinguished by different chromosomal location; linkage groups LG6 and LG16 were placed on chromosome 10, LG8 and LG18 on chromosome 14. As a result, 18 of 19 linkage groups were localized to 17 pairs of chromosomes of C. farreri. We first integrated genetic and cytogenetic map for C. farreri. These 30 chromosome specific BAC clones in the cytogenetic map could be used to identify chromosomes of C. farreri. The integrated map will greatly facilitate molecular genetic studies that will be helpful for breeding applications in C. farreri and the upcoming genome projects of this species.     

Integrated karyotyping of sorghum by in situ hybridization of landed BACs.

The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.     

A physical and genetic linkage map of the distal long arm of human chromosome 5.

By in situ hybridization of probes for three cloned genes and eight genetically-linked polymorphic DNA markers, we have prepared a physical map of the distal long arm of chromosome 5. These results, together with the localizations of 11 genes and the genetic linkage map reported previously by us and by other investigators, represent a map that spans 55 cM.     

A molecular cytogenetic map of sorghum chromosome 1. Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans approximately 60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific "paints" from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant "chromonomics," germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.     

Identification and Applications of Repetitive Probes for Gene Mapping in the Mouse

Interspecific mouse hybrids that are viable and fertile provide a wealth of genetic variation that is useful for gene mapping. We are using this genetic variation to develop multilocus linkage maps of the mouse genome. As an outgrowth of this work, we have identified three repetitive probes that collectively identify 28 loci dispersed on 16 of the 19 mouse autosomes and the X chromosome. These loci establish a skeleton linkage map that can be used to detect linkage over much of the mouse genome. The molecular probes are derived from the mouse mammary tumor virus envelope gene, the ornithine decarboxylase gene, and the triose phosphate isomerase gene. The ability to scan the mouse genome quickly and efficiently in an interspecific cross using these three repetitive probes makes this system a powerful tool for identifying the chromosomal location of mutations that have yet to be cloned, mapping multigenic traits, and identifying recessive protooncogene loci associated with murine neoplastic disease. Ultimately, interspecific hybrids in conjunction with repetitive and single-copy probes will provide a rapid means to access virtually any gene of interest in the mouse genome at the molecular level.     

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.     

The application of FISH techniques for physical mapping in the dog (Canis familiaris)

The abundance of CA/GT repeats in the DNA of the dog (Canis familiaris) has established the importance of polymorphic microsatellites in the development of a low density map of the canine genome. The assignment of linkage groups of markers to chromosomes by physical mapping requires reliable cytogenetic techniques for routine production of metaphase cells. The dog has 78 chromosomes, many of which are smaller and more contracted than those of other mammals. Although the molecular study of inherited disease in dogs has important implications for both improved welfare in dogs and the provision of animal models for human diseases, the small size and large number of chromosomes in the canine genome has discouraged the inclusion of cytogenetic analysis in the planning of relevant research protocols. In this report, Fluorescence In Situ Hybridization (FISH) techniques have been optimized for the physical mapping of probes in C. familiaris. A method to obtain a good yield of early and midmetaphases from short-term peripheral blood cultures and the optimal conditions for hybridization and detection of probes is described. Thirteen microsatellite-containing cosmid probes from a canine genomic library in pWE15, a highly repetitive probe (human ribosomal DNA pHr14E3), and a human X Chromosome (Chr) paint have been mapped. Six microsatellites, two ribosomal sites, and the human paint have been assigned to specific chromosomes. Received: 5 June 1995 / Accepted: 1 September 1995     

Ten families with fragile X syndrome: linkage relationships with four DNA probes from distal Xq

Summary We present clinical, cytogenetic, and linkage data of four DNA probes from the terminal long arm of the X chromosome in ten new families with fragile X syndrome. A prior/posterior method of multipoint linkage analysis is employed to combine these results with published data to refine the linkage map of terminal Xq. Ten possible probe/disease orderings were tested. The order with the greatest posterior probability (0.78) of the five loci is 52a-F9-fragile X gene-DX13-St14, although the order with reversal of the positions of 52a and F9 has a posterior probability 0.15. The mean estimates of the distances between the probes and the fragile X gene are 38cM and 33cM for the proximal probes 52a and F9, and 8 cM and 12 cM for the distal probes DX13 and St14. Although the current method of choice in the prenatal diagnosis and carrier detection of the fragile X syndrome remains detailed cytogenetic analysis, consideration is given to the potential role of these DNA probes, both singly and in pairs.     

Establishment of a molecular genetic map of distal mouse chromosome 1: further definition of a conserved linkage group syntenic with human chromosome 1q

A linkage map of distal mouse chromosome 1 was constructed by restriction fragment length polymorphism analysis of DNAs from seven sets of recombinant inbred (RI) strains. The data obtained with seven probes on Southern hybridization combined with data from previous studies suggest the gene order Cfh, Pep-3/Ren-1,2, Ly-5, Lamb-2, At-3, Apoa-2/Ly-17,Spna-1. These results confirm and extend analyses of a large linkage group which includes genes present on a 20-30 cM span of mouse chromosome 1 and those localized to human chromosome 1q21-32. Moreover, the data indicate similar relative positions of human and mouse complement receptor-related genes REN, CD45, LAMB2, AT3, APOA2, and SPTA. These results suggest that mouse gene analyses may help in detailed mapping of human genes within such a syntenic group.     

Analysis of human chromosome 21: correlation of physical and cytogenetic maps; gene and CpG island distributions.

Human chromosome 21 has been analyzed by pulsed-field gel electrophoresis using somatic cell hybrids containing limited regions of the chromosome and greater than 60 unique sequence probes. Thirty-three independent NotI fragments have been identified, totalling 43 million bp. This must account for essentially the entire long arm, and therefore gaps remaining in the map must be small. The extent of the pulsed-field map has allowed the direct correlation of the physical map with the cytogenetic map: translocation breakpoints can be unambiguously positioned along the long arm and the distances between them measured in base pairs. Three breakpoints have been identified, providing physical confirmation of cytogenetic landmarks. Information on sequence organization has been obtained: (i) 60% of the unique sequence probes are located within 11 physical linkage groups which can be contained in only 20% of the long arm; (ii) 9/21 genes are clustered within 4%; (iii) translocation breakpoints appear to occur within CpG island regions, making their identification difficult by pulsed-field techniques. This analysis contributes to the human genome mapping effort, and provides information to guide the rapid investigation of the biology of chromosome 21.     

Gene-based anchoring of the rat genetic linkage and cytogenetic maps: new regional localizations, orientation of the linkage groups, and insights into mammalian chromosome evolution

In order to generate anchor points connecting the rat cytogenetic and genetic maps, the cytogenetic position of 62 rat markers (including 55 genes) already localized genetically was determined by fluorescence in situ hybridization. Whenever possible, markers located near one end of the linkage groups were included. These new localizations allowed us to unambiguously orient the 20 autosomal and the X chromosome linkage groups. The position of the centromere in the linkage map could also be determined in the case of several metacentric chromosomes. In addition, the regional localization of 15 other rat genes was determined. These new data bring useful information with respect to comparative mapping with the mouse and the human and to mammalian evolution. They illustrate, for instance, that groups of genes can remain syntenic during mammalian evolution while being subjected to intrachromosomal rearrangements in some lineages (synteny is conserved while gene order is not). This analysis also disclosed cases of synteny conservation in one the two rodent species and the human, while the synteny is split in the other rodent species: such configurations are likely examples of lineage-specific interchromosomal rearrangements associated with speciation. Received: 20 April 1998 / Accepted: 26 May 1998     

Identification and mapping of six microdissected genomic DNA probes to the proximal region of mouse chromosome 1.

Six independent DNA probes, lambda Mm1C-150, lambda Mm1C-153, lambda Mm1C-156, lambda Mm1C-162, lambda Mm1C-163, and lambda Mm1C-165, have been isolated from a library of microdissected fragments from mouse chromosome 1, spanning cytogenetic bands C2 to C5. These DNA probes have been mapped by restriction fragment length polymorphism analysis with respect to 12 marker loci previously assigned to this portion of mouse chromosome 1, in a panel of 251 segregating Mus spretus x C57BL/6J interspecific backcross mice. The gene order and intergene distances were determined by segregation analysis to be centromere- lambda Mm1C-162-11.1 cM-Col3a1-8.8 cM-Len-2-2.6 cM-lambda Mm1C-163-1.6 cM-Fn-1-1.6 cM-Tp-1-0.8 cM-lambda Mm1C-165/Vil-0.4 cM-Inha-2.8 cM-lambda Mm1C-153-2.4 cM-lambda Mm1C-156-1.2 cM-Pax-3-5.6 cM-Akp-3-0.8 cM-Acrg-2.0 cM-Sag-0.5 cM-Col6a3-1.8 cM-lambda Mm1C-150-15.4 cM-Ren1,2. Four of these probes map within a chromosome 1 segment that is homologous to human chromosome 2q. Southern blotting analyses indicate that one of these anonymous probes, lambda Mm1C-165, detects DNA fragments highly conserved across species. These novel polymorphic probes should prove useful for linkage and physical mapping of this chromosomal region.     

Physical location of homoeologous groups 5 and 6 molecular markers mapped in Triticum aestivum L

In situ hybridization was used to map 21 restriction fragment length polymorphism (RFLP) probes to linkage groups 5 and 6 of hexaploid wheat (Triticum aestivum L. em Thell.) in order to compare physical distances and genetic distances between adjacent markers. All 21 probes hybridized to the corresponding homoeologous chromosome arms. The linear order and linkage relationships among the DNA probes on the in situ-based physical maps were generally the same as those on the RFLP-based genetic maps. However, significant differences were observed between the centiMorgan distances on a linkage map and the physical distances of the probes using in situ-based techniques. The results indicated a clustering of polymorphic RFLP markers in the middle of all of the homoeologous group 5 and 6 chromosome arms. This suggests that the available linkage maps do not completely cover the physical length of the chromosomes. As with the genetic maps, the physical map clearly showed the presence of nonhomoeologous rearrangements in the terminal regions of chromosome arms 5AL and 6BS. However, the physical mapping gave an indication of the physical size of the rearrangements as well as their arm location.     

Rat Chromosome 1: Regional localization of seven genes (Slc9a3, Srd5a1, Esr, Tcp1, Grik5, Tnnt3, Jak2) and anchoring of the genetic linkage map to the cytogenetic map

Seven genes were regionally localized on rat Chromosome (Chr) 1, from 1p11 to 1q42, and two of these genes were also included in a linkage map. This mapping work integrates the genetic linkage map and the cytogenetic map, and allows us to orient the linkage map with respect to the centromere, and to deduce the approximate position of the centromere in the linkage map. These mapping data also indicate that the Slc9a3 gene, encoding the Na⁺/H⁺ exchanger 3, is an unlikely candidate for the blood pressure loci assigned to rat Chr 1. These new localizations expand comparative mapping between rat Chr 1 and mouse or human chromosomes. Received: 21 March 1997 / Accepted: 3 May 1997     

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.     

Isolation and characterization of DNA probes from a flow-sorted human chromosome 8 library that detect restriction fragment length polymorphism (RFLP).

We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes detect rare variants, while the other two detect RFLPs with PIC values of .36 and .16. Informative probes will be useful for the construction of a linkage map for chromosome 8 and for the localization of mutant alleles to this chromosome.