Comparative cytogenetics and chromosomal characteristics of ribosomal DNA in the fish genus Vimba (Cyprinidae)

Karyotypic and cytogenetic characteristics of Vimba vimba and V. elongata were investigated using differential staining techniques (sequential C-banding, Ag- and CMA₃-staining) and fluorescent in situ hybridization (FISH) with 28S rDNA probe. The diploid chromosome number in both species was 2n = 50 with 8 pairs of metacentrics, 14 pairs of submetacentrics to subtelocentrics and 3 pairs of subtelo- to acrocentrics. The largest chromosome pair of the complements was characteristically subtelo- to acrocentric. The nucleolar organizer regions (NORs) in both species were detected in the telomeres of a single, middle-sized subtelocentric chromosome pair, a pattern common in a number of other Leuciscinae. FISH with rDNA probe produced consistently positive hybridization signals detected in the same regions indicated by Ag-staining and CMA₃-fluorescence. The distribution of C-positive heterochromatin was identical in both species, including a conspicuous size polymorphism of heterochromatic blocks in the largest metacentric and subtelo- to acrocentric chromosomal pairs. No heteromorphic sex chromosomes were detected. A single analyzed individual of V. melanops possessed the same karyotype and NOR phenotype as V. vimba and V. elongata. The apparent karyotype homogeneity and chromosomal characteristics of ribosomal DNA in all three species of the genus Vimba is consistent to that found in most other representatives of the European leuciscine cyprinid fishes.     

Molecular cytogenetics of <Emphasis Type="Italic">Oligosarcus hepsetus</Emphasis> (Teleostei,Characiformes) from two Brazilian locations

Karyotype and cytogenetic markers of Oligosarcus hepsetus from two Brazilian locations in the Paraíba do Sul River Basin (Brazil) were investigated using differential staining techniques (C-banding, silver (Ag)- and chromomycin A₃ (CMA₃)-staining) and fluorescent in situ hybridization (FISH) using 18 S rDNA and 5 S rDNA probes. The diploid chromosome number was invariably 2n = 50 with 3 pairs of metacentric, 5 pairs of submetacentric, 8 pairs of subtelocentric and 9 pairs of acrocentric chromosomes. No heteromorphic sex chromosomes were observed. The nucleolar organizer regions (NORs) were detected in the short arms of the largest acrocentric pair using Ag-, CMA₃- stainings and FISH with 18 S rDNA probe, the latter showing also positive labeling in the short arms of a small acrocentric pair, not visualized by the former methods. FISH with 5 S rDNA probe showed positive labeling in the two chromosome pairs. While the CMA₃-staining exhibited GC-rich heterochromatin segments in two pairs of chromosomes, including those coincided with Ag-NORs, the DAPI staining did not reveal any signal, indicating the absence of AT-rich heterochromatin. FISH with an As-51 satellite DNA probe derived from the closely related Astyanax scabripinnis did not reveal any positive signal, demonstrating the absence of this class of DNA in the genome of the specimens under study.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi, Cobitidae) detected by different banding techniques and fluorescence in situ hybridization (FISH)

When surveying the karyotype diversity of European loaches of the genus Cobitis to identify species involved in hybrid polyploid complexes, an extensive polymorphism in number and location of NORs was discovered in C. vardarensis using Ag-staining, C-banding, CMA₃-fluorescence and fluorescence in situ hybridization (FISH). This species had 2n=50, the karyotype contained 13 pairs of metacentric, 10 pairs of submetacentric and two pairs of subtelocentric chromosomes. The NOR-bearing chromosomes included one medium-sized metacentric pair with a large CMA₃-positive heterochromatic pericentromeric block, one small metacentric as well as one large submetacentric pairs. Ribosomal sites were always located in telomeres of these chromosomes. Each of the pair of NOR-bearing chromosomes occurred in three variants – (1) presence and/or (2) absence of NORs on both homologues and (3) heterozygous combination where only one of the homologues bears NORs. Altogether, 10 different NOR cytotypes from 27 theoretically possible ones were discovered among 20 indviduals examined. The number of NORs ranged from two to five per specimen. The results regarding the number and locations of NORs as revealed by banding techniques were confirmed using FISH with rDNA probe. NOR sites were of CMA₃-positive, suggesting that ribosomal sites are associated with GC-rich DNA. Very similar structural polymorphism with multiple NORs is expressed in the Danubian loach C. elongatoides indicating a close relationship between both species.     

Chromosome analysis of walking catfish,Clarias magur (Teleostei,Siluriformes, Clariidae), using staining,FISH with 18S, 5S and BAC probes

The chromosomal characteristics of Clarias magur were examined using conventional (Giemsa-staining, Ag-impregnation and CMA₃ + DAPI fluorescence) and molecular/ FISH (18S & 5S rDNA probes + one BAC DNA probe) cytogenetic tools. The diploid chromosome number was 50 and the karyotype consisted of 14 metacentric, 20 sub-metacentric, 8 sub-telocentric, 8 acrocentric chromosomes with 84 chromosome arms without any heteromorphic pair. The C-heterochromatic blocks were located on centromeric position of 13 pairs of chromosomes. The NOR sites, visualized by AgNO₃- and CMA₃- staining, were situated at p arms of chromosome pair No. 21, which also corresponded to 18S rDNA site visualized by FISH. The FISH signal of ICF_001_D19 clone probe was observed on 18th chromosome pair. The findings of the present study on C. magur provided valuable markers for the chromosome identification and locations of genes of the BAC clone on the chromosome will lead to the construction of physical map of genome of this species.     

Chromosome banding and FISH with rDNA probe in the diploid and tetraploid loach Misgurnus anguillicaudatus

The chromosomes of the diploid and tetraploid loach Misgurnus anguillicaudatus were analyzed by staining with Ag, chromomycin A₃ (CMA₃)/distamycin A (DA), and DA/4′,6-diamidino-2-phenylindole (DAPI), and using fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Nucleolus organizer regions (NORs) were mapped to the telomeric region of the short arms of the largest (first) metacentric chromosome pair in the diploid loach with 2n = 50 and the homologous quartet in the tetraploid loach with 4n = 100. The NORs were positive at the same region of the first metacentric chromosome for Ag and CMA₃/DA stainings, but negative for DA/DAPI staining. Four signals at the homologs within the same quartet suggest the duplication of the entire genome from diploid to tetraploid status. However, a size difference was detected between the rDNA signals by FISH and CMA₃ banding.     

Study of the Nucleolar Organizer Regions in Palinurus elephas (Crustacea: Decapoda)

Using fluorescence in situ hybridization (FISH), chromomycin (CMA₃) staining and silver staining, we studied the nucleolar organizer regions in the spiny lobster Palinurus elephas in order to extend our knowledge on the karyology of this commercially important species. Multiple NORs have been detected by FISH, and CMA₃ showed a good correspondence between the localization of GC-rich heterochromatin and the ribosomal genes mapped by FISH. In contrast, the number of Ag-positive regions was higher than the number of FISH and CMA₃ signals, which may be explained by silver staining of the kinetochores. A variability in the number of FISH and CMA₃ signals has been detected in metaphases I and II which is probably due to the occurrence of rDNA cistrons on B chromosomes.     

Diversification of a ZZ/ZW sex chromosome system in Characidium fish (Crenuchidae, Characiformes)

Karyotype and other chromosomal markers of Characidium cf. gomesi were analyzed using conventional (Giemsa-staining, Ag-NOR and C-banding) and molecular (Fluorescent in situ hybridization (FISH) with 18S and 5S rDNA biotinylated probes) techniques. Both sexes had invariably diploid chromosome number 2n = 50 while karyotypes of males and females differed. That of male consisted of 32 metacentric + 18 submetacentric chromosomes and that of female consisted 31 metacentric + 18 submetacentric + 1 subtelocentric chromosomes. The Z chromosome was medium-sized metacentric, while W was highly heterochromatinized subtelocentric element. NORs as revealed by Ag-staining were situated at 2–7 telomeric regions while FISH with 18S probes showed consistently 10 signals at telomeric regions. FISH with 5S rDNA probe showed constantly signals at one metacentric pair. Distribution of centromeric heterochromatin was mostly in all chromosome pairs, besides some telomeric sites. The common origin of the sex chromosome system of ZZ/ZW type in the karyotypes of other representatives of the genus analyzed so far might be hypothesized based on biogeography and partial phylogeny of the group.     

Classical and molecular cytogenetics and DNA content in Maihuenia and Pereskia (Cactaceae)

We studied cacti species of the subfamilies Pereskioideae (five species of the southern clade) and both species of Maihuenioideae using molecular cytogenetic techniques and DNA content. Mitotic chromosomes were analyzed for Pereskia aculeata, P. bahiensis, P. grandifolia, P. nemorosa, P. sacharosa, Maihuenia poeppigii, and M. patagonica, using the Feulgen stain, CMA/DAPI fluorescent chromosome banding, fluorescence in situ hybridization (FISH, probes of 5S rDNA and pTa71 for 18-5.8-26S rDNA), and DNA content by flow cytometry technique. The karyotypes were highly symmetrical, most of the pairs being metacentric (m). CMA/DAPI banding revealed the presence of CMA⁺/DAPI^? bands associated with NORs in the first m pair of all species. The co-localization of 18-5.8-26S rDNA loci with CMA⁺/DAPI^?/NORs blocks allowed the identification of homeologous chromosome pairs between species of both subfamilies. FISH using probe 5S rDNA was applied for the first time in both subfamilies. Diploid species had always one m pair carrying 5S rDNA genes, with pericentromeric location in different chromosome pairs. In the tetraploid cytotype of M. patagonica, the 5S rDNA probe hybridized to two pairs. The 2C DNA content obtained by FC varied twofold (from 1.85 to 2.52 pg), with significant differences between species. Mean chromosome length, karyotype formula, percentage of heterochromatin position of 5S rDNA locus, and nuclear Cx DNA content vary among Maihuenia and Pereskia species and allowed to differentiate them. Both genera are closely related and that the differences found are not strong enough to separate Maihuenioideae from Pereskioideae.     

Karyotype analysis of the pumpkinseed Lepomis gibbosus (Actinopterygii,Centrarchidae) by chromosomal banding and in situ hybridization

Sunfish are widely distributed in their native North American freshwaters and in many other geographic regions, including Europe. In this work the cytogenetics of L. gibbosus were studied. In particular, the authors localized the heterochromatic regions and the major and minor ribosomal gene families. The nucleolar organizer region was localized terminally in the short arm of only one pair, a large acrocentric pair, using both FISH and silver staining. Furthermore, the 5S ribosomal gene family was also localized by FISH in only one pair, in the centromeric region of the smallest chromosome pair. NOR and 5S rDNA regions were both C‐positive and CMA₃ positive. The CMA₃ positivity of the 5S ribosomal cluster is uncommon in fish, however, a similar situation has been found in M. salmoides, the only other centrarchid species studied with the same techniques. Moreover, the 5S ribosomal gene was sequenced and its molecular structure analysed.     

Multiple nucleolus organizer regions in Leptodactylus mystacinus (Amphibia, Anura) and comments on its systematic position in the L. fuscus group based on cytogenetic and molecular analyses

Specimens of Leptodactylus mystacinus from Brazil were karyotyped with conventional and differential staining. The 2n = 22 karyotype is similar to that found for the majority of the Leptodactylus, the karyotypic conservatism also confirmed by the similarity of the replication banding patterns with those previously described. L. mystacinus has a small amount of C-banded heterochromatin, located mainly at the centromeres, although telomeric or interstitial bands have also been noticed. With DA/CMA₃ some chromosome regions showed slightly bright fluorescence, and with DA/DAPI, no particular AT-rich repetitive region was observed. Silver staining showed an extensive inter- and intraindividual variation in the number and position of Ag-positive regions, in 1p, 4p, 8p, 8q, and 11p. Nevertheless, FISH using rDNA probes confirmed only the signals on the short arms of chromosomes 4 and 8 as true NORs. The remaining silver stained regions are probably due to the heterochromatin with some affinity to the Ag-staining. Phylogenetic analysis based on partial cytochrome b sequence revealed that L. mystacinus forms a basal branch, so that the presence of multiple NORs in pairs 4 and 8 in this species indicates an autapomorphy. Supported by FAPESP and CNPq.     

Constitutive heterochromatin, 5S and 18S rDNA genes in Apareiodon sp. (Characiformes, Parodontidae) with a ZZ/ZW sex chromosome system

Karyotype, sex chromosome system and cytogenetics characteristics of an unidentified species of the genus Apareiodon originating from Piquiri River (Paraná State, Brazil) were investigated using differential staining techniques (C-banding and Ag-staining) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The diploid chromosome number was 2n = 54 with 25 pairs of meta- (m) to submetacentric (sm) and 2 pairs of subtelocentric (st) chromosomes. The major ribosomal rDNA sites as revealed by Ag-staining and FISH with 18S rDNA probe were found in distal region of longer arm of st chromosome pair 26, while minor 5S sites were observed in the interstitial sites on chromosome pairs 2 (smaller cluster) and 7 (larger one). The C-positive heterochromatin had pericentromeric and telomeric distribution. The heteromorphic sex chromosome system consisted of male ZZ (pair 21) and female middle-sized m/st Z/W chromosomes. The pericentric inversion of heterochromatinized short arm of ancestral Z followed by multiplication of heterochromatin segments is hypothesized for origin of W chromosome. The observed karyotype and chromosomal markers corresponded to those found in other species of the genus.     

Localization of 5S rRNA Loci in Three Coregonid Species (Salmonidae)

In the present study the chromosome distribution of the 5S rDNA loci and its relation to the major rDNA genes were investigated in three Coregonid species (Salmonidae): Coregonus lavaretus, Coregonus peled and Coregonus albula, a family which has experienced large karyotype rearrangements along its evolution starting from a tetraploid ancestor. 5S PRINS/CMA₃ sequential staining together with previous data enabled us to locate 5S rRNA genes and nucleolar organizer regions (NORs) in the three species analyzed. PRINS revealed the 5S rDNA cluster at the distal part of the long arm of a similar submetacentric chromosome pair in the three species. Our data indicate that 5S rDNA clusters have probably conserved chromosomal location in the genus Coregonus, whereas 45S rDNA (NOR) sites are clearly differentiated, from a single locus in C. peled, to multiple loci in C. lavaretus and highly polymorphic multichromosomal location in C. albula.     

Frequency analyses of active NORs in nuclei of artificially induced triploid fishes

Summary The correspondence between increased numbers of both chromosomal and nuclear NORs and artificially induced triploidy in three fish species (rainbow trout, Oncorhynchus mykiss; common carp, Cyprinus carpio; and tench, Tinea tinea) has been confirmed by CMA₃ fluorescence and Ag-staining. The frequencies of cell nuclei with one, two and three active NORs, as revealed by Ag-staining, has been analyzed statistically to find the minimum cell number which verifies the increased ploidy level. A minimum sample size of about 80 cells exhibiting three active NORs is sufficient to confirm triploidy in all three species and may be of use for categorising other ploidy-manipulated fish species.     

A Population Analysis of the Structure and Variability of NOR in Salmo Trutta by Ag, CMA3 and ISH

An analysis of the variation in the number and location of rDNA genes has been carried out in two populations of brown trout (Salmo trutta) from Poland by using Ag and CMA₃-staining, and rDNA in situ hybridisation. We observed an interindividual variation in arm number with NF = 100, 101, and 102. This variation was connected with the size polymorphism of the short (NOR-bearing) arm of the chromosome pair 11. The population studied showed a multichromosomal distribution of active NORs. Atypical Ag-NORs consisted of rDNA genes, as evidenced by rDNA-ISH. In addition to individuals with standard NORs, specimens with extra NORs as well as others with only one active NOR and single interphase nucleolus were observed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chromosome banding in Amphibia

The structure of the nucleolus organizer regions (NORs) in mitotic chromosomes, diploid nuclei and spermatogenesis was studied in 260 individual animals from 23 genera of the Anura. The analyses were performed with conventional cytogenetic methods as well as with Ag-staining, GC- and AT-specific fluorochromes (mithramycin, chromomycin A₃, quinacrine) and C-banding. Most of the species have only one pair of NORs in their karyotypes. The majority of individuals of all species exhibited considerable differences in the sizes of their homologous NORs. Most of these heteromorphisms are due to tandem duplications or triplications in one of the two NORs. However, duplicated or triplicated NORs never occur in a homozygous form, but are instead always in combination with a normal-structured NOR in the homologous chromosome. In three animals, a complete deletion of one NOR and its closely associated constitutive heterochromatin was determined. The cytochemistry of the specific NOR-stainings are discussed. The size differences of the Ag-, mithramycin- and chromomycin A₃-stained NORs can be traced to differences in the rDNA content in these NORs.     

Karyology of the Antarctic scallop Adamussium colbecki, with some comments on the karyological evolution of pectinids

Karyotype, location of the nucleolar organiser region (NOR) and heterochromatin presence and composition were studied in the Antarctic scallop Adamussium colbecki Smith, 1902. The karyotype exhibits 2n = 38 chromosomes with 11 pairs of metacentrics, 5 of submetacentrics, one subtelocentric and two telocentrics. Ag–NOR, CMA₃, DA/MM and NOR–FISH evidenced paracentromeric NORs on the short arm of 2nd pair chromosomes. Digestion with three restriction endonucleases followed by sequential staining with Giemsa, CMA₃ and DAPI evidenced on all chromosomes centromeric heterochromatin positive for both DAPI and CMA₃. In situ hybridisation analysis showed the presence of an AT-rich satellite DNA in the centromeric heterochromatin of several chromosomes. A mosaicism was detected in the germinal cell lines of one specimen, as in six of the 20 plates examined the set had 37 chromosomes with a missing pair of telocentrics and an unpaired metacentric. Comparison of the chromosome sets of all the pectinids studied to date and comparison with a phyletic tree obtained from molecular mitochondrial genes studies yielded good agreement between karyotype morphology and taxonomic classification.     

Chromosomal localization of 18S and 5S rDNA using FISH in the genus <Emphasis Type="Italic">Tor</Emphasis> (Pisces,Cyprinidae)

Dual color fluorescence in situ hybridization (FISH) was performed to study the simultaneous chromosomal localization of 18S and 5S ribosomal genes in the genus Tor for the first time. The 18S and 5S rDNAs in four Tor species were amplified, sequenced and mapped on the metaphase chromosomes. The number and distribution of 18S and 5S rDNA clusters were examined on metaphase chromosome spreads using FISH. The specimens of T. chelynoides, T. putitora and T. progeneius showed six bright fluorescent signals of 18S rDNA and T. tor exhibited ten such signals. The 5S rDNA signals were present only on one pair of chromosomes in all the four Tor species. Ag-NORs were observed on two pairs of chromosomes in T. chelynoides, T. putitora, T. progeneius and four pairs in T. tor. Comparison of the observed 18S rDNA FISH signals and Ag-NORs strongly suggested a possible inactivation of NORs localized at the telomeres of a subtelocentric and telocentric chromosome pairs in all four species. The 5S rDNA contained an identical 120 bp long coding region and 81 bp long highly divergent non-transcribed spacers in all species examined. 18S and 5S rDNA sequencing and chromosomal localization can be a useful genetic marker in species identification as well as phylogenetic and evolutionary studies.     

Karyotype and chromosomal characteristics of Ag–NOR sites and 5S rDNA in European smelt, Osmerus eperlanus

Karyotype and cytogenetic characteristics of European smelt Osmerus eperlanus were investigated using different staining techniques (sequential Ag-, CMA3 and DAPI banding) and PRINS to detect 5S rDNA and telomeric sites. The diploid chromosome number was invariably 2n = 56 and karyotype composed of 5 pairs of metacentrics, 9 pairs of subtelocentrics and 14 pairs of subtelo- to acrocentrics. The DAPI-positive heterochromatic regions were found in centromeric positions on bi-armed chromosomes and few acrocentrics. Additionally, some interstitial DAPI-positive bands were identified on three pairs of submetacentric chromosomes. The nucleolar organizer regions (NORs) were detected in the short (p) arms of the largest metacentric pair of chromosomes No. 1. Sequential banding (Giemsa-, AgNO₃ and CMA₃ stainings) revealed NOR sites corresponding to achromatic regions but not associated with CMA₃-positive blocks of heterochromatin located on either side of NORs. Individuals from the analyzed population had this conspicuous pair of chromosomes always in heterozygous combination. A complex inversion system was hypothesized to be involved in the origin of the observed variation but analysis with telomeric PRINS and PNA-FISH did not reveal any Interstitial Telomeric Sites (ITS). Hybridization signals were confined exclusively to terminal chromosomal regions. The 5S ribosomal sites as revealed by PRINS were found to be invariably located in the short (p) arms of four pairs of subtelocentric chromosomes. Cytotaxonomic comparisons of the present results with the voluminous available cytogenetic data-set from salmoniform and esociformes fishes appear to support the recent view, based on robust molecular-based phylogeny, that salmoniform and osmeriform fishes are not as closely related as previously assumed.