Physical mapping of translocation breakpoints in rye by means of synaptonemal complex analysis

A physical map including 40 translocation breakpoints has been constructed in rye by means of synaptonemal complex (SC) analysis of well-paired pachytene quadrivalents. The chromosome arms involved in such translocations were previously identified either from mitotic C-banding analysis or from the meiotic configurations observed in the progenies of crosses with a rye line having multiple chromosome rearrangements. The synaptonemal complexes formed by some translocation homozygotes were also analyzed, the relative pachytene SC length of their translocated chromosomes being compared to that observed in the corresponding translocation heterozygotes. In the translocations in which the position of the breakpoint could be well defined from mitotic C-banding analysis, a good correspondence between the relative position of the point showing partner exchange in the pachytene quadrivalents and the actual location of the breakpoint was established. It is concluded that the mapping of translocation breakpoints by SC analysis of pachytene quadrivalents provides a more accurate estimate of the position of the breakpoints than that obtained from mitotic C-banding analysis, due to the lack of evenly-distributed interstitial C-bands in most rye chromosomes. The distribution of the breakpoints along the chromosomes in relation to their spontaneous or induced origin is also discussed.     

Male mouse recombination maps for each autosome identified by chromosome painting

Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure.     

Localization of single-copy sequences on chicken synaptonemal complex spreads using fluorescence in situ hybridization (FISH)

Synaptonemal complex (SC) spreads from bird oocytes and spermatocytes show the complete chromosome complement and can be observed at the light microscope using immunostaining of the proteins that compose the lateral elements. To investigate the use of avian SC spreads as substrates for fluorescent in situ hybridization (FISH) in combination with immunostaining, we applied two single-copy sequences to chicken oocyte spreads. Signals for both target sequences were consistently observed on the short arm of bivalent 1 in a large number of nuclei. Based on previous data about the size of chromosome 1 and from measurements on probed SC spreads, an estimate of the physical distance in Mb between each sequence and the telomere was calculated. The crossover frequencies along SC 1 obtained by immunolocalization of MLH1 foci during pachytene were used to calculate the distances in cM to the target sequences and to compare this cytogenetic SC map with the consensus linkage map for GGA1. The combination of SC-FISH and immunostaining could be generally applied to obtain high-resolution mapping of single-copy sequences in birds and, coupled with MLH1 crossover maps, it could be a reliable approach to obtain genetic distances between markers to test the genetic linkage maps generated from molecular markers.     

Mapping of the nodulation loci sym9 and sym10 of pea ( Pisum sativum L.)

Several mutants defective in the nodulation process during rhizobial or endomycorrhizal endosymbiosis of pea have been identified previously. We have integrated the map positions of two such nodulation mutations, sym9 and sym10, into the molecular map of pea by applying molecular-marker techniques combined with bulked segregant analysis (BSA). Lines P2 and P54 were found to carry alleles of sym9, line P56 carried an allele of sym10. F2 populations were derived from crosses of P2, P54 and P56, to JI281 and JI15, two of the parental lines that have been used previously to generate a molecular map of pea. sym9 was located on linkage group IV by AFLP-BSA analysis and subsequently mapped by RFLP in both F2 populations, P2 2 JI281 and P54 2 JI281. RFLP-BSA analysis was applied to assign sym10 to linkage group I. The RFLP marker locus, chs2, co-segregates with sym10 in the F2 population of P56 2 JI15.     

Integration of the FISH pachytene and genetic maps of Medicago truncatula

A molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was constructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meiotic prophase stage are 20 times longer than at mitotic metaphase, and display a well differentiated pattern of brightly fluorescing heterochromatin segments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochromatin patterns, and the positions of three repetitive sequences (5S rDNA, 45S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybridization (FISH). We determined the correlation between genetic linkage groups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive sequences on the pachytene chromosomes, and compared the genetic and cytological distances between markers. The mapping resolution was determined in a euchromatic part of chromosome 5 by comparing the cytological distances between FISH signals of clones of a BAC contig with their corresponding physical distance, and showed that resolution in this region is about 60 kb. The establishment of this FISH pachytene karyotype, with a far better mapping resolution and detection sensitivity compared to those in the highly condensed mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.     

Recombination is suppressed over a large region of the rainbow trout Y chromosome

The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus ( SEX ), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy - 163 marker, which maps close to SEX , was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome.     

An integrated map of Oryza sativa L. chromosome 5

The developments of molecular marker-based genetic linkage maps are now routine. Physical maps based on contigs of large insert genomic clones have been established in several plant species. However, integration of genetic, physical, and cytological maps is still a challenge for most plant species. Here we present an integrated map of rice (Oryza sativa L.) chromosome 5, developed by fluorescence in situ hybridization mapping of 18 bacterial artificial chromosome (BAC) clones or PI-derived artificial chromosome (PAC) clones on meiotic pachytene chromosomes. Each BAC/PAC clone was anchored by a restriction fragment length polymorphism marker mapped to the rice genetic linkage map. This molecular cytogenetic map shows the genetic recombination and sequence information of a physical map, correlated to the cytological features of rice chromosome 5. Detailed comparisons of the distances between markers on genetic, cytological, and physical maps, revealed the distributions of recombination events and molecular organization of the chromosomal features of rice chromosome 5 at the pachytene stage. Discordance of distances between the markers was found among the different maps. Our results revealed that neither the recombination events nor the degree of chromatin condensation were evenly distributed along the entire length of chromosome 5. Detailed comparisons of the correlative positions of markers on the genetic, cytological, and physical maps of rice chromosome 5 provide insight into the molecular architecture of rice chromosome 5, in relation to its cytological features and recombination events on the genetic map. The prospective applications of such an integrated cytogenetic map are discussed.     

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.     

Genetic mapping in pea. 1. RAPD-based genetic linkage map of Pisum sativum

 A genetic linkage map of Pisum sativum L. was constructed based primarily on RAPD markers that were carefully selected for their reproducibility and scored in a population of 139 recombinant inbred lines (RILs). The mapping population was derived from a cross between a protein-rich dry-seed cultivar ‘Térèse’ and an increased branching mutant (K586) obtained from the pea cultivar ‘Torsdag’. The map currently comprises nine linkage groups with two groups comprising only 6 markers (n=7 in pea) and covers 1139 cM. This RAPD-based map has been aligned with the map based on the (JI281×JI399) RILs population that currently includes 355 markers in seven linkage groups covering 1881 cM. The difference in map lengths is discussed. For this alignment 7 RFLPs, 23 RAPD markers, the morphological marker le and the PCR marker corresponding to the gene Uni were used as common markers and scored in both populations. Received: 13 March 1998 / Accepted: 29 April 1998     

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.     

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.     

Genetic linkage analysis and homology relationships of genes located on human chromosome 11q.

We have used DNA polymorphisms detected by probes for 11q to order 16 genes and to determine the genetic distances between them. Our map includes the genes for CD20, tyrosinase, progesterone receptor, stromelysin, collagenase, N-CAM, dopamine-D2 receptor, apolipoproteins AI-CIII-AIV, CD3-epsilon, -delta, and -gamma, porphobilinogen deaminase, thy-1, and ets-1. These genes have previously been sequenced as well as placed on the 11q cytogenetic map, which now makes them anchor points between the cytogenetic, genetic, and physical maps of this region. The ordering and distances between these genes are of immediate use in testing hypotheses of candidate genes for human genetic diseases associated with chromosome 11q. A comparison between our genetic map and similar maps from other species defines regions of homologous synteny that may be useful in mapping human genetic disease genes localized to the 11q region. Analysis of such homology provides additional bases for speculation of the evolutionary histories of gene families in this region.     

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2

Background

Integration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.).

Results

In this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed.

Conclusions

Recombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.     

An integrated physical, genetic and cytogenetic map around the sunn locus of Medicago truncatula.

The sunn mutation of Medicago truncatula is a single-gene mutation that confers a novel supernodulation phenotype in response to inoculation with Sinorhizobium meliloti. We took advantage of the publicly available codominant PCR markers, the high-density genetic map, and a linked cytogenetic map to define the physical and genetic region containing sunn. We determined that sunn is located at the bottom of linkage group 4, where a fine-structure genetic map was used to place the locus within a approximately 400-kb contig of bacterial artificial chromosome (BAC) clones. Genetic analyses of the sunn contig, as well as of a second, closely linked BAC contig designated NUM1, indicate that the physical to genetic distance within this chromosome region is in the range of 1000 -1100 kb.cM-1. The ratio of genetic to cytogenetic distance determined across the entire region is 0.3 cM.microm(-1). These estimates are in good agreement with the empirically determined value of approximately 300 kb.microm(-1) measured for the NUM1 contig. The assignment of sunn to a defined physical interval should provide a basis for sequencing and ultimately cloning the responsible gene.     

Physical organization of the major duplication on Brassica oleracea chromosome O6 revealed through fluorescence in situ hybridization with Arabidopsis and Brassica BAC probes.

The close relationship between Brassica oleracea and Arabidopsis thaliana has been used to explore the genetic and physical collinearity of the two species, focusing on an inverted segmental chromosome duplication within linkage group O6 of B. oleracea. Genetic evidence suggests that these segments share a common origin with a region of Arabidopsis chromosome 1. Brassica oleracea and Arabidopsis bacterial artificial chromosome probes have been used for fluorescence in situ hybridization analysis of B. oleracea pachytene chromosomes to further characterize the inverted duplication. This has been highly effective in increasing the local resolution of the cytogenetic map. We have shown that the physical order of corresponding genetic markers is highly conserved between the duplicated regions in B. oleracea and the physical lengths of the regions at pachytene are similar, while the genetic distances are considerably different. The physical marker order is also well conserved between Arabidopsis and B. oleracea, with only one short inversion identified. Furthermore, the relative physical distances between the markers in one segment of B. oleracea and Arabidopsis have stayed approximately the same. The efficacy of using fluorescence in situ hybridization, together with other forms of physical and genetic mapping, for elucidating such issues relating to synteny is discussed.     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

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.     

A genetic linkage map of human chromosome 5 with 60 RFLP loci.

A genetic map of human chromosome 5 that contains 60 restriction fragment length polymorphism (RFLP) loci in one linkage group has been constructed. Segregation data using these markers and 40 large multigenerational families supplied by the Centre d'Etude du Polymorphisme Humain have been collected. Linkage analyses were performed with the program package CRI-MAP; using odds greater than 1000:1, 30 RFLP loci could be placed on the map. This genetic map spans 289 cM sex-equal, 353 cM in females, and 244 cM in males. While the relative rate of recombination for female meioses is nearly twice that of males over much of the chromosome, several instances of statistically significant excess male recombination were observed. The order of probes on the genetic map has been confirmed by their physical order as determined by somatic cell hybrid lines containing deletions of normal chromosome 5. There is concordance between the physical positions of markers and their genetic positions. Our most distal probes on the genetic map are cytologically localized to the most distal portions of the chromosome. This suggests that our genetic map spans most of chromosome 5.     

Development of Chromosome-specific Cytogenetic Markers and Merging of Linkage Fragments in Papaya

Carica papaya L. is a tropical and sub-tropical fruit-tree crop with a small genome and nine pairs of chromosomes. The transgenic cultivar ‘SunUp’ has been sequenced and three high-density genetic maps are available for mapping agronomically and economically-important traits. However, the small size and similar morphology of papaya chromosomes hinder their identification and few cytological resources are available for integration of genetic and cytogenetic information. Fluorescence in situ hybridization (FISH) was performed on mitotic metaphase chromosomes using BAC clones harboring mapped simple sequence repeat (SSR) markers as probes. A total of 104 BAC clones covering all 12 linkage groups (LGs) were tested and 12 of them, that gave a single specific signal, were chosen as representative of the 12 LGs of the SSR genetic map. This set of chromosome-specific DNA markers acted as a foundation for papaya chromosome karyotyping and re-assigning orientation of LGs. Chromosome-specific markers allowed us to assign the minor LGs 10, 11, and 12 to major LGs 8, 9, and 7, respectively. We thus reduced the number of LGs in the genetic map to nine, corresponding to the haploid number of papaya chromosomes. We also tested the relative order of DNA markers on minor LGs 10 and 11 to place them on top of LGs 8 and 9 in the correct orientation. Ribosomal DNAs (rDNAs), a set of major cytogenetic markers, were positioned on specific papaya chromosomes. The 25S rDNA showed strong signals at the constriction site of a single pair of chromosomes identified as LG 2 by LG 2-specific BAC clone. The 5S rDNA showed strong signals on two pairs of chromosomes that are syntenic with LG 4- and LG 5-specific BAC clones. This integrated map will facilitate genome assembly, quantitative trait locus (QTL) mapping, and the study of cytological, physical and genetic distance relationships between papaya chromosomes.     

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.