Cytological analysis of hybrids among triticales and trigopiros

WE STUDIED THREE DIFFERENT TRICEPIROS: (Don Santiago x Don Noé), (Cumé x Horovitz) and (Cumé x Don Noé). The tricepiro (Don Santiago x Don Noé) was obtained by crossing the triticale Don Santiago INTA (AABBRR, 2n = 6x = 42) with the trigopiro Don Noé INTA (AABBDDJJ, 2n = 8x = 56). The number of chromosomes for the F(1) was 2n = 49, the most frequent meiotic configuration being 14 bivalents and 21 univalents. The univalents were situated in the periphery of the equatorial plane, whereas the bivalents were located in the central zone. The chromatids in some of the univalents split when bivalents underwent reductional division in anaphase I. There were few laggard chromosomes or chromatids at this phase. The number of chromosomes (2n = 48-58) was high and variable, and the number of bivalents per cell (18-23) also high in F (3) individuals. In all F (8) tricepiros (Don Santiago x Don Noé), F (12) tricepiros (Cumé x Horovitz) and F (12) tricepiros (Cumé x Don Noé), the number of chromosomes (2n = 42) was the same, these retaining the rye genome, as demonstrated by GISH and FISH. These new synthesized allopolyploids constitute interesting models for investigating the evolutionary changes responsible for diploidization, and the chromosomal and genomic re-ordering that cannot be revealed in natural allopolyploids.     

The fate of ribosomal genes in three interspecific somatic hybrids of Medicago sativa: three different outcomes including the rapid amplification of new spacer-length variants

We have characterized the genetic consequences of somatic hybridization within the ribosomal DNA (rDNA) of three interspecific hybrids, each involving M. sativa as one of the parents. Restriction-fragment-length-polymorphisms (RFLPs) of rDNA spacers and fluorescent-in-situ-hybridization (FISH) of an 18S-gene probe to mitotic chromosomes were used to compare parental and hybrid species. The M. sativa-coerulea hybrid retained all six parental nucleolar-organizing regions (NORs) and all parental RFLPs representing a complete integration of rDNA. The M. sativa-arborea hybrid retained five of six parental NORs while losing half of the arborea-specific RFLPs, indicating that simple chromosome loss of one arborea NOR accounted for the RFLP losses. Dramatic alterations occurred within the M. sativa-falcata hybrid where five of six parental NORs were retained and new rDNA RFLPs were created and amplified differentially among somaclonal-variant plants. The molecular basis of the new RFLPs involved increased numbers of a 340-bp subrepeating element within the rDNA intergenic spacer (IGS), suggesting that recurrent cycles of unequal recombination occurred at high frequency within the rDNA in somatic lineages.This paper was supported by the National Research Council of Italy, Special Project RAISA, Sub-project No. 2, Paper No. 1077     

Cytogenetic study in Corydoras britskii (Siluriformes: Callichthyidae), using different chromosomal banding and fluorescence hybridization in situ with rDNA probes

Cytogenetic analyses were performed on Corydoras britskii from the Miranda River basin, an important river located in the Pantanal, Mato Grosso do Sul State, Brazil. The karyotype of this species comprises 90 chromosomes and a karyotype formula of 4m + 10 sm + 22 st + 54a. The nucleolus organizer regions were detected by impregnation with silver nitrate and FISH with an 18S rDNA probe on the short arm of three acrocentric chromosomes. The constitutive heterochromatin is distributed in pericentromeric and interstitial positions, and also associated with the NORs. The HinfI restriction endonuclease was used and showed homology with practically all types of heterochromatin observed in C. britskii, except for two interstitial heterochromatic blocks present in a subtelocentric pair. The fluorochrome staining evidenced six chromosomes with chromomycin-positive signals indicating that both the heterochromatin interspersed with NORs and some heterochromatic blocks were rich in GC base pairs. FISH using a 5S rDNA probe revealed the presence of these regions in only one subtelocentric pair in the interstitial position. The obtained data substantiate the karyotype diversity of the genus Corydoras and provide novel information about the composition of heterochromatin and location of 5S and 18S rDNA sites.     

Considerations on karyotype evolution in the genera Imparfinis Eigenmann and Norris 1900 and Pimelodella Eigenmann and Eigenmann 1888 (Siluriformes: Heptapteridae)

Heptapteridae is one of the fish families of the order Siluriformes with a wide distribution throughout the basins of the Neotropical region. The genera Imparfinis and Pimelodella comprise few species and/or populations with some chromosome information. Specimens of Imparfinis schubarti, Imparfinis mirini, and Pimelodella meeki from different sites located in the Paranapanema River Basin/PR/Brazil were cytogenetically analyzed. The two species of the genus Imparfinis exhibited 2n = 58 and FN = 116: I. schubarti, with a karyotypic formula of 30m + 28sm and I. mirini with a karyotype formula of 36m + 22sm. P. meeki presented a karyotype of 2n = 46 characterized by 26m + 14sm + 6st and FN = 92, confirming a variability in the 2n of the Heptapteridae family. Both Imparfinis species exhibited interstitial NORs in pair 1, coincident with a secondary constriction; P. meeki presented NORs located in the terminal position on the short arm of pair 17. All AgNORs were coincident with 18S rDNA probe and CMA₃ positive. P. meeki showed a small amount of heterochromatin rich in AT and GC bases. The heterochromatin in I. schubarti was CMA₃ positive. In I. mirini the heterochromatin was DAPI-positive. Furthermore, the long arm of one of the chromosomes of pair 19 revealed the presence of heterochromatic heteromorphism only in male individuals. After meiotic analyses, this heteromorphism could be easily identified in the pachytene and metaphase I stages, and was heteropyknotic and DAPI positive. This feature may be an indication of initial differentiation of sex chromosomes in I. mirini, increasing the great karyotypic variability within this family of fish.     

Distribution of rDNA loci in the genus Glycine Willd.

The objective of this study was to examine the distribution of rDNA loci in the genus Glycine Willd. by fluorescent in situ hybridization (FISH) using the internal transcribed spacer (ITS) region of nuclear ribosomal DNA as a probe. The hybridized rDNA probe produced two distinct yellow signals on reddish chromosomes representing two NORs in 16 diploid (2n=40) species. Aneudiploid (2n=38) and aneutetraploid (2n=78) Glycine tomentella Hayata also exhibited two rDNA sites. However, the probe hybridized with four chromosomes as evidenced by four signals in two diploid species (Glycine curvata Tind. and Glycine cyrtoloba Tind.) and tetraploid (2n=80) G. tabacina (Labill.) Benth. and G. tomentella. Synthesized amphiploids (2n=80) of Glycine canescens F. J. Herm. (2n=40) and the 40-chromosome G. tomentella also showed four signals. This study demonstrates that the distribution of the rDNA gene in the 16 Glycine species studied is highly conserved and that silence of the rDNA locus may be attributed to amphiplasty during diploidization and speciation. Received: 10 October 2000 / Accepted: 6 December 2000     

Assessment of intergenomic recombination through GISH analysis of F1, BC1 and BC2 progenies of Tulipa gesneriana and T. fosteriana

Using 23 F1 hybrids, 14 BC1 and 32 BC2 progenies, the genome composition of Darwin hybrid tulips was analysed through genomic in situ hybridisation (GISH) of somatic chromosomes. All plants were diploids (2n = 2x = 24) with the exception of one tetraploid BC1 (2n = 4x = 48) and one aneuploid BC2 (2n = 2x + 1 = 25) hybrid. Morphometric analysis in F1 hybrids revealed a difference in the total length of chromosomes representing genomes of T. gesneriana and T. fosteriana, where the percentage of each genome equaled 55.18 ± 0.8 and 44.92 ± 0.6% respectively. GISH distinguished chromosomes from both parent genomes although there was a lack of consistent chromosome labelling in some cases. In both T. gesneriana and T. fosteriana chromosomes some segments of heterochromatin in the telomeric and intercalary regions exhibited a higher intensity of fluorescence. In situ hybridisation with 5S rDNA and 45S rDNA probes to metaphase chromosomes of F1 hybrids showed that these regions are rich in rDNA. A notable feature was that, despite genome differences, there was a considerable amount of intergenomic recombination between the parental chromosomes of the two species as estimated in both BC1 and BC2 offspring. The number of recombinant chromosomes ranged from 3 to 8 in BC1 and from 1 to 7 in BC2 progenies. All recombinant chromosomes possessed mostly a single recombinant segment derived from either a single crossover event or in a few cases double crossover events. This explains the fact that, unlike the situation in most F1 hybrids of other plant species, certain genotypes of Darwin hybrid tulips behave like normal diploid plants producing haploid gametes and give rise to mostly diploid sporophytes.     

Molecular cytogenetic study of the European bitterling Rhodeus amarus (Teleostei: Cyprinidae: Acheilognathinae)

The European bitterlings (Rhodeus amarus) from the Eastern locations were cytogenetically examined by conventional and molecular techniques. All analyzed individuals presented invariably the same chromosomal constitution of 2n = 48, with 8 metacentrics + 20 submetacentrics + 20 subtelo-acrocentrics and C-banding positive heterochromatin at the pericentromeric regions in most of the chromosomes. Moreover, some of the chromosomes had short arms entirely built with heterochromatin. GC-rich Ag-NORs (nucleolus organizer regions) were located at the short arms of two submetacentric chromosomes, and the length polymorphism of these regions was found. Multiple location of 28S rDNA sequences with fluorescence in situ hybridization signals was observed on the long and/or short arms of three submetacentric chromosomes including NOR regions and short arms of three to five acrocentric chromosomes in the studied fish. 5S rDNA sites were found on the short arms of two subtelocentric chromosomes, and telomeric repeats were localized at the ends of all chromosomes. Provided results have expanded our knowledge concerning genetic characteristics of the European bitterlings that may be profitable in the conservation programs of this endangered species.     

XX/XO, a rare sex chromosome system in Potamotrygon freshwater stingray from the Amazon Basin, Brazil

Potamotrygonidae is a representative family of South American freshwater elasmobranchs. Cytogenetic studies were performed in a Potamotrygon species from the middle Negro River, Amazonas, Brazil, here named as Potamotrygon sp. C. Mitotic and meiotic chromosomes were analyzed using conventional staining techniques, C-banding, and detection of the nucleolus organizing regions (NOR) with Silver nitrate (Ag-NOR). The diploid number was distinct between sexes, with males having 2n = 67 chromosomes, karyotype formula 19m + 8sm + 10st + 30a, and fundamental number (FN) = 104, and females having 2n = 68 chromosomes, karyotype formula 20m + 8sm + 10st + 30a, and FN = 106. A large chromosome, corresponding to pair number two in the female karyotype, was missing in the male complement. Male meiotic cells had 33 bivalents plus a large univalent chromosome in metaphase I, and n = 33 and n = 34 chromosomes in metaphase II. These characteristics are consistent with a sex chromosome system of the XX/XO type. Several Ag-NOR sites were identified in both male and female karyotypes. Positive C-banding was located only in the centromeric regions of the chromosomes. This sex chromosome system, which rarely occurs in fish, is now being described for the first time among the freshwater rays of the Amazon basin.     

Cytogenetics characterization of <Emphasis Type="Italic">Crenuchus spilurus</Emphasis> (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 (Caeté 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.     

Cytogenetic characterization of the ant Trachymyrmex fuscus Emery, 1934 (Formicidae: Myrmicinae: Attini) with the description of a chromosomal polymorphism

The genus Trachymyrmex is a key group in the tribe Attini because of its close phylogenetic relationship to leaf-cutter ants, Acromyrmex and Atta. Cytogenetic data are only available for five taxa of Trachymyrmex, with chromosome numbers of 2n = 12, 18, 20 and 22, and morphology with predominantly metacentric chromosomes. The aim of the present study was to characterize the karyotype of the ant Trachymyrmex fuscus Emery, 1934, by means of the number and morphology of its chromosomes, heterochromatin pattern, CMA₃ and DAPI fluorochromes in the population of two nests collected at Paraopeba, state of Minas Gerais, Brazil. Nineteen females presented 2n = 18 chromosomes (16m + 2sm) and a single male presented n = 9 (8m + 1sm). A size chromosomal polymorphism involving the short arm of the submetacentric pair was confirmed by statistical analysis, with three character conditions: heterozygous SB (with a difference in size between the short arms), standard SS (smaller short arms) and homozygote BB (bigger short arms). In the first nest, both SB and SS workers were observed. The other nest contained heterozygous (SB), homozygous (BB), and a male carrying the B chromosome (larger size). The presence of heterochromatin on all centromeric and pericentromeric chromosomes of T. fuscus suggests that the size difference observed in the submetacentric pair in the SB and BB workers is not related to the heterochromatin but to a duplication of euchromatic regions through intra- or inter- chromosomal rearrangements. The fluorochrome CMA₃ matched the C-banding markings, indicating that the heterochromatin is rich in GC base pairs. As far as we know, this is the first chromosomal polymorphism reported in the tribe Attini.     

B chromosomes in Nierembergia aristata (Solanaceae): nucleolar activity and competition with the A chromosomes

B chromosomes are additional dispensable chromosomes that may be present in some individuals, populations, or species, which have probably arisen from the A chromosomes but follow their own evolutionary pathway. Supposedly, B chromosomes do not contain major genes except for ribosomal DNA (rDNA) sequences that have been mapped on the supernumerary chromosomes of many plants and animals. This paper is a new report of B chromosome occurrence in plants. B chromosomes with nucleolar organizing regions (NORs) were found in a diploid sample of Nierembergiaaristata D. Don (sub nom. N. stricta Miers) (2n = 2x = 16). This is an extreme case in which B chromosomes possess not only strong nucleolar activity, as revealed by conventional staining methods, AgNOR and fluorescence banding, and fluorescent in situ hybridization (FISH), but also show nucleolar competition with the A chromosomes. The observed phenomenon could be analogous to the nucleolar dominance or 'differential amphiplasty' phenomenon that occurs in interspecific hybrids.     

Multigenerational outbreeding effects in Chinook salmon (Oncorhynchus tshawytscha)

Outbreeding, mating between genetically divergent individuals, may result in negative fitness consequences for offspring via outbreeding depression. Outbreeding effects are of notable concern in salmonid research as outbreeding can have major implications for salmon aquaculture and conservation management. We therefore quantified outbreeding effects in two generations (F₁ hybrids and F₂ backcrossed hybrids) of Chinook salmon (Oncorhynchus tshawytscha) derived from captively-reared purebred lines that had been selectively bred for differential performance based on disease resistance and growth rate. Parental lines were crossed in 2009 to create purebred and reciprocal hybrid crosses (n = 53 families), and in 2010 parental and hybrid crosses were crossed to create purebred and backcrossed hybrid crosses (n = 66 families). Although we found significant genetic divergence between the parental lines (F_ST = 0.130), reciprocal F₁ hybrids showed no evidence of outbreeding depression (hybrid breakdown) or favorable heterosis for weight, length, condition or survival. The F₂ backcrossed hybrids showed no outbreeding depression for a suite of fitness related traits measured from egg to sexually mature adult life stages. Our study contributes to the current knowledge of outbreeding effects in salmonids and supports the need for more research to better comprehend the mechanisms driving outbreeding depression.     

Chromosome investigations in annual Medicago species (Fabaceae) with emphasis on the origin of the polyploid Medicago rugosa and M. scutellata

Chromosome number variations play an important role in the genus Medicago. In addition to polyploidy there are cases of dysploidy as evidenced by two basic numbers, x = 8 and x = 7, the latter limited to five annual species having 2n = 14. Annuals are diploid with the exception of Medicago scutellata and Medicago rugosa which have 2n = 30 and are considered the result of crosses between the 2n = 16 and 2n = 14 species. However, this hypothesis has never been tested. This study was carried out to investigate the 2n = 14 and 2n = 30 karyotypes and verify the allopolyploid origin of M. scutellata and M. rugosa. Fluorescence in situ hybridization (FISH) of rDNA probes and genomic in situ hybridization (GISH) were performed. FISH showed that all five diploids with 2n = 14 have one pair of 45S and one pair of 5S rDNA sites. M. scutellata displayed four sites of 45S and four sites of 5S rDNA, while in M. rugosa only one pair of each of these sites was found. GISH did not produce signals useful to identify the presumed progenitors with 14 chromosomes. This result suggests alternative evolutionary pathways, such as the formation of tetraploids (2n = 32) and subsequent dysploidy events leading to the chromosome number reduction.     

Characterization of B chromosomes in Lilium hybrids through GISH and FISH

Supernumerary (B) chromosomes and small aberrant chromosomes were detected in Lilium hybrids and characterized through genomic in situ hybridization (GISH) and florescence in situ hybridization (FISH). Two small, supernumerary or B chromosomes were detected as extra chromosomes in a tetraploid plant derived from chromosome doubling of a hybrid (2n = 2x = 24) between a cultivar of the Longiflorum (L) and the Trumpet (T) group. When this tetraploid LLTT hybrid was crossed with a triploid LLO hybrid (O = Oriental), the B chromosome was transmitted to 73.4 % of the progenies. Based on GISH and FISH characterization, it was shown that the B chromosome consisted of two identical arms, with 5S rDNA hybridizing to the majority of it, which were flanked by normal telomeres, suggesting that this is an isochromosome. In another population, which is a backcross progeny between a F1 hybrid of Longiflorum × Asiatic (LA) and its Asiatic parent, the former produced functional 2n gametes which resulted in a triploid LAA progeny (2n = 3x = 36), in which three exceptional plants possessed 35 normal chromosomes and a small aberrant chromosome instead of the expected normal number of 36. In all three cases, the small aberrant chromosomes were isochromosomes which had obviously originated during the first backcross generation. These three chromosomes showed normal telomeres and mitosis. In addition, one of the new generated chromosomes possessed two 45S rDNA sites in the proximal positions. These new arisen isochromosomes were proposed to originate from centric breakage and fusion of two short arms of the missing chromosome in three genotypes, respectively, based on the comparison of arm lengths as well as rDNA loci. Their relevance to the origin of Bs is discussed.     

Distribution of 45S and 5S rDNA sites in 23 species of Eleocharis (Cyperaceae)

Studies of rDNA location in holocentric chromosomes of the Cyperaceae are scarce, but a few reports have indicated the occurrence of multiple 45S rDNA sites at terminal positions, and in the decondensed state of these regions in prometaphase/metaphase. To extend our knowledge of the number 45S and 5S rDNA sites and distribution in holocentric chromosomes of the Cyperaceae, 23 Brazilian species of Eleocharis were studied. FISH showed 45S rDNA signals always located in terminal regions, which varied from two (E. bonariensis with 2n = 20) to ten (E. flavescens with 2n = 10 and E. laeviglumis with 2n = 60). 5S rDNA showed less variation, with 16 species exhibiting two sites and 7 species four sites, preferentially at terminal positions, except for four species (E. subarticulata, E. flavescens, E. sellowiana and E. geniculata) that showed interstitial sites. The results are discussed in order to understand the predominance of terminal rDNA sites, the mechanisms involved in the interstitial positioning of 5S rDNA sites in some species, and the events of amplification and dispersion of 45S rDNA terminal sites.     

Chromosomal localization of rDNA genes and genomic organization of 5S rDNA in <Emphasis Type="Italic">Oreochromis mossambicus,O. urolepis hornorum</Emphasis> and their hybrid

In this study, classical and molecular cytogenetic analyses were performed in tilapia fishes, Oreochromis mossambicus (XX/XY sex determination system), O. urolepis hornorum (WZ/ZZ sex determination system) and their hybrid by crossing O. mossambicus female × O. u. hornorum male. An identical karyotype ((2n = 44, NF (total number of chromosomal arms) = 50) was obtained from three examined tilapia samples. Genomic organization analysis of 5S rDNA revealed two different types of 5S rDNA sequences, 5S type I and 5S type II. Moreover, fluorescence in situ hybridization (FISH) with 5S rDNA probes showed six positive fluorescence signals on six chromosomes of all the analysed metaphases from the three tilapia samples. Subsequently, 45S rDNA probes were also prepared, and six positive fluorescence signals were observed on three chromosome pairs in all analysed metaphases of the three tilapia samples. The correlation between 45 rDNA localization and nucleolar organizer regions (NORs) was confirmed by silver nitrate staining in tilapia fishes. Further, different chromosomal localizations of 5S rDNA and 45S rDNA were verified by two different colour FISH probes. Briefly, the current data provide an insights for hybridization projects and breeding improvement of tilapias.     

Genomic relationships between hexaploid Helianthus resinosus and diploid Helianthus annuus (Asteraceae)

Genus Helianthus comprises diploid and polyploid species. An autoallopolyploid origin has been proposed for hexaploid species but the genomic relationships remain unclear. Mitotic and meiotic studies in annual Helianthus annuus (2n = 2x = 34) and perennial Helianthus resinosus (2n = 6x = 102) as well as the F₁ hybrids between both species were carried out. Chromosome counting confirmed the hybrid origin of the latter plants and their tetraploid condition. Bivalents in hybrids ranged from 12 to 28 ( $ \bar{x} $  = 20.8). Univalents, trivalents and quadrivalents were also observed. Meiotic products comprised dyads, triads and normal tetrads and pollen grains were heterogeneous in size. These observations suggest the occurrence of 2n pollen in addition to the expected n. Genomic in situ hybridization (GISH) of total H. annuus DNA on H. resinosus chromosomes rendered weak but uniform signals; similar hybridization pattern was observed using three other annual species. Hybridization with H. annuus probe performed on root tip cells of F₁ H. annuus × H. resinosus hybrids revealed 17 chromosomes with a strong hybridization signal. GISH in hybrid meiocytes distinguished chromosomes from parental species and revealed autosyndetic pairing of H. resinosus chromosomes, allosyndetic pairing in bivalents, trivalents and quadrivalents, and the presence of univalents derived from parents, H. annuus and H. resinosus. Results obtained from classical and molecular cytogenetics do not support H. annuus as a direct ancestor of H. resinosus. The occurrence of allosyndetic pairing and the relatively high fertility of the F₁ hybrids point to the possibility that useful genes could be transferred from H. resinosus to cultivate sunflower, although the effective rate of recombination has not been evaluated. GISH method proved effective to recognize parental chromosomes in H. annuus × H. resinosus progeny.     

Homoploid hybrid speciation in Doronicum L. (Asteraceae)? Morphological,karyological and molecular evidences

Abstract

The first unambiguous documentation of hybridism in the genus Doronicum (Senecioneae – Asteraceae) is reported. All our morphological, karyological and molecular data concur to indicate that Doronicum × minutilloi Peruzzi hybr. nov. (2n = 60) is a hybrid growing in Monti Aurunci (Central Italy), originated from the spontaneous crossing D. orientale Hoffm. (2n = 60) × D. columnae Ten. (2n = 60). This new hybrid shows a slightly higher morphological, karyotypic and ribotypic affinity with D. columnae, but shares a trnL-trnF IGS haplotype with D. orientale, and co-occurs with the latter species only; it has reduced fertility and a high potential for vegetative propagation through rhizome fragmentation. Our results led us to suspect in fieri homoploid hybrid speciation.     

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.