5S rRNA Gene Clusters Research Articles

Cytogenetics characterization of Crenuchus spilurus (Günther, 1863): a remarkable low diploid value within family Crenuchidae (Characiformes)

Conventional and molecular cytogenetic analyses were performed in specimens of the Neotropical Crenuchus spilurus freshwater fish species from a single location (Caete River, Brazil). All specimens presented diploid values of 2n = 38 chromosomes (12 m + 4sm + 2st + 20a), the lowest reported for family Crenuchidae up to now. A single pair of nucleolar organizing regions (NORs) was detected in the subtelocentric chromosome pair no. 9 by silver-staining and fluorescence in situ hybridization (FISH) with 18S rDNA sequence-specific probe. Two pairs of 5S rRNA gene clusters were found either interstitial or terminally located in the long arms of the acrocentric chromosome pairs nos. 10 and 13. Heterochromatic regions were clearly observed in the short arms of the NOR-bearing chromosome pair and weakly-positive to the pericentromeric regions of most acrocentric chromosomes. Additionally, no sex chromosomes were identified in the surveyed specimens. Crenuchidae have signals of several mechanisms involved in karyotype diversification within this family: differential location of heterochromatin-rich regions, multiplication, and translocation of rDNA clusters, presence/absence of sex chromosomes, macrostructural changes in morphology and number of chromosomes. This variety of karyotype patterns reveals the importance of widening cytogenetic studies to more taxa for better know the chromosomal evolution occurred in this group.

Are Pericentric Inversions Reorganizing Wedge Shell Genomes?

Wedge shells belonging to the Donacidae family are the dominant bivalves in exposed beaches in almost all areas of the world. Typically, two or more sympatric species of wedge shells differentially occupy intertidal, sublittoral, and offshore coastal waters in any given locality. A molecular cytogenetic analysis of two sympatric and closely related wedge shell species, Donax trunculus and Donax vittatus, was performed. Results showed that the karyotypes of these two species were both strikingly different and closely alike; whilst metacentric and submetacentric chromosome pairs were the main components of the karyotype of D. trunculus, 10–11 of the 19 chromosome pairs were telocentric in D. vittatus, most likely as a result of different pericentric inversions. GC-rich heterochromatic bands were present in both species. Furthermore, they showed coincidental 45S ribosomal RNA (rRNA), 5S rRNA and H3 histone gene clusters at conserved chromosomal locations, although D. trunculus had an additional 45S rDNA cluster. Intraspecific pericentric inversions were also detected in both D. trunculus and D. vittatus. The close genetic similarity of these two species together with the high degree of conservation of the 45S rRNA, 5S rRNA and H3 histone gene clusters, and GC-rich heterochromatic bands indicate that pericentric inversions contribute to the karyotype divergence in wedge shells.

Distinct classical and molecular cytogenetics of Astyanax marionae and A. fasciatus (Characiformes: Characidae): a comparative study of the organization of heterochromatin and repetitive genes.

Genus Astyanax is well distributed in Neotropical freshwater environments and its taxonomic position is uncertain, as is the case with other Characidae genera allocated in the group incertae sedis. This study aimed to analyse the karyotype of different populations of Astyanax fasciatus (Corumbataí River basin) using Giemsa staining, C-band technique, and fluorescence in situ hybridization for the H3 histone and 5S rRNA genes, in addition we describe for the first time the chromosomal organization of H3 histone and 5S rRNAgenes in A. marionae (ParaguayRiver basin). Chromosomes of three A. fasciatus populations were analysed (two with 2n = 50 and one with 2n = 48) and the heterochromatin was organized in two forms (blocks with blurred boundaries and distinct blocks). H3 histone and 5S rRNA genes were observed in all the three populations of A. fasciatus on two chromosome pairs (one metacentric chromosome showing H3 histone and 5S rRNA gene clusters). In A. marionae (2n = 48), H3 histone and 5S rRNA genes were observed in one acrocentric chromosome pair (different pairs). Further, differences between karyotypes and heterochromatin, as well as the chromosomal organization of H3 histone and 5S rRNA genes in Astyanax species, focussing on chromosome evolution in the group are discussed.

Molecular and Cytogenetic Characterization of Wild Musa Species.

The production of bananas is threatened by rapid spreading of various diseases and adverse environmental conditions. The preservation and characterization of banana diversity is essential for the purposes of crop improvement. The world's largest banana germplasm collection maintained at the Bioversity International Transit Centre (ITC) in Belgium is continuously expanded by new accessions of edible cultivars and wild species. Detailed morphological and molecular characterization of the accessions is necessary for efficient management of the collection and utilization of banana diversity. In this work, nuclear DNA content and genomic distribution of 45S and 5S rDNA were examined in 21 diploid accessions recently added to ITC collection, representing both sections of the genus Musa. 2C DNA content in the section Musa ranged from 1.217 to 1.315 pg. Species belonging to section Callimusa had 2C DNA contents ranging from 1.390 to 1.772 pg. While the number of 45S rDNA loci was conserved in the section Musa, it was highly variable in Callimusa species. 5S rRNA gene clusters were found on two to eight chromosomes per diploid cell. The accessions were genotyped using a set of 19 microsatellite markers to establish their relationships with the remaining accessions held at ITC. Genetic diversity done by SSR genotyping platform was extended by phylogenetic analysis of ITS region. ITS sequence data supported the clustering obtained by SSR analysis for most of the accessions. High level of nucleotide diversity and presence of more than two types of ITS sequences in eight wild diploids pointed to their origin by hybridization of different genotypes. This study significantly expands the number of wild Musa species where nuclear genome size and genomic distribution of rDNA loci is known. SSR genotyping identified Musa species that are closely related to the previously characterized accessions and provided data to aid in their classification. Sequence analysis of ITS region provided further information about evolutionary relationships between individual accessions and suggested that some of analyzed accessions were interspecific hybrids and/or backcross progeny.

Chromosome Mapping of H1 Histone and 5S rRNA Gene Clusters in Three Species of Astyanax (Teleostei, Characiformes)

We report here on the physical mapping of the H1 histone genes (hisDNA) and the 5S ribosomal DNA (rDNA) in 3 Neotropical fish species of the genus Astyanax(A. altiparanae, A. bockmanni and A. fasciatus) and the comparative analysis of the chromosomes bearing these genes. Nucleotide analyses by sequencing of both genes were also performed. The distribution of the H1 histone genes was more conserved than that of the rRNA genes, since these were always located in the pericentromeric regions of 2 chromosome pairs. 5S rDNA was found on one of the pairs that presented an H1 histone cluster; this seems to be a conserved chromosomal feature of the genus Astyanax. In addition, individuals of A. bockmanni and A. fasciatus showed clusters of 5S rDNA on 1 pair of acrocentric chromosomes, not found in A. altiparanae. The results obtained by chromosome mapping as well as by sequencing of both genes showed that A.bockmanni is more closely related to A. fasciatus than to A. altiparanae. The results allow the characterization of cytogenetic markers for improved elucidation of the processes involved in karyotype differentiation of fish genomes.

CHROMOSOMAL MAPPING OF 5S RIBOSOMAL RNA GENES IN THE EASTERN OYSTER, CRASSOSTREA VIRGINICA GMELIN BY FLUORESCENCE IN SITU HYBRIDIZATION

Chromosomal location of the 5S ribosomal RNA gene was studied in the eastern oyster, Crassostrea virginica Gmelin. using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos, and the FISH probe was made by PCR (polymerase chain reaction) amplification of the 5S rRNA gene and labeled by incorporation of digoxigenin-1 1-dUTP during PCR. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. Two pairs of FISH signals were observed on metaphase chromosomes. Karyotypic analysis showed that the 5S rRNA gene cluster is interstitially located on short arms of chromosomes 5 and 6. On chromosome 5, the 5S rRNA genes were located immediately next to the centromere, whereas on chromosome 6, they were located approximately half way between the telomere and the centromere. Chromosomes of C. virginica are difficult to identify because of their similarities in size and arm ratio, and the chromosomal location of 5S rRNA genes provides unambiguous identification of chromosomes 5 and 6. Previous studies have mapped the major rRNA gene cluster (18S-5.8S-28S) to chromosome 2. and this study shows that the 5S rRNA gene cluster is not linked to the major rRNA genes and duplicated during evolution.

Nuclear genome size and genomic distribution of ribosomal DNA in Musa and Ensete (Musaceae): taxonomic implications

Nuclear DNA content and genomic distributions of 5S and 45S rDNA were examined in nineteen diploid accessions of the genus Musa representing its four sections Eumusa, Rhodochlamys, Callimusa and Australimusa, and in Ensete gilletii, which was the outgroup in this study. In the Eumusa (x = 11), 2C DNA content ranged from 1.130 to 1.377 pg, M. balbisiana having the lowest DNA content of all sections. M. beccarii (x = 9), a representative of Callimusa, had the highest 2C nuclear DNA content (1.561 pg). Species belonging to Rhodochlamys (x = 11) and Australimusa (x = 10) had 2C DNA contents ranging from 1.191 to 1.299 pg and from 1.435 to 1.547 pg, respectively. E. gilletii (x = 9) had 2C DNA content of 1.210 pg. The number of 5S rDNA loci in Musa varied from 4 to 8 per diploid cell. While different numbers of 5S rDNA loci were observed within Eumusa and Rhodochlamys, four 5S rDNA loci were observed in all accessions of Australimusa. M. beccarii (Callimusa) and E. gilletii contained 5S rRNA gene clusters on five and six chromosomes, respectively. The number of 45S rDNA loci was conserved within individual sections. Hierarchical cluster analysis of genome size, number of chromosomes and 45S rDNA sites suggested a close relationship between Rhodochlamys and Eumusa; Australimusa was clearly separated as were M. beccarii and E. gilletii. Within the Eumusa-Rhodochlamys group, M. balbisiana, M. schizocarpa and M. ornata formed distinct subgroups, clearly separated from the accessions of M. acuminata, M. mannii, M. laterita and M. velutina, which formed a tight subgroup. The results expand the knowledge of genome size and genomic distribution of ribosomal DNA in Musa and Ensete. They aid in clarification of the taxonomical classification of Musa and show a need to supplement the analyses on the DNA sequence level with cytogenetic studies.

Evolution of rRNA gene clusters and telomeric repeats during explosive genome repatterning in Taterillus X (Rodentia, Gerbillinae)

A survey of 28S and 5S rRNA gene clusters, and telomeric repeats was performed using single and double FISH in the Taterillus genus (Rodentia, Muridae, Gerbillinae). Taterillus was previously demonstrated to have undergone a very recent and extensive chromosomal evolution. Our FISH results demonstrate that rRNA genes can vary in location and number irrespective of the phylogenetic relationships. Telomeric repeats were detected in pericentromeric and interstitial regions of several chromosomes, thus providing nonambiguous evolutionary footprints of Robertsonian and tandem translocation events. These footprints are discussed in reference to the molecular process of these karyotypical changes. Also, examples of colocation of rDNA clusters and telomeric repeats lend support to their possible involvement in nucleolus formation. Finally, the presence of rRNA genes, and the extensive amplification of telomeric repeats at specific loci within a double X-autosome translocated element which were not observed on the homologous Y1 and Y2, served as basis for an epigenomic hypothesis on X-autosome translocation viability in mammals.

Canine 5S rRNA: nucleotide sequence and chromosomal assignment of its gene cluster in four canid species

The purpose of this study was to determine the nucleotide sequence of canine 5S rRNA and use this information to develop a molecular probe to assign the gene locus to chromosomes of the dog and three other related canid species using fluorescence in situ hybridization. The nucleotide sequence of canine liver 5S rRNA is 120 base pairs long and identical to the 5S rRNA nucleotide sequence of all other mammalian species investigated so far. A single 5S rRNA gene cluster was localized pericentromerically on chromosomes of four canid species: dog 4q1.3, red fox 4q1.3, blue fox 3q1.3 and Chinese raccoon dog 8q1.3. Chromosome arms carrying the 5S rRNA gene cluster showed striking similarities in their QFQ banding patterns, suggesting high conservation of these chromosome arms among the four species studied. The chromosomal assignments of 5S rRNA genes are among the first gene mapping results for the blue fox and the Chinese raccoon dog, and are in accordance with published data on comparative chromosome maps from human, dog, red fox, blue fox and raccoon dogs.

Trans-spliced leader RNA, 5S-rRNA genes and novel variant orphan spliced-leader of the lymphatic filarial nematode Wuchereriabancrofti, and a sensitive polymerase chain reaction based detection assay

A genomic library of Wuchereriabancrofti was examined for the presence of the 22 nucleotide spliced leader (SL) which plays a vital role in the maturation of the 5′ end of certain mRNAs through the addition of a small spliced leader (SL) exon and also in the generation of monocistronic mRNA from initial polycistronic transcripts in nematodes. Here, we report the characterization of three SL RNA genes (SLG1, SLG2 and SLG3), an internal copy of a novel variant SL1 sequence (SL1v) with 23 nucleotides within an open reading frame of 75 amino acid residues of an unknown gene and two 5S-rRNA genes (5SR2 and 5SR3) from two genomic clones (TZP/11, TZP/91) of W.bancrofti. Our results revealed that the genes for the spliced leader RNA of W.bancrofti (SL RNA) is reiterated within the 5S-rRNA gene cluster and are in the same orientation. The genes SLG1, SLG2 and SLG3 were identical in nucleotide sequence except for an additional nucleotide at position 43 on SLG2. Sequence analysis of the three genes indicated that the 22-nt sequence is invariably adjacent to the dinucleotide GT, characteristic of a potential spliced donor site. The Sm-binding sequence AATTTTGG was conserved in SLG1, SLG2 and SLG3. Further, both 5′ and 3′ flanking regions of genes SLG1, SLG2 and SLG3 shared considerable sequence similarity. Two 5S-rRNA genes characterized from the genomic clone TZP 11 were shown to have sequence heterogeneity. Genomic southern showed that the spliced leader sequence is multicopy within the W.bancrofti genome and is also encoded in the region of DNA unlinked to the 5S rRNA gene cluster. Primers designed to amplify intergenic regions between 5S-rRNA and SL RNA genes in a PCR assay were found to be specific for W.bancrofti and was sensitive enough to detect 1 pg of W.bancrofti DNA or 1/8th of a microfilariae in infected blood samples. The high specificity and sensitivity of the optimised PCR assay makes it an ideal diagnostic tool for the identification of W.bancrofti in both the host and the vector.

Molecular phylogeny and rearrangement of rRNA genes in Rickettsia species.

It has previously been observed that Rickettsia prowazekii has an unusual arrangement of the rRNA genes. In this species, the three rRNA genes, 16S (rrs), 23S (rrl), and 5S (rrf), are not linked in the typical arrangements for bacteria. Rather, the 16S rRNA gene has been separated from the 23S and 5S rRNA gene cluster, and the 23S rRNA gene is preceded by a gene which codes for methionyl-tRNAf(Met) formyltransferase (fmt). In this study, we screened the genus Rickettsia for the fmt-rrl motif in order to examine the phylogenetic depth of this unusual rRNA gene organization. A rearranged operon structure was observed in Rickettsia conorii, Rickettsia parkeri, Rickettsia sibirica, Rickettsia rickettsii, Rickettsia amblyomii, Rickettsia montana, Rickettsia rhipicephali, Rickettsia australis, Rickettsia akari, Rickettsia felis, Rickettsia canada, and Rickettsia typhi. There is also evidence for a divided operon in Rickettsia belli, but in this species, the fmt gene could not be identified upstream of the 23S rRNA gene. In order to place the rearrangement event in the evolutionary history of the Rickettsia, phylogenetic analyses were performed based on the fmt-rrl spacer regions and the 23S rRNA genes. Based on these phylogenies, we suggest that the genomic rearrangement of the rRNA genes preceded the divergence of the typhus group and the spotted fever group Rickettsia. The unique organization of the 23S rRNA genes provides a simple diagnostic tool for identification of Rickettsia species.

The 5S rRNA gene clusters have a defined orientation toward the nucleolus in Petunia hybrida and Crepis capillaris.

The 3D localization of the 5S ribosomal RNA genes was studied in cells of the cortex zone of roots in the plant species Petunia hybrida inbred line V26 and in Crepis capillaris. The analysis was carried out on interphase nuclei (both species) and on prophase nuclei (C. capillaris). The 5S ribosomal RNA genes were detected by fluorescence in-situ hybridization and 3D images were obtained by confocal scanning laser microscopy. In both plant species, the 5S ribosomal genes were localized at the short arm of chromosome 2, which, in both plants, also possesses a satellite at its end. Statistical and visual analysis of interphase nuclei showed that: (1) there is a preference for an association of the 5S rRNA gene clusters of the two homologous chromosomes, and (2) the 5S rRNA gene clusters in both species had a preserved spatial position within the interphase nucleus and they tended to be polarized with respect to their neighbouring cells (i.e. a relic telophase orientation). Moreover, tracing of the chromosomal segment between the 5S loci and the active NOR revealed that the homologous chromosomes during early/mid prophase were aligned and that they entered the nucleolus side by side, at least for these chromosome segments. We interpret our data to mean that location of 5S rRNA near the nucleolus favours their functioning in ribosome biogenesis.

Mapping of 5-methylcytosine residues in Nicotiana tabacum 5S rRNA genes by genomic sequencing.

Genomic sequencing was used to localise 5-methylcytosine residues in individual DNA strands of 5S rRNA genes in tobacco. The density of methylation along the sequence was high in both strands, exceeding the average methylation density of the tobacco genome. Besides methylation of CG and CNG sequences, considerable amounts of mC were found in non-symmetrical sites. Among 69 sequenced clones obtained from leaf DNA we did not detect any non-methylated clone, and Southern blot hybridisation analysis also failed to suggest the presence of methylation-free 5S rDNA units in the tobacco genome. Differences were observed among methylation patterns of individual sequenced clones. This heterogeneity reflects either heterogeneity among individual members of 5S rRNA gene cluster or differences among individual cells. Methylation of CNG and non-symmetrical sites can be efficiently reduced by treatment with dihydroxypropyladenine, an inhibitor of S-adenosylhomocysteine hydrolase.

Investigation of DNA Loop Domains using Fluorescent in Situ Hybridization (FISH) and Epifluorescence Microscopy

Abstract The average sperm nucleus is only 8 micrometers long, but contains 1 meter of DNA. We have previously described how the sperm cell is able to package this DNA into the small volume of the sperm head. The only DNA structure that exists in both somatic cells and sperm nuclei is the organization of DNA loop domains that are attached at their bases to the nuclear skeleton, the nuclear matrix. We compared the structure of one gene in sperm nuclei to that of spermatogenic and adult cells. Individual loop domains of the 5S rRNA gene cluster were visualized by fluorescent in situ hybridization (FISH). Nuclei were isolated from hamster spermatozoa, spermatogonia, pachytene spermatocytes, round spermatids, and adult liver cells. They were then extracted to remove the protamines or histones, fixed, hybridized to a biotinylated 5S rDNA probe, then viewed by epifluorescence microscopy.

Investigation of DNA Loop Domains using Fluorescent in Situ Hybridization (FISH) and Epi-Fluorescence Microscopy

Abstract The average sperm nucleus is only 8 micrometers long, but contains 1 meter of DNA. We have previously described how the sperm cell is able to package this DNA into the small volume of the sperm head. The only DNA structure that exists in both somatic cells and sperm nuclei is the organization of DNA loop domains that are attached at their bases to the nuclear skeleton, the nuclear matrix. We compared the structure of one gene in sperm nuclei to that of spermatogenic and adult cells. Individual loop domains of the 5S rRNA gene cluster were visualized by fluorescent in situ hybridization (FISH). Nuclei were isolated from hamster spermatozoa, spermatogonia, pachytene spermatocytes, round spermatids, and adult liver cells. They were then extracted to remove the protamines or histones, fixed, hybridized to a biotinylated 5S rDNA probe, then viewed by epifluorescence microscopy. The 5S rDNA in liver nuclei was organized into a single large loop domain (Fig. 1),

Comparison of Molecular Cytogenetic and Genetic Analyses in Accessions of the Two Biotypes ofVicia benghalensisL.

In Vicia benghalensis two different biotypes, named A and B, had been observed which differed for several cytological, genetical and biochemical characters. In the present study 27 accessions of this species belonging to the two biotypes were investigated using different molecular analyses, supported by an attempt to produce hybrids between selected accessions. RAPD fingerprinting of the 27 accessions demonstrated that accessions belonging to biotype A show a high degree of genetic similarity, while the opposite is true for the accessions of biotype B. The total genomic DNA from one reference accession was used as a probe to Southern blots of the DNA extracted from all the accessions in use. This analysis demonstrated that cross hybridization among the DNA of the two biotypes occurs only to a limited extent. The chromosomal localization of 18S-5.8S-25S and 5S rRNA gene clusters was determined by in situ hybridization. The results indicate that the two biotypes differ in the position of one of the 5S rRNA gene clusters. This indicates a major chromosomal rearrangement. Cell synchronization experiments on two reference accessions suggested that the two biotypes might differ in the duration of the single phases of the cell cycle. Finally, in crossing experiments three pods were obtained which developed for only a few days and proved to bear no viable seeds. These results confirm the existence of two divergent gene-pools and demonstrate the reproductive barrier between the two biotypes, thus suggesting the possibility of a specific ranking.

Localization of three genes in the hook-shaped hamster sperm nucleus by fluorescent in situ hybridization.

We mapped the positions of three different genes in the flat, hook-shaped hamster sperm nucleus to determine the specificity of sperm DNA positioning. The positions of the 5S rRNA gene cluster, the CAD gene, and the class I 1.6 gene were determined by fluorescent in situ hybridization (FISH) in over 50 hamster sperm nuclei for each gene. We first demonstrated by FISH with mitotic chromosomes that the latter two genes were localized on the same chromosome. Within the sperm nuclei, we found that the precise position was variable for each of the three genes, but that there were two areas of preferred localization that contained 26-31% of the nuclear area and within which 80% of the signals were located. Nuclei were then hybridized to two genes simultaneously, using either two genes located on the same chromosome or two genes located on different chromosomes. We found no preference for orientation of one gene relative to the other for either pair of genes examined. This suggested that the relative arrangements of chromosomes within the sperm nucleus are flexible. These data demonstrate that the topographical arrangements of genes within the hamster sperm nucleus have a limited plasticity allowing for a relatively large range of possible localization.

Cell-specific organization of the 5S ribosomal RNA gene cluster DNA loop domains in spermatozoa and somatic cells.

DNA in eucaryotic cells is organized into loop domains, ranging in size from 25 to 100 kb, that are attached at their bases to the structural component of the nucleus termed the nuclear matrix. These DNA loop domains have been shown to be important in the regulation of both DNA replication and RNA transcription. In this study we have compared the structural organization of the DNA loop domains of the 5S rRNA gene cluster in sperm, liver, and brain nuclei in the Syrian golden hamster. The individual loop domains were visualized by fluorescent in situ hybridization to protamine (sperm)- and histone (somatic)-depleted nuclei, termed nuclear matrix halo preparations. We found that in sperm nuclei, the 5S rRNA gene cluster was organized into three small loop domains that were approximately 48 kb each. In both types of somatic cell nuclei examined, the 5S rRNA gene cluster was organized into a single, much larger loop domain that was up to 480 kb in length. The data suggest that at least some of the compaction that sperm DNA undergoes during spermiogenesis is mediated by the nuclear matrix independent of protamine binding. Additionally, this sperm-specific DNA organization may be involved in the specific patterns of DNA replication and transcription of the paternal genome in the embryo.

5S rRNA gene hybridizes to human chromosome 1 at two sites (1q42.11→q42.13 and1q43)

Hybridization of a loach (Misgurnus fossilis) oocyte 5S rRNA gene repeat with human chromosomes was carried out to refine the localization of the 5S RNA gene in the human genome. Preliminary in situ hybridization analysis showed that this repeat hybridized with human chromosome region 1q42-->q43, the same location previously reported for the human 5S rRNA gene. High-resolution banding revealed the presence of two sites of hybridization, with a main peak in chromosome region 1q42.1 and a second peak in 1q43. These results are consistent with our data on the existence in the human genome of different 5S rRNA gene clusters specified by BamHI and HindIII restriction sites in spacer DNA (Timofeeva et al., 1993) and suggest a multicluster organization of the human 5S rRNA genes.

Physical mapping of rRNA genes by fluorescent in-situ hybridization and structural analysis of 5S rRNA genes and intergenic spacer sequences in sugar beet (Beta vulgaris)

A digoxigenin-labelled 5S rDNA probe (pTa-794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet,Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sizedB. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. TwoXbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.