Chromosomal localization and evolution of satellite DNAs and heterochromatin in grasses (Poaceae), especially tribe Aveneae

The physical mapping of three abundant tandemly repeated DNA sequences, CON1, CON2, and COM2, and the distributional pattern of AT- and GC-rich regions in the chromosomes of 32 species of the grass family Poaceae have been established by means of fluorescence in situ hybridization and fluorochrome banding with chromomycin and DAPI. Additionally, locations of 5S, 35S rDNA, and the C-banding pattern were examined. All satellite DNAs (satDNA) tested are situated predominantly subtelomerically in the chromosomes, but occur also colocalized with 35S and 5S ribosomal DNAs (rDNA). Especially, CON2 is most often colocalized with the 5S rDNA, but is evolutionarily not derived from it. Subtelomeric heterochromatin bands are frequently, but not always correlated with satDNA bands. Moreover, the DAPI- or rarely chromomycin-positive stainability of heterochromatin is not caused by these satDNAs as revealed by their sequence organization, showing too few clusters of AT or GC base pairs as required for binding of the fluorochromes. The occurrence of satDNAs is not correlated with that of other components of the heterochromatin. Proportions of satDNAs and other sequences of the heterochromatin relative to the entire genome appear subjected to a much faster evolutionary change than the rather stable proportions of the rDNAs. Heteromorphism in banding patterns found in many species is related in most instances with breeding system and life form. The independent evolution and amplification of different satDNAs is discussed in relation to molecular phylogenetic data. The value and limitations of satDNA data in addressing systematic questions in grasses is exemplified for several grass subfamilies and tribes.     

Karyotype differentiation among Spondias species and the putative hybrid Umbu-cajá (Anacardiaceae)

Spondias L. comprises at least nine Neotropical species, including the widely cultivated S. monbim and S. tuberosa. Umbu‐cajá, a putative hybrid between these two species, is also grown. In this paper, the karyotypes of five Spondias species and Umbu‐cajá were analysed for evidence of this hybridization. Chromosome banding with chromomycin A₃ and the distribution of 5S and 45S rDNA sites were used to characterize the plants, also genomic in situ hybridization using nuclear DNA from both putative parents and the hybrid as probes. All material presented the same chromosome number (2n = 32) and morphology, but differed in the number and distribution of bands. Spondias monbim and S. tuberosa, the supposed relatives of Umbu‐cajá, displayed similar banding patterns, with five to six chromosome pairs having terminal bands, whereas Umbu‐cajá exhibited bands on both members of nine chromosome pairs. The three other species, S. venulosa, S. cytherea and S. purpurea, showed less closely related karyotypes, with bands in 12–18 chromosome pairs. In situ hybridization with 5S and 45S rDNA probes revealed one site of each probe per haploid chromosome complement in all material. However, in S. tuberosa, the location of 5S rDNA was different from the other species and found no counterpart in Umbu‐cajá. Several tests with total DNA from S. mombin and S. tuberosa against metaphase chromosomes of Umbu‐cajá failed to differentiate the individual genomes in the hybrid. From the chromosome banding and the distribution of rDNA sites, as well as from the genomic in situ hybridization, it seems clear that Umbu‐cajá is related closely to S. monbim and S. tuberosa, but it is karyotypically homozygous and distinct from theses other species. Karyotypically, the three other investigated species were related less closely to Umbu‐cajá. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155 , 541–547.     

CPD staining: an effective technique for detection of NORs and other GC-rich chromosomal regions in plants

Mitotic chromosome spreads of 16 plant species belonging to six families were analyzed using an improved combined PI and DAPI (CPD) staining procedure. Fluorescence in situ hybridization (FISH) with 45S rDNA probe was conducted sequentially on the same spreads to evaluate the efficiency and sensitivity of the technique. Fluorochrome staining with chromomycin A₃ (CMA)-DAPI also was conducted to clarify the properties of the sequences involved in the CPD banded regions. Our results revealed that all of the NORs (rDNA sites) in the species tested were efficiently shown as red bands by CPD staining, and the number and position of the bands corresponded precisely to those of the 45S rDNA FISH signals, indicating that the detection sensitivity of CPD staining is similar to that of FISH. In 10 of the species tested including Aegilops squarrosa, Allium sativum, Oryza sativum ssp. indica, Oryza officinalis, Pisum sativum, Secale cereale, Setaria italica, Sorghum vulgare, Vicia faba and Zea mays, CPD bands were exhibited exclusively in their NORs, while in other six species including Hordeum vulgare, Allium cepa, Psophocarpus tetragonolobus, Arabidopsis thaliana, Brassica oleracea var. capitata and Lycopersicon esculentum, CPD bands appeared in chromosomal regions other than their NORs. The CPD bands were in accordance with the CMA bands in all species tested, indicating GC-rich sequences in the CPD bands and that the improved CPD staining procedure is specific for GC-rich regions in plant genomes. Our investigation not only elucidated the banding mechanisms of CPD, but also demonstrated that the CPD staining technique, which may be preferable to CMA staining, is an effective tool for detecting NORs and other GC-rich chromosomal regions in plants.     

A new species of Urvillea (Sapindaceae) from northwestern Venezuela

The new speciesUrvillea venezuelensis, from northwestern Venezuela, is described, illustrated, and compared to its putative closest relativeU. dasycarpa. Additionally micromorphological characters of pollen grains are described. The new species belongs toUrvillea sectionStenelytron, which is characterized by compressed fruit locules.     

Cytogenetics of Alismataceae and Limnocharitaceae: CMA/DAPI banding and 45S rDNA sites

Species belonging to the Alismataceae (Echinodorus) and Limnocharitaceae (Hydrocleys and Limnocharis) families were analysed by banding with CMA/DAPI fluorochromes, C/CMA/DAPI banding, and in situ hybridization (FISH) with probes that recognise 45S rDNA. All species of Echinodorus presented 2n = 22, but only in E. lanceolatus were DAPI+ telomeric bands in seven chromosome pairs observed. A bimodal karyotype and GC-rich heterochromatin preferably located in two smaller acrocentric pairs that generally corresponded to the number of sites of 45S rDNA. A similar pattern of bands was observed in both Limnocharis species (2n = 20), but the two differed with respect to 45S rDNA, with L. laforestii showing only two sites. Hydrocleys nymphoides and H. martii had a chromosome number of 2n = 16, but the position of the GC-rich heterochromatin associated with the satellite differed among chromosomal types. In this work, the cytotaxonomic implications of these patterns are discussed and correlated with previous data from the literature.     

Chromosomal differentiation and genome size in three European mountain Lilium species

 Three related and taxonomically close species of the genus Lilium (L. pyrenaicum Gouan, L. pomponium L. and L. carniolicum Bernh.), all of them with 2n=24 chromosomes, have been studied for chromosomal differentiation, using fluorochrome banding and fluorescence in situhybridization (FISH), and for genome size and GC percentage using flow cytometry. The total DNA content of L. pomponium (2C=70.26 pg) was about 5% higher than that of L. pyrenaicum (2C=67.74) and L. carniolicum (2C=67.37 pg), while GC percentage was higher in this last species (36.60%) than in L. pomponium (35.56%) and lower than in L. pyrenaicum (37.92%). Silver staining, fluorochrome banding with chromomycin A₃ (CMA) and fluorescence in situ hybridization (FISH) clearly pointed out the number of nucleoli, the number and position of GC-rich bands and the number and location of rDNA sites thus permitting distinction of the three species at chromosomal level. Two families of ribosomal genes, 18S-5.8S-26S (18S) and 5S rRNA genes, were separated onto different pairs in chromosome complements of examined species. Chromosome regions containing both kinds of rRNA genes were also GC-rich regions. The results revealed a clear interspecific differentiation at the chromosomal level and permitted the discussion about relationships among the species. Received June 21, 2002; accepted October 4, 2002 Published online: Febraury 7, 2003     

Characterization of diploid,tetraploid and hexaploid Helianthus species by chromosome banding and FISH with 45S rDNA probe

Comparative karyotype analyses of five diploid, two tetraploid, and three hexaploid species of Helianthuswere performed using Feulgen staining, Giemsa C and CMA₃ (C-CMA) staining, and FISH with 45S rDNA probe. The karyotypes are composed by a basic number of x=17 with a predominance of meta- and submetacentric chromosome types. A polyploid series is associated with the basic number. Giemsa C- and C-CMA banding revealed terminal or interstitial heterochromatin according to the species, suggesting the existence of a mechanism that may be acting in the dispersion of heterochromatic segments in Helianthus. The nucleolar organizer regions were located at terminal chromosome positions by FISH with 45S rDNA probe. Diploid species presented four, six, and eight rDNA sites, tetraploid species showed eight sites and hexaploid species presented 12 rDNA sites. Karyomorphological differences include variation in number, size and chromosome morphology, suggesting that rearrangements involving small heterochromatic and rDNA segments played a major role in karyotype evolution.     

Cytogenetic characterization and nuclear DNA content of three North African endemic Centaurea species

Three endemic Centaurea species from North Africa are investigated for the first time by chromomycin fluorochrome banding for GC-rich DNA distribution, fluorescence in situ hybridization for physical mapping of rRNA genes, and flow cytometry for genome-size assessment. Investigated species belong to three different sections and possess three basic chromosome numbers: C. tougourensis subsp. tougourensis 2n = 4x = 36 (x = 9), C. musimonum 2n = 2x = 20 (x = 10), and C. maroccana 2n = 2x = 24 (x = 12). The number and distribution of chromomycin positive bands (CMA⁺) and 18S-5.8S-26S (35S) rDNA loci were different among investigated species and ranged from 6 to 80 chromomycin bands and from 2 to 6 35S rDNA loci. The three species have just one 5S rDNA locus at intercalary position on a separate chromosome pairs, except in the case of C. musimonum in which both rDNA loci were localized on the same chromosome. All rDNA loci were co-localized with CMA⁺ bands, except three 35S in C. musimonum. Genome size ranged from 2C = 1.66 to 2C = 2.86 pg in diploid species (C. musimonum and C. maroccana, respectively) and to 2C = 4.51 pg in tetraploid C. tougourensis subsp. tougourensis.     

Cytogenetic studies in South American species of Serjania (Sapindaceae: Paullinieae)

Abstract

Serjania Mill. (Paullinieae) is considered the most important neotropical genus of Sapindaceae due to species number and its widespread distribution. In this study, 14 species belonging to three sections were analyzed using conventional staining, C/CMA/DAPI banding, and fluorescence in situ hybridization (FISH) with a 18S-5.8S-26S rDNA probe. New chromosome counts are reported for Serjania crassifolia, Serjania platycarpa, and Serjania regnellii, all with 2n = 24, which is remarkably constant for Serjania. The karyotypes are moderately asymmetric, and variations observed in A1 and A2 indices show resemblances between S. platycarpa, Serjania hebecarpa, and S. crassifolia, and between Serjania communis, Serjania gracilis, and S. regnellii. The banding pattern was homogeneous in Serjania. C/DAPI bands (AT-rich sites) were not clearly evidenced, but changes in the number and position of GC-rich sites (CMA bands) were observed. These segments were associated with 18S-5.8S-26S rDNA sites. The significance of the results is discussed in relation to chromosomal data available for the genus and in regard to the infrageneric treatment of Serjania.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Molecular cytogenetics of the genus Artemisia (Asteraceae, Anthemideae): fluorochrome banding and fluorescence in situ hybridization. I. Subgenus Seriphidium and related taxa

The distributional pattern of AT- and GC-rich regions and the physical mapping of ribosomal DNA (location of 18S-5.8S-26S and 5S rDNA) in the chromosomes of seven Artemisia species have been established by means of fluorochrome banding and fluorescence in situ hybridization (FISH). This is the first study in the large genus Artemisia using FISH. Five species (A. barrelieri, A. caerulescens subsp. gallica, A. fragrans, A. herba-alba subsp. valentina, A. herba-alba subsp. herba-alba) belong to the subgenus Seriphidium, one of the most homogeneous in the genus; one (A. tridentata susbp. spiciformis) belongs to the small subgenus Tridentatae, classically included in Seriphidium; and one (A. annua) belongs to the subgenus Artemisia, but shows some affinities with Seriphidium. Genome organization is relatively constant in all the species studied. AT- and GC-rich DNA is predominantly terminal, but some intercalary and centromeric bands also exist. The rDNA loci are also most often terminal and usually located in GC-rich regions. 5S rDNA sites are present in a lower number than 18S-5.8S-26S sites, and are always colocated with some of them. In the light of these cytogenetic features, subgenus Seriphidium is clearly placed within the genus Artemisia, so that it does not make sense to segregate it as a genus; on the other hand, subgenus Tridentatae must not be classified within Seriphidium, but kept as an independent subgenus.     

Ribosomal DNA,tri- and bi-partite pericentromeres in the permanent translocation heterozygote <Emphasis Type="Italic">Rhoeo spathacea</Emphasis>

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A₃-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.     

Nuclear DNA content,base composition,heterochromatin and rDNA in Picea omorika and Picea abies

Two closely related spruces, Picea abies and Picea omorika, a Balkan paleoendemic species, often share habitats, yet never hybridize in nature. The present study adresses their characteristics such as nuclear DNA content, base composition, heterochromatin and rDNA pattern. The genome size of P. abies was 10% larger than that of P. omorika when assessed by flow cytometry, respectively 2C=37.2 pg and 33.8 pg; although when estimated as total chromosome length it was virtually the same. The heterochromatin Chromomycin-A (CMA)/ DAPI fluorochrome banding patterns of both P. abies and P. omorika are given here for the first time. Simultaneous FISH (fluorescent in situ hybridization) using 18S-26S and 5S rDNA probes revealed 16 18S rDNA sites in P. omorika, 12 18S rDNA sites in P. abies, and a single 5S rDNA locus in both species. The genomes have about 41% GC. The number and position of CMA/DAPI bands and rDNA loci provide good chromosome markers to clarify the karyotypes of the two species. Received: 18 October 2000 / 14 June 2001     

Comparative cytogenetic analysis of Cestrum (Solanaceae) reveals different trends in heterochromatin and rDNA sites distribution

Species of Cestrum L. (Solanaceae) exhibit large variability in the accumulation of repetitive DNA, although their species possess a stable diploid number with 2n = 16. In this study, we used chromosome banding and fluorescence in situ hybridization (FISH) to characterize the karyotypes and populations of two species, Cestrum nocturnum L. and C. mariquitense Kunth. We also performed a karyotype comparison using 16 idiograms, of which 4 were developed in this study and 12 were obtained from the literature. Cestrum nocturnum displayed more bands than C. mariquitense, but the latter exhibited greater interpopulational variation in the band patterns. There was a tendency for large bands to be located at intercalary/terminal regions and for small bands to be located at intermediate/proximal regions. The idiogram comparison revealed a large variation in the amount, distribution, and size of heterochromatic bands. FISH with rDNA probes revealed stability in the number and location of 5S sites, while 45S was more variable in size and number of sites. Although 45S rDNA always appeared in the subterminal regions, this DNA family exhibited a mobility among chromosome pairs. These data highlight the dynamic of repetitive DNA families in these genomes, as well as the contribution for intra- and interspecific karyotype differentiation in Cestrum.     

Reduction of chromosome number in Eleocharis subarticulata (Cyperaceae) by multiple translocations

Eleocharis subarticulata is recorded as the third species of Cyperaceae with a reduced chromosome number ( n  = 3), following reports on Rhynchospora tenuis ( n  = 2) and Fimbristylis umbellaris ( n  = 3). For Eleocharis, the numbers recorded to date vary from 2 n  = 10 to 2 n  = c. 196, with x  = 5 as the possible basic number. The karyotype of E. subarticulata was studied using conventional staining (mitosis and meiosis), C-CMA₃/DAPI banding, and FISH with 45S rDNA and telomere probes. The chromosomes showed no primary constrictions, as expected in the holocentric chromosomes of Cyperaceae. The meiotic behaviour was abnormal, with a single multivalent ring of six chromosomes at metaphase I, resulting from multiple translocations. At anaphase I six chromatids migrated to each pole, evidencing the inverted meiosis, and these groups were also visible at metaphase II. The C-CMA₃/DAPI banding technique showed only four terminal GC-rich blocks. FISH with 45S rDNA probes revealed four terminal signals, probably associated with GC-rich blocks. The telomeric probe located terminal signals in all the chromosomes, besides a hybridization site in the middle of the large pair. The occurrence of ectopic telomeric sites has not been described previously for plants with holokinetic karyotypes and with reduced chromosome numbers. These data reinforce the hypothesis of the reduction in chromosome number by multiple translocations.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 149 , 457–464.     

Comparative cytogenetics between the species Passiflora edulis and Passiflora cacaoensis

Passiflora edulis Sims is the most economically important species of the genus Passiflora. A new species was described recently, Passiflora cacaoensis Bernacci & Souza, which displayed morphologic characteristics very similar to P. edulis. Due to the need for delimitation of the two species, karyomorphological and banding analyses were carried out. Both species have 2n = 18, with the same karyotype formula 16 m + 2sm. There was variation between the species regarding the location of satellites and the width of chromosome pairs 2, 4 and 8. C banding revealed the presence of constitutive heterochromatin in the centromeric and telomeric regions of all chromosomes in both species. However, only in P. cacaoensis did chromosomes 3 and 9 have a large quantity of heterochromatin. Fluorochrome banding revealed CMA⁺ bands only in the satellites, but no DAPI⁺ bands. Fluorescence in situ hybridisation (FISH) showed that in P. cacaoensis the rDNA 5S probe is located in a single site in the subterminal position of the long arm of chromosome 5. However, for the rDNA 45S probe, two sites were detected in terminal positions of the long arms of chromosome 7, with a bigger and stronger signal, and of chromosome 9. According to the asymmetry index and the quantity of heterochromatin, P. cacaoensis is a more basal species than P. edulis. The cytogenetic data indicate that P. cacaoensis is closely related to P. edulis, but is a different species.     

X-linked heterochromatin distribution in the holocentric chromosomes of the green apple aphid <Emphasis Type="Italic">Aphis pomi</Emphasis>

Chromatin organization in the holocentric chromosomes of the green apple aphid Aphis pomi has been investigated at a cytological level after C-banding, NOR, Giemsa, fluorochrome staining and fluorescent in situ hybridization (FISH). C-banding technique showed that heterochromatic bands are exclusively located on X chromosomes. This data represents a peculiar feature that clearly contradicts the equilocal distribution of heterochromatin typical of monocentric chromosomes. Moreover, silver staining and FISH carried out with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; CMA₃ staining reveals that these silver positive telomeres are the only GC-rich regions among A. pomi heterochromatin, whereas all other C-positive bands are DAPI positive thus containing AT-rich DNA.     

基于荧光原位杂交的藜属植物核型分析

为精确地识别藜属植物染色体组的核型特征,该文研究了4种来自青海高原的野生藜属植物(灰绿藜、藜、菊叶香藜及杂配藜)和1种从美国引进的栽培藜麦品种PI614932-HX(3)基于染色体荧光原位杂交(rDNA FISH)的核型。利用5S rDNA和45S rDNA对5种藜属植物有丝分裂中期的染色体进行FISH研究。藜属植物的核型分析结果表明:(1)藜属植物中存在二倍体(2n=2x=18)和四倍体(2n=4x=36)两种倍性,藜麦和灰绿藜为四倍体,其余3种为二倍体。(2)藜麦、灰绿藜、藜、菊叶香藜及杂配藜的核型公式分别为2n=4x=36=34m(2AST)+2sm,2n=4x=36=32m(4AST)+4sm,2n=2x=18=16m(4AST)+2sm,2n=2x=18=18m及2n=2x=18=16m+2sm。(3)染色体由大部分的中部着丝粒染色体(m)和少部分近中部着丝粒染色体(sm)组成。(4)核型类型除了菊叶香藜为1B以外,其余均属于2B类型。(5)在藜麦、灰绿藜及藜中具有分布位置不同、数量不等的双随体。5S rDNA、45S rDNA FISH结果表明:(1)藜麦和灰绿藜的染色体上存在2对5S rDNA位点和1对45S rDNA位点,藜、杂配藜的染色体上存在1对5S rDNA位点和1对45S rDNA位点,菊叶香藜的染色体上只存在1对5S rDNA位点。(2)5S rDNA和45S rDNA位点均位于染色体的短臂上。该研究首次获得了藜属植物基于5S rDNA和45S rDNA荧光原位杂交核型,为藜属植物亲缘关系研究和细胞生物学研究提供了分子细胞遗传学依据。     

Comparative molecular cytogenetic analysis of three <Emphasis Type="Italic">Leuciscus</Emphasis> species (Pisces,Cyprinidae) using chromosome banding and FISH with rDNA

A comparative molecular cytogenetic analysis was performed on three species of the genus Leuciscus viz. ide L. idus, chub L. cephalus and dace L. leuciscus distributed in Poland, using C-, Ag- and chromomycin A₃ (CMA₃)-stainings and fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Although the three species examined shared 2n = 50 chromosomes and the largest acrocentric chromosome pair in the complement, they were characterized with karyotypic differences in terms of the number of uni- and biarmed chromosomes and the localization of nucleolar organizer regions (NORs) revealed by Ag-staining and FISH. L. idus and L. cephalus showed the rDNA sites on the long arms of one submetacentric (SM) chromosome pair and on the short arms of one subtelocentric (ST) chromosome pair, respectively. These NORs were CMA₃-positive, GC-rich and C-positive heterochromatic sites in both species. Such chromosome banding features were also true for four NORs localizing on one of each SM and ST pair in L. leuciscus, but considerable numerical NOR polymorphism became apparent with Ag-staining and FISH due to a different combination of these NOR-bearing SMs and STs in this dace. The present results indicate that the molecular cytogenetic analysis applied herein may become useful to elucidate the karyotype evolution and phylogenetic relationships among the species in the genus Leuciscus and other related groups.