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.     

rDNA site number polymorphism and NOR inactivation in natural populations ofAllium schoenoprasum

Nucleolar-organiser activity has been studied by silver staining and byin situ hybridization with an rDNA probe in two populations ofAllium schoenoprasum. One population is monomorphic with NORs and rDNA sites terminal on the short arm of pair 8 in all individuals. The other populations is monomorphic for pair 8 NORs but is also polymorphic for NORs on the long arm of pair 7. All plants in this population carry ribosomal cistrons on both chromosomes of pair 7 but 0, 1 or 2 of these sites can be active in rRNA synthesis. Cis-acting nucleolar-suppression affects the pair 7 locus. We suggest that there has been progressive reduction in the number of NORs during the evolution ofA. schoenoprasum.     

High Ag-NOR-site variation associated to a secondary contact in brown trout from the Iberian Peninsula

The analysis of nucleolar organizer regions (NORs) using silver (Ag-) staining and in situ hybridization (ISH) in brown trout (Salmo trutta) from various river basins in the Iberian Peninsula revealed high variation in the number and location of NORs. A total of 17 different Ag-NOR sites were revealed in 10 different chromosome pairs. Three different Ag-NOR patterns clustered by river basins and strongly associated to the internal transcribed spacer 1 (ITS1) variation were detected. The main variability in NOR-sites was found in a secondary contact between two divergent lineages of brown trouts at Duero basin. Our results confirmed the abrupt break in the spatial distribution of genetic variation of brown trout populations previously reported at Duero basin. We hypothesize that NOR-site variation might be a consequence of hybridization between divergent lineages of brown trouts and that NORs could play a major role in the maintenance of a hybrid zone in Duero basin via post-zygotic isolation mechanisms.     

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.     

Gene mapping of 28S and 5S rDNA sites in the spined loach Cobitis taenia (Pisces, Cobitidae) from a diploid population and a diploid–tetraploid population

We compare the chromosomal 28S and 5S rDNA patterns of the spined loach C. taenia (2n = 48) from an exclusively diploid population and from a diploid–polyploid population using 28S and 5S rDNA probe preparation and labelling, and fluorescence in situ hybridization (FISH). The 5S rDNA was located in two to three chromosome pairs, and separated from the 28S loci for the males and one female (F1) from the diploid population. Loaches from a diploid–polyploid population, and one female (F2) from the diploid population were characterized by at least one chromosome pair with 5S and 28S overlapping signals. The fishes differed mainly in their number of 28S rDNA loci, located on 3–6 chromosomes. All individuals from both populations were characterized by one acrocentric chromosome bearing a 28S rDNA signal on the telomeres of its long arm. The number of major ribosomal DNA in the karyotype of C. taenia by FISH was always higher than the number of Ag-NORs. Our data confirm the extensive polymorphism of NORs in both populations, as already has been observed in closely related Cobitis species, and less polymorphic 5S rDNA pattern. However, this preliminary result highlights the need for a wider scale study.     

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.     

Low genetic variation in Swedish populations of the rare speciesVicia pisiformis (Fabaceae) revealed with rflp (rDNA) and RAPD

Nine Swedish populations, 1–5 individuals/population, and one cultivated individual of the rare speciesVicia pisiformis were investigated for genetic variation. In hybridizations with two rDNA probes using 8 restriction enzymes, only two individuals belonging to one population were polymorphic. A map of the rDNA gene cluster was constructed for four of the restriction enzymes used. Two of the polymorphic sites were mapped and were found to be located outside regions coding for rRNA, presumably caused by single point mutations or small deletions. The repeat length of the rDNA region was c. 10,000 bp, which corresponds well with the size found for other species belonging toFabaceae. No length polymorphism was found in the intergenic spacer, contrary to the situation found for most other plant species investigated for rDNA variation. The haplotype diversity for the species (Hsp Shannon) was very low (0.055). Within-population values (Hpop) was 0 for all populations except the variable one, which had 0.301. PCR amplification with 6 random primers also revealed very low levels of genetic diversity. A polymorphism was observed in a limited number of individuals for four populations. Hsp was 0.065 and was 0.050. The average D value (Wetton) for the PCR haplotypes was 0.99.     

Chromosomal Characteristics of rDNA in European Grayling <Emphasis Type="Italic">Thymallus thymallus</Emphasis> (Salmonidae)

The chromosomal characteristics, locations and variations of two classes of ribosomal DNA (5S and 18S) were studied in European grayling karyotype (Thymallus thymallus, Salmonidae). Major rDNA sites as revealed by sequential CMA₃/Ag staining and confirmed by in situ hybridization with a 18S rDNA probe were situated in two loci and were found to be polymorphic in size and displaying several distinct forms. The 5S rDNA was located by PRINS on three pairs of subtelocentric chromosomes, additional minor signal was present at the centromere of one metacentric element. 5S sites were not associated with NORs. The dosage compensation mechanism was proposed as an explanation of high frequency of lethal rDNA-deleted forms of the NOR-bearing chromosomes. Double variable pattern in the number and location of NORs supported the bi-directional evolution of salmonid rDNA loci.     

Ribosomal DNA variation within and among individuals ofLisianthius (Gentianaceae) populations

Restriction endonuclease fragment analysis of nuclear ribosomal DNA (rDNA) was completed on 25 individuals each from seven populations of theLisianthius skinneri (Gentianaceae) species complex in Panama. Seven restriction enzymes were used to determine the amount and type of rDNA variation within and among individuals of the populations. No restriction site variation was seen within populations or individuals although site differences were seen among populations. Spacer length variation within and among individuals of populations was mapped to the internal transcribed spacer (ITS) region between the 18S and 5.8S rRNA genes, a region inLisianthius rDNA that previously was shown to exhibit length differences among populations. This is the first reported case of such variation within and among individuals of populations for the ITS region. Presence or absence of ITS spacer length variation is not correlated with levels of isozymic heterozygosity within populations. No detectable length variation within individuals or populations was seen in the larger intergenic spacer (IGS). Although populations varied with respect to IGS length, all individuals of a given population had a single and equivalent IGS length.     

Chromosomal characteristics of ribosomal DNA in two extant species of North American mudminnows Umbra pygmaea and U. limi (Euteleostei: Umbridae)

The locations and chromosomal characteristics of ribosomal DNA (rDNA) sites in the karyotypes of two extant North American species of mudminnows, Umbra pygmaea and U. limi (2n = 22, NF = 44), were analyzed sequentially by conventional Giemsa staining, Ag staining, CMA(3) fluorescence and fluorescence in situ hybridization (FISH). The nucleolar organizer regions (NORs) were located in the fourth chromosomal pair in both species (pericentromeric region in U. pygmaea and subtelomeric in U. LIMI). These sites were strongly CMA(3)-positive suggesting that the rDNA sites in these species are associated with GC-rich DNA. FISH with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA(3)-fluorescence. However, both species also had additional CMA(3)-positive/Ag-negative heterochromatic blocks at pericentrometric regions of several chromosomal pairs (three in U. pygmaea and five in U. limi). FISH revealed additional rDNA clusters in both species. It is hypothesized that a paracentric inversion of the chromosome arm carrying the NORs might be one of the rearrangements differentiating the karyotypes of two North American species. The presence of additional rDNA sites is indicative of more complex rearrangements. The pericentromeric NOR phenotype of Umbra pygmaea is similar to that seen in U. krameri and in the distantly related genus Esox.     

Population analysis of the kelpCostaria costata (Phaeophyta) using a polymorphic ribosomal DNA probe

Nuclear DNAs extracted from individuals of the kelpCostaria costata (Phaeophyta) were studied using a small-subunit ribosomal DNA probe (pCc 18). On the basis of annealing analysis of rDNA variation within and among individuals and between populations, polymorphisms detected by pCc 18 appeared to indicate population identity. These rDNA polymorphisms were used to chart the distribution ofCostaria breeding groups in the northeast Pacific. 20 sites were sampled and eight distinct populations resolved. One variant (Variant A) identified a southern group with plants from 13, loosely clustered sites sharing this banding pattern. The other seven variants, found in the northern portion of the study area, were unique and population-specific.     

A Comparative Cytogenetic Analysis between the Grasshopper Species Chromacris nuptialis and C. speciosa (Romaleidae): Constitutive Heterochromatin Variability and rDNA Sites

The chromosomes of Chromacris nuptialis and C. speciosa were comparatively analyzed using different cytogenetic techniques, in order to determine the level of karyotypic similarities and differences between the species. The results show similarities in chromosome number (2n = 23,X0) and acrocentric morphology. In some C. nuptialis individuals meiotic irregularities were detected involving the L₂ bivalent. This bivalent was delayed and presented anaphasic bridges and other aberrations. Differences in constitutive heterochromatin (CH) patterns and composition were observed through C-banding and fluorochromes staining. Silver nitrate staining revealed a single medium nucleolar organizer regions (NORs) pair, per species. Differences were also observed in NORs location, which was pericentromeric in C. nuptialis and proximal in C. speciosa. FISH using an rDNA probe confirmed the existence of ribosomal sites coinciding with active regions visualized by silver nitrate. The possible implications of the karyotype differences observed between both species are discussed.     

Conservation of rDNA inPolystichum (Dryopteridaceae)

Restriction site variation in the nuclear 18S–25S ribosomal RNA genes (rDNA) was analyzed hierarchically in a species complex in the fern genusPolystichum. Two distinct rDNA repeat types were present in all individuals ofPolystichum examined. No variation was detected among individuals within a population ofP. munitum, among populations ofP. munitum orP. imbricans, or among the six diploid species ofPolystichum from North America, including the circumborealP. lonchitis. The identity of rDNA repeats across all six North American species ofPolystichum may reflect an overall similarity of the nuclear genomes of these species, an observation supported by isozyme data as well. However, this nuclear similarity contrasts sharply with the highly divergent chloroplast genomes of these six species. The conservative nature of the rDNA inPolystichum also is in contrast to the much more variable rDNAs of most angiosperms investigated. Perhaps the tempo and mode of evolution of rDNA in ferns differ from those of angiosperms; however, the data base for fern rDNA is very small. Furthermore, the number of repeat types per individual is consistent with a diploid, rather than polyploid, condition despite the high chromosome number (n = 41) of these plants, although homogenization of multiple, divergent rRNA genes cannot be disproven.     

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 organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae)

The position and the number of 18S-5.8S-26S and 5S rDNA loci, characterization of nucleolar organizing region (NOR)-associated heterochromatin and NOR activity assessment are given for six south-eastern Adriatic populations of Allium commutatum Guss. The karyotype characteristics were identical for all the populations studied, even those of distant islands. Diploid karyotypes (2 n = 16) always possessed two NOR-bearing chromosome pairs with pericentric and median secondary constrictions (SCs) on the short arm of the chromosomes VII and VIII. Fluorescent in situ hybridization (FISH) confirmed that these were the only sites of 18S-5.8S-26S rRNA genes. NOR-associated heterochromatin was of the constitutive character as shown after C-banding. Differential fluorochrome banding with Chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) revealed that this heterochromatin comprises both GC- and AT-rich DNA segments. Heteromorphism of C- and CMA-bands was noticed between homologous NOR-bearing chromosomes. The maximum number of four active NORs was correlated with the maximum number of four nucleoli in interphase. Variability of NOR-activity, expressed as number and size of silver stained NORs, existed between cells and between individuals of the same population. The different size of homologous and nonhomologous silver stained NORs was correlated with the extension of SCs. The only 5S rDNA locus was in an intercalary position on short arm of the chromosome VI, at the region of AT-rich constitutive heterochromatin. Dimorphism of C-bands and DAPI/Hoechst(H)-fluorescent bands was noticed between homologous chromosomes VI. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 99–108.     

Ribosomal DNA repeat unit polymorphism in 49 Vicia species

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of Vicia ribosomal DNA (rDNA), more particularly among V. faba and its close relatives and the taxa within three (Narbonensis, Villosa, Sativa) species' complexes. Total genomic DNA of 90 accessions representing 49 Vicia species was restricted with 11 enzymes, and the restriction fragments were probed with three ribosomal clones. Twenty-eight repeat unit length classes were identified. The number of length classes (1–2) per accession did not correspond to the number of nucleolar organizing regions (NORs). The number of rRNA genes was independent of the 2C nuclear DNA amount present in the taxon. Each of the 90 accessions had 2 (rarely 1)-4 DraI sites. Those taxa with the same number of DraI sites generally could be distinguished from each other by different configurations. Probing of the DNA samples digested with tetranucleotide recognition restriction endonucleases emphasized differences between divergent spacer regions and enabled relative homologies between the coding regions to be established. Overall, rDNA restriction site variation among the species showed a good correlation with taxonomic classification. The rDNA analysis indicated evolutionary relatedness of the various taxa within the Narbonensis species complex. rDNA diversity within two other species complexes (Villosa, Sativa), on the other hand, was more extensive than expected. With few exceptions, data on the two complexes give evidence of taxon-specific divergences not seen with other approaches. The restriction site variability and repeat length heterogeneity in the rDNA repeat exhibited startling differences between V.faba and its close wild relatives included in the Narbonensis species complex. This analysis provides new evidence that none of the species within the complex can be considered to be putative allies of broad bean.     

Nucleolar behavior during meiosis in four species of phyllostomid bats (Chiroptera, Mammalia)

We analyzed the behavior of the nucleolus, nucleolar structures and nucleolus organizer regions (NORs) during meiotic division in four species of phyllostomid bats that have different numbers and locations of NORs. Nucleoli began disassembly at leptotene, and the subcomponents released from the nucleolus were dispersed in the nucleoplasm, associated with perichromosomal regions, or they remained associated with NORs throughout division. In Phyllostomus discolor, a delay in nucleolus disassembly was observed; it disassembled by the end of pachytene. The RNA complexes identified by acridine orange staining were observed dispersed in the nucleoplasm and associated with perichromosomal regions. FISH with rDNA probe revealed the number of NORs of the species: one NOR in Carollia perspicillata, one pair in Platyrrhinus lineatus and P. discolor, and three pairs in Artibeus lituratus. During pachytene, there was a temporary dissociation of the homologous NORs, which returned to pairing at diplotene. The variation in the number (from one to three pairs) and location of NORs (in sex or autosomal chromosomes, at terminal or interstitial regions) did not seem to interfere with the nucleolar behavior of the different species because no variation in nucleolar behavior that could be correlated with the variation in the number and chromosomal location of NORs was detected.     

Localisation of NORs and counterstain-enhanced fluorescence studies in Salmo gairdneri and Salmo trutta (Pisces,Salmonidae)

Summary The karyotypes of the rainbow trout (Salmo gairdneri R.) and the brown trout (Salmo trutta L.) were analyzed by means of silver staining and the chromomycin A₃/distamycin A/DAPI fluorescence banding technique. The nucleolus organizer regions (NORs) were localized at the secondary constrictions of chromosome no. 14 in S. gairdneri and of chromosome no. 10 in S. trutta. Additional silver positive dots were observed at or close to several centromeres in S. gairdneri. Brilliant chromomycin A₃ (CMA₃) fluorescence heterochromatin blocks were localized on both sides of the nucleolar constrictions in S. gairdneri. A polymorphic CMA₃ positive band was detected close to the NORs of S. trutta. No distamycin A/DAPI intense heterochromatin blocks were detected in the genomes of the two Salmo species investigated.     

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.     

Accentuated polymorphism of heterochromatin and nucleolar organizer regions in Astyanax scabripinnis (Pisces, Characidae): tools for understanding karyotypic evolution

Astyanax scabripinnis has been considered a species complex because it presents high karyotypic and morphological variability among its populations. In this work, individuals of two A. scabripinnis populations from different streams in the same hydrographic basin were analyzed through C‐banding and AgNOR. Although they present distinct diploid numbers, they show meta and submetacentric chromosome groups highly conserved (numerically and morphologically). Other chromosomal characteristics are also shared by both populations, as the pattern of constitutive heterochromatin distribution (large blocks in the telomeric regions of subtelocentric and acrocentric chromosomes) and some nucleolar chromosomes. Inter‐individual variations both in the number and size of heterochromatic blocks, and in the number and localization of NORs were verified in the studied populations, characterizing them as polymorphics for these regions. The mechanisms involved in the dispersion of heterochromatin and NORs through the karyotypes, as well as the possible events related to the generation of polymorphism of those regions are discussed. Furthermore, relationships between these populations and within the context of the scabripinnis complex are also approached. This revised version was published online in July 2006 with corrections to the Cover Date.