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.     

Population differences in the expression of nucleolus organizer regions in the grasshopperEyprepocnemis plorans

Summary Fluorescence in situ hybridization revealed the presence of ribosomal RNA genes in paracentromeric regions of all A chromosomes and in the distal half of B chromosomes in embryonic cells from Moroccan specimens of the grasshopperEyprepocnemis plorans. The expression of these genes was monitored by the presence of nucleoli attached to each chromosome bivalent in diplotene cells from males collected from two different Moroccan populations and was compared to previous data of Spanish populations. Whereas only the nucleolus organizer regions (NORs) on S₉–S₁₁ and X chromosomes were active in the Spanish specimens. Moroccan individuals showed NOR activity in all chromosomes. The rRNA genes on the B chromosome were inactive in both populations. The S₉ and S₁₀ NORs were less active in Moroccan specimens than in Spanish specimen, which might be partly explained by the negative interdependence for expression of the S₁₀ NOR with respect to those on L₂ and X chromosomes. On the other hand, the X NOR was more active in Moroccan specimens than in Spanish specimens, and this might be partly due to the positive effect that the presence of B chromosomes has on the expression of this NOR. The implications of these observations on current models of NOR activity regulation are discussed.Abbreviation NOR nucleolus organizer region     

Physical mapping of ribosomal DNA on several species of the subgenus Rosa

The localization of NORs by fluorescent in situ hybridization on metaphase spreads of five diploid (Rosa gigantea Coll., Rosa moschata Herrm., Rosa multiflora Thunb., Rosa rugosa Thunb. and Rosa sempervirens L., 2n=2x=14), one triploid (Rosa chinensis’semperflorens’ Koehne., 2n=3x=21) and one tetraploid (Rosa gallica ’versicolor’ L., 2n=4x=28) ancestral species of modern roses was studied. Two terminal hybridization signals were observed in all diploid species corresponding to a single NOR per genome in these species. Triploid R. chinensis showed three hybridization sites on the short arm of three morphologically similar chromosomes. Six hybridization sites, located at terminal positions of the short arms of three chromosome pairs, were observed in the tetraploid species. These signals corresponded to three pairs of NORs and all of them were located in chromosome pairs of different size. These observations, together with the analysis of meiotic pairing in PMCs, support the view that our specimen of R. chinensis is an autotriploid and that R. gallica’versicolor’ has an alloploidy nature. Received: 27 November 2000 / Accepted: 12 March 2001     

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.     

Polymorphism of major ribosomal gene chromosomal sites (NOR-phenotypes) in the hybridogenetic fish <Emphasis Type="Italic">Squalius alburnoides</Emphasis> complex (Cyprinidae) assessed through crossing experiments

Chromosomal locations of major ribosomal sites, i.e. NOR-phenotypes, were assigned in Squalius alburnoides complex using sequential chromomycin A₃ (CMA₃)- and silver (Ag)-staining. This hybridogenetic Iberian minnow comprises diploid, triploid and tetraploid forms that arose by interspecific hybridisation between S. pyrenaicus and an unknown species. Inheritance of NOR patterns was studied by means of crossing experiments involving most diploid–polyploid forms of the S. alburnoidescomplex with identified specific genotype constitution. In all the specimens studied, the NORs were localised in the short arms of submetacentric chromosomes. Although S. pyrenaicus presented only one pair of NOR-bearing chromosomes, the data from experimental crosses evidenced that S. alburnoides complex was characterised by a multiple NOR phenotype composed of one chromosome pair with stable NORs and two chromosome pairs with NOR site polymorphism of presence/absence type. These data suggest that the karyotype of the unknown parental species of the S. alburnoidescomplex should have a multiple NOR pattern and emphasised the role of the all-male diploid linage in the dynamics and evolutionary potential of the S.alburnoidescomplex allowing the preservation of the missing ancestor genome. Cross-analyses evidenced that in spite of the high polymorphic nature of NORs in this fish complex, we have no reason to reject the hypothesis that their inheritance patterns were in accordance with Mendelian segregation.     

Occurrence of macro B chromosomes in Astyanax scabripinnis paranae (Pisces,Characiformes, Characidae)

B chromosomes occur in several Neotropical fish species. Cytogenetic analysis of 27 specimens (15 females and 12 males) of Astyanax scabripinnis paranae from the Araquá river (a small headwater tributary of the Tietê river) shows that this population has 2n=50 chromosomes (4M+30 SM+4ST+12A), two chromosome pairs with NORs and conspicuous C-band positive blocks in the terminal position of the long arm of four chromosome pairs. In this population, eight females presented 2n=51 chromosomes and the extra chromosome was a large metacentric similar in size and morphology to the first chromosome pair in the karotype. This accessory chromosome is entirely heterochromatic in C-banded metaphases and shows a late replication pattern evidenced by BrdU incorporation. There was no significant correlation between the presence of B chromosomes and increased NOR activity at the P>0.05 level. Some aspects related to these B chromosomes are discussed.     

Comparative cytogenetics in four species of Palinuridae: B chromosomes, ribosomal genes and telomeric sequences

The evolutionary pathway of Palinuridae (Crustacea, Decapoda) is still controversial, uncertain and unexplored, expecially from a karyological point of view. Here we describe the South African spiny lobster Jasus lalandii karyotype: n and 2n values, heterochromatin distribution, nucleolar organizer region (NOR) location and telomeric repeat structure and location. To compare the genomic and chromosomal organization in Palinuridae we located NORs in Panulirus regius, Palinurus gilchristi and Palinurus mauritanicus: all species showed multiple NORs. In J. lalandii NORs were located on three chromosome pairs, with interindividual polymorphism. In P. regius and in the two Palinurus species NORs were located on two chromosome pairs. In the two last species 45S ribosomal gene loci were also found on B chromosomes. In addition, the nature and location of telomeric repeats were investigated by FISH in J. lalandii, P. gilchristi, P. mauritanicus Palinurus elephas, and P. regius (Palinuridae, Achelata), and in Scyllarus arctus (Scyllaridae, Achelata): all these Achelata species showed the (TTAGG)n pentameric repeats. Furthermore, in J. lalandii these repeats occurred in all the telomeres and in some interstitial chromosomal sites, associated with NORs.     

Conservation of chromosomal location of nucleolus organizer in American marsupials (Didelphidae)

The distribution and expression of nucleolus organizer regions (NORs) were analyzed in seven species of marsupials representative of the three karyotypes (2n = 14, 18 and 22) found in the American family Didelphidae. Analyses comprised silver-staining of NORs and fluorescence in situ hybridization with an rDNA probe. In addition to confirming the variability in number and distribution of NORs in Didelphidae, we demonstrated the conserved location of NORs on one autosome pair in the three karyotypes. In Monodelphis domestica (2n = 18), the NOR on the X chromosome was not inactivated in females.     

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.     

Nucleolar organiser region (NOR) location in karyotypes of Australian ground frogs (family Myobatrachidae)

Nucleolar organiser regions (NORs) were examined in over 90% of the species of Australian ground frogs (familiy Myobatrachidae), including representatives from all twenty currently recognised genera and the three subfamilies. Throughout the family, location of the NOR within the karyotype showed considerable variation yet karyotype morphology showed uniformity. The precise mechanism(s) whereby variation in NOR location evolved while karyotype morphology was unchanged remains uncertain. Comparison of the two major subfamilies showed that the Limnodynastinae had a greater diversity of NOR location than the Myobatrachinae. The limnodynastine genus,Heleioporus, was the only one to show multiple NOR sites in several species. NOR location was particularly stable within most polytypic genera. Differences in NOR location within the remaining polytypic genera (Heleioporus, Limnodynastes, Neobatrachus, Philoria andRanidella) pointed to taxonomic discriminations that were generally consistent with recent proposals based on other criteria.     

Chromosome mapping of ribosomal genes and histone H4 in the genus Radacridium (Romaleidae)

In this study, two species of Romaleidae grasshoppers, Radacridium mariajoseae and R.nordestinum, were analyzed after CMA₃/DA/DAPI sequential staining and fluorescence in situ hybridization (FISH) to determine the location of the 18S and 5S rDNA and histone H4 genes. Both species presented karyotypes composed of 2n = 23, X0 with exclusively acrocentric chromosomes. CMA₃⁺ blocks were detected after CMA₃/DA/DAPI staining in only one medium size autosome bivalent and in the X chromosome in R. mariajoseae. On the other hand, all chromosomes, except the L1 bivalent, of R. nordestinum presented CMA₃⁺ blocks. FISH analysis showed that the 18S genes are restricted to the X chromosome in R. mariajoseae, whereas these genes were located in the L₂, S₉ and S₁₀ autosomes in R. nordestinum. In R. mariajoseae, the 5S rDNA sites were localized in the in L₁ and L₂ bivalents and in the X chromosome. In R. nordestinum, the 5S genes were located in the L₂, L₃, M₄ and M₅ pairs. In both species the histone H4 genes were present in a medium size bivalent. Together, these data evidence a great variability of chromosome markers and show that the 18S and 5S ribosomal genes are dispersed in the Radacridium genome without a significant correlation.     

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.     

Mapping of the nucleolus organizer region on chromosome 4 inArabidopsis thaliana

InArabidopsis thaliana the ribosomal RNA genes (rRNA genes or rDNA) are clustered in tandemly repeated blocks in two nucleolus organizer regions (NORs). Cytogenetic analysis has shown that the NORs are localized on chromosome 2 (NOR 2) and 4 (NOR 4). Recently the map position of NOR 2 was determined using a RFLP which was larger than 100 kb. In the course of a fingerprint analysis of differentArabidopsis ecotypes we have detected four rDNA polymorphisms between the ecotypes Landsberg (La) and Niederzenz (Nd). Mapping of these polymorphisms using established segregating F₂ populations reveals that all polymorphisms detected are dominant. Three of them map to the locus on the second chromosome that has been shown to harbour the NOR 2. The fourth polymorphism can be unambigously assigned to the upper arm of the fourth chromosome. This is the first polymorphism found which originates in the second rDNA cluster ofArabidopsis thaliana. It enables localization of NOR 4 and thus completes the mapping of rDNA genes in the NORs ofArabidopsis.     

Chromosomal banding in three species of the genus Rineloricaria (Siluriformes, Loricariidae, Loricariinae)

Cytogenetic analyses were conducted in three species: Rineloricaria cadeae, Rineloricaria strigilata and Rineloricaria pentamaculata. The nucleolar organizing regions (NORs) were localized at the terminal position, on the ninth pair of the complement for R. cadeae and R. strigilata, on the eighth and sixth pair of st–a, respectively; and on the fifth pair of the complement for R. pentamaculata, the first pair of st–a. Rineloricaria strigilata and R. pentamaculata showed NOR heteromorphism and polymorphism, respectively. All species showed heterochromatin in the pericentromeric regions and associated with NORs, which were chromomycin A₃ (CMA₃) positive, demonstrating to be GC rich. The results show that NOR is nonconservative in this genus.     

Position-dependent NOR activity in barley

Two types of intraspecific nucleolar dominance/suppression are described for barley,Hordeum vulgare L. When the nucleolus organizing regions (NORs) originally belonging to chromosomes 6 and 7 are combined by translocation in one chromosome, NOR 6 is dominant over NOR 7. Neither significant loss of rDNA nor its hypermethylation is the reason for the reduced nucleolus forming activity of NOR 7. Intrachromosomal NOR suppression probably does not occur in isochromosome 6s, which has two NORs 6 in one chromosome. Meiotic and somatic pairing of the homologous arms might be the reason for early fusion of their nucleoli and thus for the lower than expected maximum number of interphase nucleoli. Variable suppression of a partial NOR (6³) is described for descendants of crosses between translocation lines with split NORs 6 and 7. In these cases also, the reduced activity of the partial NOR 6³ is not due to deletion of rDNA as shown by in situ hybridization. Unstable methylation of NOR 6³ in heterozygous F₁ individuals is probably the cause of this phenomenon.     

Chromosome banding and essential oils composition of Brazilian accessions of <Emphasis Type="Italic">Lippia alba</Emphasis> (Verbenaceae)

The essential oil components and a karyotypic analysis of five Lippia alba (Verbenaceae) accessions from Brazil were performed with the objective of investigating the variation among different populations. The chemistry analysis allowed the grouping of the accessions in two main chemotypes: neral chemotype (LaCat, LaJF and LaRJ) and linalool chemotype (LaGua and LaVC). However, large karyotypic differences, verified by different chromosome banding techniques, were not detected. The results presented the same chromosome number for all accessions (2n = 30) with 10 metacentric chromosomes and 5 submetacentric. The chromosome banding showed great blocks of constitutive heterochromatin (C-bands) around the centromeric region, which was rich in AT bases (DAPI₊), while the CMA bands were observed only in terminal regions of six chromosomes. Through Ag-NOR techniques, only two active pairs of NORs were detected on the three pairs of secondary constrictions (the NOR activity is discussed). This work relates the pattern of heterochromatin for Lippia alba for the first time.     

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.     

Analysis of a centric shift in the S11 chromosome of Aiolopus strepens (Orthoptera: Acrididae)

A centric shift in the S₁₁ chromosome of Aiolopus strepens, previously described as a pericentric inversion (Cabrero & Camacho, 1982), was analyzed by using the C-banding procedure. The location of breakage points as well as the posibilities of pericentric inversion and three-break transposition are discussed. The relationship between C-bands and NOR location as revealed by both silver staining and a double procedure Ag-NOR C-banding is also discussed.