Number Of 5S rDNA Sites Research Articles

Uneven Levels of 5S and 45S rDNA Site Number and Loci Variations across Wild Chrysanthemum Accessions.

Ribosomal DNA (rDNA) is an excellent cytogenetic marker owing to its tandem arrangement and high copy numbers. However, comparative studies have focused more on the number of rDNA site variations within the Chrysanthemum genus, and studies on the types of rDNA sites with the same experimental procedures at the species levels are lacking. To further explore the number and types of rDNA site variations, we combined related data to draw ideograms of the rDNA sites of Chrysanthemum accessions using oligonucleotide fluorescence in situ hybridization (Oligo-FISH). Latent variations (such as polymorphisms of 45S rDNA sites and co-localized 5S-45S rDNA) also occurred among the investigated accessions. Meanwhile, a significant correlation was observed between the number of 5S rDNA sites and chromosome number. Additionally, the clumped and concentrated geographical distribution of different ploidy Chrysanthemum accessions may significantly promote the karyotype evolution. Based on the results above, we identified the formation mechanism of rDNA variations. Furthermore, these findings may provide a reliable method to examine the sites and number of rDNA variations among Chrysanthemum and its related accessions and allow researchers to further understand the evolutionary and phylogenetic relationships of the Chrysanthemum genus.

Identification of 5S and 45S rDNA sites in Chrysanthemum species by using oligonucleotide fluorescence in situ hybridization (Oligo-FISH).

Fluorescence in situ hybridization (FISH) is a conventional method used to visualize the distribution of DNA elements within a genome. To examine the relationships within the Chrysanthemum genus, ribosomal DNA (rDNA), a popular cytogenetic marker, was utilized as a probe for FISH within this genus. Based on the genome data of Chrysanthemum nankingense, C. seticuspe and its allied genera in the Compositae(Asteraceae), we explored rDNA sequences to design oligonucleotide probes and perform oligonucleotide fluorescence in situ hybridization (Oligo-FISH) in eight Chrysanthemum accessions. The results showed that the majority of 5S rDNA signals were located in subterminal chromosome regions and that the number of 5S rDNA sites might be tightly associated with ploidy. For 45S rDNA sites, the number and intensity of signals differed from those of previously investigated Chrysanthemum resources. These findings may provide an optimally reliable method of examining the chromosome composition and structural variation of Chrysanthemum and its related species and allow researchers to understand the evolutionary history and phylogenetic relationships of Chrysanthemum.

Karyotype and leaf epidermis histology traits of Digitaria abyssinica (Hochst. Ex A. Rich.) (Poaceae)

Digitaria abyssinica (African couchgrass) has economic potential as forage. This species is taxonomically complex, with a high number of synonyms, basic chromosome number x = 9, and diploid and tetraploid cytotypes. Anatomical, histological, and cytotaxonomic studies may contribute to the taxonomic description of this species and reveal its polyploidization and hybridization events. To this end, this study aimed to characterize the karyotype and leaf epidermis histology of two D. abyssinica genotypes. Chromosome characterization was performed based on chromosome banding using chromomycin A (CMA) and 4,6-diamidino-2-phenylindole (DAPI) and identification of 35S and 5S rDNA sites by fluorescent in situ hybridization. Nuclear DNA quantification was performed by flow cytometry. Paradermal sections were obtained for the leaf epidermis study by light and electron microscopy. The results confirmed that both genotypes are tetraploid (2n = 36). The number and distribution of 35S and 5S rDNA sites suggest the occurrence of postpolyploidization structural chromosomal changes or hybridization processes. Despite intraspecific variation in the number of 5S rDNA sites and CMA+/DAPI+ bands, no changes were identified in karyotypical symmetry or genome size. Leaf epidermis histology traits and cytogenetic data can support breeding programs and germplasm banks in identifying species or cultivars.

The 5S rRNA genes in Alexandrium: their use as a FISH chromosomal marker in studies of the diversity, cell cycle and sexuality of dinoflagellates

Chromosomal markers of the diversity and evolution of dinoflagellates are scarce because the genomes of these organisms are unique among eukaryotes in terms of their base composition and chromosomal structure. Similarly, a lack of appropriate tools has hindered studies of the chromosomal localization of 5S ribosomal DNA (rDNA) in the nucleosome-less chromosomes of dinoflagellates. In this study, we isolated and cloned 5S rDNA sequences from various toxin-producing species of the genus Alexandrium and developed a fluorescence in situ hybridization (FISH) probe that allows their chromosomal localization. Our results can be summarized as follows: 1) The 5S rDNA unit is composed of a highly conserved 122-bp coding region and an intergenic spacer (IGS), the length and sequence of which are variable even within strains. 2) Three different IGS types, one containing the U6 small nuclear RNA (snRNA) gene, were found among four of the studied species (A. minutum, A. tamarense, A. catenella and A. pacificum). 3) In all strains investigated by FISH (A. minutum, A. tamarense, A. pacificum, A. catenella, A. andersonii and A. ostenfeldii), 5S rDNA gene arrays were separate from the nucleolar organizer region, which contains the genes for the large 45S pre-ribosomal RNA. 4) One to three 5S rDNA sites per haploid genome were detected, depending on the strains/species. Intraspecific variability in the number of 5S rDNA sites was determined among strains of A. minutum and A. pacificum. 5) 5S rDNA is a useful chromosomal marker of mitosis progression and can be employed to differentiate vegetative (haploid) vs. planozygotes (diploid) cells. Thus, the FISH probe (oligo-Dino5Smix5) developed in this study facilitates analyses of the diversity, cell cycle and life stages of the genus Alexandrium.

Karyological traits related to phylogenetic signal and environmental conditions within the Hymenaea clade (Leguminosae, Detarioideae)

The Hymenaea clade is a lineage of the subfamily Detarioideae of Leguminosae. The clade is currently composed of three genera: Guibourtia, Hymenaea, and Peltogyne. The genera Guibourtia and Hymenaea present an Amphi-Atlantic distribution, whereas Peltogyne is exclusively found in the Neotropics. Previous studies have focused on the phylogeny of the group but there are no studies that demonstrate mechanisms that could be involved in the evolution of this Amphi-Atlantic clade. The present work is the largest cytogenetic analysis for the group published so far. We aimed to provide a study of karyotypic characteristics of the Hymenaea clade, interpreted using the plastidial trnK phylogenetic reconstruction as background. We analyzed karyological characteristics such as chromosome number, chromosome size, centromeric position, CMA/DAPI patterns, and banding for 5S and 45S rDNA. With the present data available, multivariate analyses (PCA) and Phylogenetic Eigenvector Regression (PVR) demonstrated that centromeric asymmetry and number of 5S rDNA sites may present a strong phylogenetic signal, whereas number of CMA+ sites is associated with geography/climate characteristics. The association of CMA bands and geography has been reported for other legumes of the Caesalpinia group. This study demonstrates that the use of statistical tools for analyzing karyotypic characteristics can result in more precise interpretation of evolutionary processes related to structural changes in chromosomes and how these changes can also be related to environmental conditions.

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae).

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation.

Dynamics of 45S rDNA sites in the cell cycle: fragile sites and chromosomal stability in Lolium and Festuca.

Analyses carried out with fluorescence in situ hybridization (FISH) in C-metaphases of the Lolium-Festuca complex have shown the occurrence of spontaneous fragile sites (FSs) in 45S rDNA regions. FSs are expressed as gaps but they do not result in breaks or chromosomal fragments in these species. These gaps have high DNA condensation observed as thin chromatin fibers that connect the apparent segments of the fragile chromosome, allowing for genomic stability. Assessing the behavior of these regions in the cell cycle of Lolium and Festuca species may lead to a better understanding of the dynamics that preserve stability during cell division. Furthermore, it is interesting to track the dynamics of chromosomes bearing 45S rDNA sites in the cell cycle as well as to observe the expression of FSs with no effect of the mitotic block. We observed variation in both the number and size of 45S FISH signals from the S/G2 phases of interphase and from prophase to anaphase where gaps in 45S rDNA sites also were observed. The change in the degree of condensation of the 45S site begins in the S/G2 phase and appears to be related to the transcriptional demand. Taking into account that the number of 45S rDNA sites tends to be re-established when cells reach telophase, we suggest that the chromatin fiber goes back to the normal condensation level to the anaphase (after segregation), allowing for the approximation of chromosome segments and ensuring dynamics that favor the genomic stability of these species.

Comparative chromosomal localization of 45S and 5S rDNAs and implications for genome evolution in Cucumis.

Ribosomal DNAs are useful cytogenetic markers for chromosome analysis. Studies investigating site numbers and distributions of rDNAs have provided important information for elucidating genome organization and chromosomal relationships of many species by fluorescence in situ hybridization. But relevant studies are scarce for species of the genus Cucumis, especially in wild species. In the present study, FISH was conducted to investigate the organization of 45S and 5S rDNA among 20 Cucumis accessions, including cultivars and wild accessions. Our results showed that the number of 45S rDNA sites varied from one to five pairs in different accessions, and most of these sites are located at the terminal regions of chromosomes. Interestingly, up to five pairs of 45S rDNA sites were observed in C. sativus var. sativus, the species which has the lowest chromosome number, i.e., 2n = 14. Only one pair of 5S rDNA sites was detected in all accessions, except for C. heptadactylus, C. sp, and C. spp that had two pairs of 5S rDNA sites. The distributions of 5S rDNA sites showed more variation than 45S rDNA sites. The phylogenetic analysis in this study showed that 45S and 5S rDNA have contrasting evolutionary patterns. We find that 5S rDNA has a polyploidization-related tendency towards the terminal location from an interstitial location but maintains a conserved site number, whereas the 45S rDNA showed a trend of increasing site number but a relatively conserved location.

Karyotype differentiation in 19 species of river loach fishes (Nemacheilidae, Teleostei): extensive variability associated with rDNA and heterochromatin distribution and its phylogenetic and ecological interpretation.

BackgroundLoaches of the family Nemacheilidae are one of the most speciose elements of Palearctic freshwater ichthyofauna and have undergone rapid ecological adaptations and colonizations. Their cytotaxonomy is largely unexplored; with the impact of cytogenetical changes on this evolutionary diversification still unknown. An extensive cytogenetical survey was performed in 19 nemacheilid species using both conventional (Giemsa staining, C- banding, Ag- and Chromomycin A3/DAPI stainings) and molecular (fluorescence in situ hybridization with 5S rDNA, 45S rDNA, and telomeric (TTAGGG)n probes) methods. A phylogenetic tree of the analysed specimens was constructed based on one mitochondrial (cytochrome b) and two nuclear (RAG1, IRBP) genes.ResultsSeventeen species showed karyotypes composed of 2n = 50 chromosomes but differentiated by fundamental chromosome number (NF = 68–90). Nemachilichthys ruppelli (2n = 38) and Schistura notostigma (2n = 44–48) displayed reduced 2n with an elevated number of large metacentric chromosomes. Only Schistura fasciolata showed morphologically differentiated sex chromosomes with a multiple system of the XY1Y2 type. Chromomycin A3 (CMA3)- fluorescence revealed interspecific heterogeneity in the distribution of GC-rich heterochromatin including its otherwise very rare association with 5S rDNA sites. The 45S rDNA sites were mostly located on a single chromosome pair contrasting markedly with a pattern of two (Barbatula barbatula, Nemacheilus binotatus, N. ruppelli) to 20 sites (Physoschistura sp.) of 5S rDNA. The cytogenetic changes did not follow the phylogenetic relationships between the samples. A high number of 5S rDNA sites was present in species with small effective population sizes.ConclusionDespite a prevailing conservatism of 2n, Nemacheilidae exhibited a remarkable cytogenetic variability on microstructural level. We suggest an important role for pericentric inversions, tandem and centric fusions in nemacheilid karyotype differentiation. Short repetitive sequences, genetic drift, founder effect, as well as the involvement of transposable elements in the dispersion of ribosomal DNA sites, might also have played a role in evolutionary processes such as reproductive isolation. These remarkable dynamics of their genomes qualify river loaches as a model for the study of the cytogenetic background of major evolutionary processes such as radiation, endemism and colonization of a wide range of habitats.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0532-9) contains supplementary material, which is available to authorized users.

Non-Random Distribution of 5S rDNA Sites and Its Association with 45S rDNA in Plant Chromosomes

5S and 45S rDNA sites are the best mapped chromosome regions in eukaryotic chromosomes. In this work, a database was built gathering information about the position and number of 5S rDNA sites in 784 plant species, aiming to identify patterns of distribution along the chromosomes and its correlation with the position of 45S rDNA sites. Data revealed that in most karyotypes (54.5%, including polyploids) two 5S rDNA sites (a single pair) are present, with 58.7% of all sites occurring in the short arm, mainly in the proximal region. In karyotypes of angiosperms with only 1 pair of sites (single sites) they are mostly found in the proximal region (52.0%), whereas in karyotypes with multiple sites the location varies according to the average chromosome size. Karyotypes with multiple sites and small chromosomes (<3 µm) often display proximal sites, while medium-sized (between 3 and 6 µm) and large chromosomes (>6 µm) more commonly show terminal or interstitial sites. In species with holokinetic chromosomes, the modal value of sites per karyotype was also 2, but they were found mainly in a terminal position. Adjacent 5S and 45S rDNA sites were often found in the short arm, reflecting the preferential distribution of both sites in this arm. The high frequency of genera with at least 1 species with adjacent 5S and 45S sites reveals that this association appeared several times during angiosperm evolution, but it has been maintained only rarely as the dominant array in plant genera.

Karyological, morphological, and phylogenetic diversification in Leucocoryne Lindl (Allioideae, Amaryllidaceae)

The genus Leucocoryne (Amaryllidaceae) comprises approximately 15 species and is the only genus of the tribe Ipheieae endemic to the western region of Chile in sclerophyll and arid winter-rainfall biomes in the Andes. To better understand the karyotype diversification of this genus, nine species of Leucocoryne were analyzed in terms of their chromosome number and morphology, CMA and DAPI heterochromatic bands, distribution of rDNA sites, and their molecular phylogenetic relationships. Based on phylogenetic analyses Leucocoryne was divided in four clades: I (L. alliacea and L. conferta); II (L. dimorphopetala and L. narcisoides); III (L. coquimbensis, L. macropetala, L. appendiculata, L. talinensis, L. angustipetala, L. purpurea, and L. violacescens); and IV (L. vittata, L. ixioides, L. foetida, and L. pauciflora). The species were diploids with 2n = 10 (6M + 4A) or tetraploids with 2n = 18 (14M + 4A)/2n = 19 (13M + 6A). 45S rDNA sites were co-localized with CMA+/DAPI− bands on the short arms of acrocentric chromosomes in all species. 5S rDNA sites were observed on one, two, or four pairs of metacentric chromosomes per species. Estimations of the ancestral karyotype suggest that 2n = 10 (6M + 2A) with one 5S rDNA site per monoploid complement was the plesiomorphic condition of the genus Leucocoryne. Increases in number of 5S rDNA sites in the same species associated with biome shifts from sclerophyll to arid winter-rainfall region occurred independently in clade II and clade III. The two species of clade II retained the six fertile stamens (plesiomorphic condition) in contrast to the other Leucocoryne species that reduced to only three functional stamens. Based on these findings, we discuss the role of environmental change on the diversification of this group.

Physical localization of rRNA genes by fluorescence in situ hybridization (FISH) and analysis of spacer length variants of 45S rRNA (slvs) genes in some species of genus Sesbania

The somatic chromosome numbers 2n = 12 in S. macrantha, S. coerulescens and 2n = 24 in S. simplicuscula were determined, additionally the contradictory chromosome numbers of S. bispinosa (2n = 12, 13, 14, 24) and S. pachycarpa (2n 12, 14) were determined as 2n = 24 and 2n = 14, respectively. The number of 5S rDNA sites in chromosome pair 1 was highly conserved in all the diploid and tetraploid species studied irrespective of their geographic distribution, suggesting that all diploid and tetraploid species/cytotypes of Sesbania analyzed in the present study are in close proximity. Cytogenetic mapping of the 45S multi-gene family was also carried out using fluorescence in situ hybridization. 45S rDNA was consistently located on short or long arms of two sub-metacentric chromosome pairs except on one chromosome pair in S. macrantha and on three chromosome pairs in S. bispinosa and S. cannabina. Out of these nine species, we observed the homogenization of intergenic spacer in six species and find only one spacer length variant (slv) located on one to three chromosome loci. However, three of the species were observed to have two slvs located on two different chromosomes. The species were grouped as per their evolutionary relationship on the basis of the results of the present study.

Sugar Cane Genome Numbers Assumption by Ribosomal DNA FISH Techniques

Conventional cytological method is limited for polyploidy plant genome study, especially sugar cane chromosomes that show unstable numbers of each cultivar. Molecular cytogenetic as fluorescent in situ hybridization (FISH) techniques were used in this study. A basic chromosome number of sugar cane was estimated with three information; 1) number of 18S rDNA sites, 2) number of 5S rDNA sites and 3) total number of chromosomes. 18S and 5S rDNA were located by FISH techniques, the number of hom (e) ologous sites were illustrated in range of 7 to 9 and 13 to 15 sites. 110 chromosome numbers were shown in tapetal cells of flower buds of sugar cane. The implications of these results can predict about 14 basic chromosomes numbers but 5S rDNA seem reliable indicate for basic chromosome number and 18S rDNA were discussed about nucleolar dominance phenomenon of the sugar cane “KPS 00-25” cultivar

Distribution of 45S rDNA sites in chromosomes of plants: Structural and evolutionary implications

Background45S rDNA sites are the most widely documented chromosomal regions in eukaryotes. The analysis of the distribution of these sites along the chromosome in several genera has suggested some bias in their distribution. In order to evaluate if these loci are in fact non-randomly distributed and what is the influence of some chromosomal and karyotypic features on the distribution of these sites, a database was built with the position and number of 45S rDNA sites obtained by FISH together with other karyotypic data from 846 plant species.ResultsIn angiosperms the most frequent numbers of sites per diploid karyotype were two and four, suggesting that in spite of the wide dispersion capacity of these sequences the number of rDNA sites tends to be restricted. The sites showed a preferential distribution on the short arms, mainly in the terminal regions. Curiously, these sites were frequently found on the short arms of acrocentric chromosomes where they usually occupy the whole arm. The trend to occupy the terminal region is especially evident in holokinetic chromosomes, where all of them were terminally located. In polyploids there is a trend towards reduction in the number of sites per monoploid complement. In gymnosperms, however, the distribution of rDNA sites varied strongly among the sampled families.ConclusionsThe location of 45S rDNA sites do not vary randomly, occurring preferentially on the short arm and in the terminal region of chromosomes in angiosperms. The meaning of this preferential location is not known, but some hypotheses are considered and the observed trends are discussed.

Evolutionary dynamics of 5S rDNA location in acridid grasshoppers and its relationship with H3 histone gene and 45S rDNA location

We analyze the chromosomal location of 5S rDNA clusters in 29 species of grasshoppers belonging to the family Acrididae. There was extensive variation among species for the number and location of 5S rDNA sites. Out of 148 sites detected, 75% were proximally located, 21.6% were interstitial, and only 3.4% were distal. The number of 5S rDNA sites per species varied from a single chromosome pair (in six species) to all chromosome pairs (in five species), with a range of intermediate situations. Thirteen chromosomes from eight species carried two 5S rDNA clusters. At intraspecific level, differences among populations were detected in Eyprepocnemis plorans, and some heteromorphisms have also been observed in some species. Double FISH for 5S rDNA and H3 histone gene DNA, performed on 17 of these 29 species, revealed that both markers are sometimes placed in a same chromosome but at different location, whereas they appeared to co-localize in five species (Calliptamus barbarus, Heteracris adpersa, Aiolopus strepens, Oedipoda charpentieri and O. coerulescens). Double fiber-FISH in A. strepens and O. coerulescens showed that the two DNAs are closely interspersed with variable relative amounts of both classes of DNA. Finally, no correlation was observed between the number of 5S and 45S rDNA clusters in 23 species where this information was available. These results are discussed in the light of possible mechanisms of spread that led to the extensive variation in the number of clusters observed for both rDNA types in acridid grasshoppers.

Chromosomal organization of the 18S and 5S rRNAs and histone H3 genes in Scarabaeinae coleopterans: insights into the evolutionary dynamics of multigene families and heterochromatin

BackgroundScarabaeinae beetles show a high level of macro-chromosomal variability, although the karyotypic organization of heterochromatin and multigene families (rDNAs and histone genes) is poorly understood in this group. To better understand the chromosomal organization and evolution in this group, we analyzed the karyotypes, heterochromatin distribution and chromosomal locations of the rRNAs and histone H3 genes in beetles belonging to eight tribes from the Scarabaeinae subfamily (Coleoptera, Scarabaeidae).ResultsThe number of 18S rRNA gene (a member of the 45S rDNA unit) sites varied from one to 16 and were located on the autosomes, sex chromosomes or both, although two clusters were most common. Comparison of the 45S rDNA cluster number and the diploid numbers revealed a low correlation value. However, a comparison between the number of 45S rDNA sites per genome and the quantity of heterochromatin revealed (i) species presenting heterochromatin restricted to the centromeric/pericentromeric region that contained few rDNA sites and (ii) species with a high quantity of heterochromatin and a higher number of rDNA sites. In contrast to the high variability for heterochromatin and 45S rDNA cluster, the presence of two clusters (one bivalent cluster) co-located on autosomal chromosomes with the 5S rRNA and histone H3 genes was highly conserved.ConclusionsOur results indicate that the variability of the 45S rDNA chromosomal clusters is not associated with macro-chromosomal rearrangements but are instead related to the spread of heterochromatin. The data obtained also indicate that both heterochromatin and the 45S rDNA loci could be constrained by similar evolutionary forces regulating spreading in the distinct Scarabaeinae subfamily lineages. For the 5S rRNA and the histone H3 genes, a similar chromosomal organization could be attributed to their association/co-localization in the Scarabaeinae karyotypes. These data provide evidence that different evolutionary forces act at the heterochromatin and the 45S rDNA loci compared to the 5S rRNA and histone H3 genes during the evolution of the Scarabainae karyotypes.

Cytogenetic analysis of two Coprophanaeus species (Scarabaeidae) revealing wide constitutive heterochromatin variability and the largest number of 45S rDNA sites among Coleoptera

The Coleopterans of Scarabaeinae clade presents Coprophanaeus ( Megaphanaeus) ensifer and C. ( Coprophanaeus) cyanescens (Scarabaeidae) when they are studied cytogenetically by different techniques. The species present symmetric karyotypes, diploid number of 2 n = 20, and meta-submetacentric chromosomes. C. ( M.) ensifer present an XY sex-determining mechanism and C. ( C.) cyanescens an XY p parachute mechanism. Analysis of constitutive heterochromatin (CH) in the two species revealed the presence of diphasic autosomes, with log arm heterochromatics. Moreover, an additional heterochromatic block in four autosomal bivalents were observed in C. ( M.) ensifer. CMA 3/DA/DAPI fluorochrome staining detected CMA 3 positive heterochromatic blocks restricted to the sex chromosomes in C. ( C.) cyanescens, whereas in C. ( M.) ensifer CMA 3 positive pericentromeric blocks were present in all autosomes, in the Y chromosome and in the four additional heterochromatic blocks. DAPI staining was neutral in both species. Silver nitrate (AgNO 3) staining was inefficient for the detection of the nucleolar organizer region (NORs), but showed affinity for the heterochromatic regions. Fluorescence in situ hybridization (FISH) revealed the presence of 45S rDNA sites in the terminal region of the three autosomal bivalents of C. ( C.) cyanescens and in seven bivalents and the Y chromosome of C. ( M.) ensifer. These results contribute to a better understanding of chromosome evolution in the genus Coprophanaeus, and demonstrate a wide CH variability and the largest number of ribosomal sites among Coleoptera.

Cytogenetic evidence of nucleolar dominance in allotetraploid species ofBrachypodium

Sequential silver staining and fluorescence in situ hybridization (FISH) were used to establish activity and number of 45S rDNA sites in meristematic root tip cells of 6 ecotypes of allotetraploid (2n = 4x = 30) species of Brachypodium and their putative ancestors, B. distachyon (2n = 2x = 10) and ABR114 (2n = 2x = 20). Using either total nuclear DNA of ABR114 or the ABR1-63-E6 BAC clone from a B. distachyon genomic library as an auxiliary probe, it was possible to distinguish by FISH between the two genomes composing the ecotypes of allotetraploid Brachypodium species and to determine unambiguously the parentage of both dominant and suppressed rRNA genes. Each of the diploid species possessed two rDNA loci, both transcriptionally active. The number of 45S rDNA sites in 6 ecotypes of allotetraploid Brachypodium species was always equal to the sum of loci present in their putative diploid parents. Two smaller sites were located in chromosomes corresponding to the ABR114 chromosomal set, and two larger ones in the chromosomes of B. distachyon origin. In all analyzed allotetraploid ecotypes, only rRNA genes belonging to the B. distachyon-like genome were transcriptionally active, while rDNA from the other parental genome was always suppressed. Thus the occurrence of nucleolar dominance in the allotetraploid (2n = 4x = 30) species of Brachypodium is demonstrated for the first time.

Comparative karyotype analysis of threePassifloraL. species and cytogenetic characterization of somatic hybrids

The present study was carried out to analyze the karyotypes of three Passiflora species with 2n = 18 (the yellow passion fruit P. edulis f. flavicarpa and the wild species P. amethystina and P. cincinnata) previously used to obtain somatic hybrids by protoplast fusion. A biometrical analysis of Feulgen stained metaphases showed that the karyotypes of these species were quite similar with subtle differences in the morphology of some chromosomes be- tween the species. These data were integrated with the physical mapping of the 45S rDNA (18S-5.8S-26S rRNA genes) and 5S rDNA loci using FISH, and with the pattern of fluorescent staining with chromomycin A3. FISH sig- nals of 45S rDNA were localized on the secondary constriction and satellite of two chromosome pairs, identified as 8 and 9, in the three species. An additional site was observed on the long arm of chromosome 1 in P. amethystina. Chromomycin A3 staining revealed highly fluorescent bands corresponding to 45S rDNA loci. One 5S rDNA site was detected on the long arm of chromosome pair 4 in the three species. Silver staining showed four positive signals on the small satellited chromosomes, and the locus on chromosome 1 of P. amethystina seemed to be inactive. Soma- clones derived from four somatic hybrid plants (P. edulis + P. amethystina) had 2n = 4x = 36, but in samples of two hybrids, 2n = 35 was observed. Variability in the number of 45S rDNA sites (10 or 12) was detected among the hy- brids, and the FISH signals were smaller than in the parental species. These findings suggest that DNA sequence loss and transposition occurred in these newly formed polyploids.

Simultaneous Visualization of 5S and 45S rDNAs in Persimmon (Diospyros kaki) and Several Wild Relatives (Diospyros spp.) by Fluorescent in situ Hybridization (FISH) and MultiColor FISH (MCFISH)

5S ribosomal DNA (rDNA) was visualized on the somatic metaphase chromosome of persimmon (Diospyros kaki) and ten wild Diospyros species by fluorescent in situ hybridization (FISH). The digoxigenin (DIG)-labeled 5S rDNA probe was hybridized onto the chromosomes and visualized by incubation with anti-DIG-fluorescein isothiocyanate (FITC). Strong signals of 5S rDNA probe were observed on several chromosomes of Diospyros species tested. Furthermore, multicolor FISH using 5S and 45S rDNA probes differently labeled with DIG and biotin, revealed separate localization of the two rDNA genes on different chromosomes of Diospyros species tested, suggesting that 5S and 45S rDNA sites can be used as chromosome markers in Diospyros. The number of 5S rDNA sites varied with the Diospyros species. More 5S rDNA sites were observed in four diploid species native to Southern Africa than in three Asian diploid species. The former had four or six 5S rDNA sites while the latter had two. Three Asian polyploidy species had four to eight 5S rDNA sites. Among the Asian species, the number of 5S rDNA sites seemed to increase according to ploidy level of species. These features of 5S rDNA sites were very similar to those of 45S rDNA sites in Diospyros. Phylogenetic relationship between D. kaki and wild species tested are discussed based on the number and chromosomal distribution of 5S and 45S rDNA.