Structure,behaviour and repetitive DNA of B-chromosomes in Cestrum nocturnum (Solanaceae)

Abstract

The Cestrum genus is karyotypically exceptional in Solanaceae. It is characterised by a basic number x?=?8, a large chromosomal and genomic size, complex heterochromatin patterns, B-chromosomes (Bs) with particular heterochromatin and distribution of 18–5.8–26S and 5S rDNA. Cestrum nocturnum L. has a diploid number of 2n?=?16 plus a variable number of B-chromosomes. The aims of work was to analyse their numerical variation, structure and behaviour of C. nocturnum B-chromosomes by classical and molecular cytogenetics. The individuals analysed had 2n?=?16?+?0?13 B-chromosomes. All B-chromosomes were metacentric and smaller than A-chromosomes. The number of B-chromosomes showed a great variability between and within individuals, thereby denoting the occurrence of events that promote mitotic and meiotic instability. Cytogenetic techniques made it possible to observe that B-chromosomes are rich in heterochromatin, probably with AT- and GC-rich regions. In addition, molecular techniques allowed to detect homologous sequences of transposable element conserved domains of Ty1-Copia and Ty3-Gypsy superfamilies. These sequences were located by FISH in all B-chromosomes and some A-chromosomes. Our results showed that repetitive DNA could play an important role in chromosomal evolution as well as in the stability of B-chromosomes in C. nocturnum.     

Karyotype differentiation of four Cestrum species (Solanaceae) revealed by fluorescent chromosome banding and FISH

The karyotypes of four South American species of Cestrum (C. capsulare,C. corymbosum,C. laevigatum and C. megalophylum) were studied using conventional staining, C-CMA/DAPI chromosome banding and FISH with 45S and 5S rDNA probes. The karyotypes showed a chromosome number of 2n = 2x = 16, with metacentric chromosomes, except for the eighth submeta- to acrocentric pair. Several types of heterochromatin were detected, which varied in size, number, distribution and base composition. The C-CMA(+) bands and 45S rDNA were located predominantly in terminal regions. The C-CMA (+) /DAPI (+) bands appeared in interstitial and terminal regions, and the C-DAPI (+) bands were found in all chromosome regions. The 5S rDNA sites were observed on the long arm of pair 8 in all species except C. capsulare, where they were found in the paracentromeric region of the long arm of pair 4. The differences in band patterns among the species studied here, along with data from other nine species reported in the literature, suggest that the bands are dispersed in an equilocal and non-equilocal manner and that structural rearrangements can be responsible for internal karyotype diversification. However, it is important to point out that the structural changes involving repetitive segments did not culminate in substantial changes in the general karyotype structure concerning chromosome size and morphology.     

Karyotype relationships among four South American species of Urvillea (Sapindaceae: Paullinieae)

A cytogenetic study was conducted on four species of the genus Urvillea (Sapindaceae, Paullinieae): U. chacoensis Hunz., U. filipes Radlk. and U. ulmacea Kunth of the Urvillea section and U. laevis Radlk. of the Stenelytron section. The chromosome numbers in U. chacoensis (2n = 22) and U. laevis (2n = 24) were confirmed, and new chromosome numbers are reported for U. filipes with 2n = 22 and U. ulmacea with 2n = 88. Additionally, data on interphase nuclear structure, chromosome banding patterns (C-Giemsa and C-CMA₃/DAPI) and FISH with rDNA probes are also presented. The distribution of AT- and GC-rich regions and the physical mapping of ribosomal genes (45S and 5S rDNA sites) were established for the first time in these Urvillea species. Sections of Urvillea are cytogenetically differentiated according to basic chromosome number, where x = 11 in the section Urvillea and x = 12 in the section Stenelytron. This first section displayed an important karyotypic feature, the occurrence of large AT- and GC-rich bands at terminal chromosomal regions. The Urvillea section showed polyploidy and its species were differentiated by their banding patterns. Urvillea chacoensis showed several terminal AT-rich bands, while terminal AT- and GC-rich bands were both found in U. ulmacea. However, the section Stenelytron did not exhibit this banding pattern. The 45S rDNA sites appeared always associated with GC-rich regions and they were numerically variable among species, being located or not the same chromosome 5S rDNA sites. Variation in the repetitive DNA distribution and their role in karyotype differentiation among these Urvillea species are discussed.     

Cold-sensitive chromosome regions and heterochromatin inCestrum aurantiacum (Solanaceae)

The karyotype ofCestrum aurantiacum was analyzed for the presence of coldsensitive regions (CSRs) and other types of constitutive heterochromatin. A range of techniques was employed including the fluorescent DAPI, chromomycin/DAPI double staining and actinomycin D/DAPI counter-staining, and the non-fluorescent C-banding applied as single or sequential staining, sequential N-banding and silver impregnation. Four classes of constitutive heterochromatin were recognized: CSRs, nucleolar organizers, non-nucleolar chromomycin-positive bands, and indifferently fluorescent bands. The banded karyotype ofC. aurantiacum is compared with those of otherCestrum species. The sectionsHabrothamnus andCestrum are not karyologically distinct.     

Molecular cytogenetics and characterization of a ZZ/ZW sex chromosome system in Triportheus nematurus (Characiformes, Characidae)

Chromosomes of Triportheus nematurus, a fish species from family Characidae, were analyzed in order to establish the conventional karyotype, location of C-band positive heterochromatin, Ag-NORs, GC- and AT-rich sites, and mapping of 18S and 5S rDNA with fluorescence in situ hybridization (FISH). The diploid number found was 2n = 52 chromosomes in both males and females. However, the females presented a pair of differentiated heteromorphic chromosomes, characterizing a ZZ/ZW sex chromosome system. The Z chromosome was metacentric and the largest one in the karyotype, bearing C-positive heterochromatin at pericentromeric and telomeric regions. The W chromosome was middle-sized submetacentric, appearing mostly heterochromatic after C-banding and presenting heterogeneous heterochromatin composed of GC- and AT-rich regions revealed by fluorochrome staining. Ag-NORs were also GC-rich and surrounded by heterochromatic regions, being located at the secondary constriction on the short arms of the second chromosome pair, in agreement with 18S rDNA sites detected with FISH. The 18S and 5S rDNA were aligned in tandem, representing an uncommon situation in fishes. The results obtained reinforce the basal condition of the ZZ/ZW sex system in the genus Triportheus, probably arisen prior to speciation in the group.     

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.     

Multicolor FISH mapping of the dioecious model plant,<Emphasis Type="Italic"> Silene latifolia</Emphasis>

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.Communicated by J.S. Heslop-Harrison     

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.     

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.     

Mapping of ribosomal DNA and (TTAGGG)n telomeric sequence by FISH in the bivalve Patinopecten yessoensis (Jay, 1857)

The chromosome set of Patinopecten yessoensis (Jay, 1857) wascharacterized using Giemsa staining, DAPI staining and fluorescencein situ hybridization (FISH) with three repetitive DNA probes[18S–28S rDNA, 5S rDNA and telomeric (TTAGGG)_n]. DAPIstaining showed that AT-rich regions were located on the centromereof almost all chromosomes and interstitial banding was not observed.FISH showed that 18S–28S rDNA spread over the short armsof two subtelocentric chromosome pairs and 5S rDNA was locatedon the long arm of one subtelocentric chromosome pair. SequentialFISH demonstrated that 18S–28S and 5S rDNA were locatedon different chromosomes. FISH also showed that the vertebratetelomeric sequence (TTAGGG)_n was located on both ends of eachchromosome and no interstitial signals were detected. Sequential18S–28S rDNA and (TTAGGG)_n FISH indicated that repeatedunits of the two multicopy families were closely associatedon the same chromosome pair. (Received 4 January 2007; accepted 1 September 2007)     

Differentiated ZZ/ZW sex chromosomes in Apareiodon ibitiensis (Teleostei,Parodontidae): cytotaxonomy and biogeography

Conventional and molecular chromosomal analyses were carried out on three populations of Apareiodon ibitiensis sampled from the hydrographic basins of the São Francisco River and Upper Paraná River (Brazil). The results reveal a conserved diploid number (2n = 54 chromosomes), a karyotype formula consisting of 50 m‐sm + 4st and a ZZ/ZW sex chromosome system that has not been previously identified for the species. C‐banding analysis with propidium iodide staining revealed centromeric and terminal bands located in the chromosomes of the specimens from the three populations and allowed the identification of heteromorphism of heterochromatin regions in the Z and W chromosomes. The number of 18S sites located through fluorescent in situ hybridization (FISH) varied between the populations of the São Francisco and Upper Paraná Rivers. The location of 5S rDNA sites proved comparable in one pair of metacentric chromosomes. Thus, the present study proposes a ZZ/ZW sex chromosome system for A. ibitiensis among the Parodontidae, and a hypothesis is presented regarding possible W chromosome differentiation stages in this species through DNA accumulation, showing geographical variations for this characteristic, possibly as a consequence of geographical reproductive isolation.     

Comparative cytogenetic study of the forms of <Emphasis Type="Italic">Macleaya cordata</Emphasis> (Willd.) R. Br. From different localities

A comparative cytogenetic study of two introduced forms of Makleaya cordata (Willd.) R. Br. = syn. Bocconia cordata Willd. grown in different ecological and geographical regions (Moscow and Donetsk areas) was carried out. In the study, a complex of methods utilizing various chromosomal markers, i.e., C- and DAPI-banding technique, fluorescence in situ hybridization (FISH) with probes of 26S and 5S rDNA, as well as estimation of the total area of C-positive regions (C-HCH) in prophase nucleoli and meiosis analysis, was used. In the karyotypes (2n = 20), each chromosome was identified on the basis of C-banding and FISH patterns and the chromosome ideograms were built. Pericentrometric and telomeric C-positive bands in chromosomes of the Moscow form karyotype were found to be smaller and intercalary bands, larger than the corresponding bands in the M. cordata form grown in Donetsk. It was found that the content of C-HCH in prophase nucleoli in the form of M. cordata grown in Donetsk was higher than in the form grown in Moscow. In both forms sites of 26S rDNA and 5s rDNA were localized on satellite chromosome 1 and on chromosome 4 respectively but the signals were more intensive in the plant form grown in Donetsk. The results of this study enable selecting M. cordata forms for use in pharmacology and recommending them for cultivation in various ecological and geographical regions.     

The karyotype and 5S rRNA genes from Spanish individuals of the bat species <Emphasis Type="Italic">Rhinolophus</Emphasis><Emphasis Type="Italic">hipposideros</Emphasis> (Rhinolophidae; Chiroptera)

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.     

New insights on the origin of B chromosomes in <Emphasis Type="Italic">Astyanax scabripinnis</Emphasis> obtained by chromosome painting and FISH

Chromosome painting (CP) with a probe of B chromosome obtained by microdissection and fluorescence in situ hybridization (FISH) with probes of As51 satellite DNA, C _o t−1 DNA, and 18S and 5S rDNA confirmed sharing of some repetitive DNA but not rDNA between A and B chromosomes in the fish Astyanax scabripinnis. Meiotic analysis revealed a pachytene B chromosome bivalent nearly half the size of its mitotic configuration, suggesting a self-pairing of B chromosome arms. Such an isochromosome nature of somatic B chromosome was further evidenced by CP and FISH. All the findings obtained suggest (i) intraspecific origin of B chromosome, and (ii) evolutionary enrichment of repetitive DNA classes, especially those contained in the C _o t−1 and the As51 probes, in B chromosome. However, the precise origin of B chromosome in the present species remains to be elucidated by further molecular cytogenetic analysis because of painting of some A chromosome regions with the B chromosome-derived probe.     

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.     

Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and (TTAGGG)n by one-color and double-color FISH in the toadfish Halobatrachus didactylus (Teleostei: Batrachoididae)

The karyotype of Halobatrachus didactylus presents 46 chromosomes, composed of eight metacentric, 18 submetacentric, four subtelocentric, and 16 acrocentric chromosomes. The results of FISH showed that the major ribosomal genes were located in the terminal position of the short arm of a large submetacentric chromosome. They also showed a high variation in the hybridization signals. The products of amplification of 5S rDNA produced bands of about 420 pb. The PCR labeled products showed hybridization signals in the subcentromeric position of the long arm of a submetacentric chromosome of medium size. Double-color FISH indicated that the two ribosomal families are not co-located since they hybridizated in different chromosomal pairs. Telomeres of all the chromosomes hybridized with the (TTAGGG) _n probe. The GATA probe displayed a strong signal in the long arm of a submetacentric chromosome of medium size, in the subcentromeric position. The double-color FISH showed that the microsatellite GATA and the 5S rDNA gene are located in different chromosomal pairs. The majority presence of GATA probes in one pair of chromosomes is unusual and considering its distribution through different taxa it could be due to evolutionary mechanisms of heterochromatine accumulation, leading to the formation of differentiated sex chromosomes.     

Ribosomal DNA distribution in somatic chromosomes of Stangeria eriopus (Stangeriaceae, Cycadales) and molecular-cytotaxonomic relationships to some other cycad genera

Somatic chromosomes ofStangeria eriopus (Stangeriaceae, Cycadales) were investigated by fluorescentin situ hybridization (FISH) using an 18S ribosomal DNA (rDNA) probe.Stangeria eriopus showed a chromosome number of 2n=16 with a karyotype of 12 median-, 2 subterminal-, and 2 terminal-centromeric chromosomes. FISH study ofS. eriopus revealed 16 signals made up of rDNA sites located on the terminal regions of the long arms of the 7 median- and 2 subterminal-centromeric chromosomes, on terminal region of the short arm of the 1 median-centromeric chromosome, on the terminal regions of the long and the short arms of 1 median- and 2 terminal-centromeric chromosomes. This result suggests that, not only karyomorphologically but also molecular-cytologically, the genusStangeria may be more closely related to the genusCeratozamia than the genusBowenia or the genusMicrocycas previously hypothesized.     

Heterochromatin patterns and ribosomal DNA loci distribution in diploid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolution

Most Crotalaria species display a symmetric karyotype with 2n = 16, but 2n = 14 is found in Chrysocalycinae subsection Incanae and 2n = 32 in American species of the section Calycinae. Seven species of the sections Chrysocalycinae, Calycinae, and Crotalaria were analyzed for the identification of heterochromatin types with GC- and AT-specific fluorochromes and chromosomal location of ribosomal DNA loci using fluorescent in situ hybridization (FISH). A major 45S rDNA locus was observed on chromosome 1 in all the species, and a variable number of minor ones were revealed. Only one 5S rDNA locus was observed in the species investigated. Chromomycin A(3) (CMA) revealed CMA(+) bands colocalized with most rDNA loci, small bands unrelated to ribosomal DNA on two chromosome pairs in Crotalaria incana, and CMA(+) centromeric bands that were quenched by distamycin A were detected in species of Calycinae and Crotalaria sections. DAPI(+) bands were detected in C. incana. The results support the species relationships based on flower specialization and were useful for providing insight into mechanisms of karyotype evolution. The heterochromatin types revealed by fluorochromes suggest the occurrence of rearrangements in repetitive DNA families in these heterochromatic blocks during species diversification. This DNA sequence turnover and the variability in number/position of rDNA sites could be interpreted as resulting from unequal crossing over and (or) transposition events. The occurrence of only one 5S rDNA locus and the smaller chromosome size in the polyploids suggest that DNA sequence losses took place following polyploidization events.     

The 5S rDNA related repetitive sequences in the sex chromosomes of the spiny eel (Mastacembelus aculeatus)

The karyotype of the spiny eel (Mastacembelus aculeatus) has highly evolved heteromorphic sex chromosomes. X and Y chromosomes differ from each other in the distribution of heterochromatin blocks. To characterize the repetitive sequences in these heterochromatic regions, we microdissected the X chromosome, constructed an X chromosome library, amplified the genomic DNA using PCR and isolated a repetitive sequence DNA family by screening the library. All family members were clusters of two simple repetitive monomers, MaSRS1 and MaSRS2. We detected a conserved 5S rDNA gene sequence within monomer MaSRS2; thus, tandem-arranged MaSRS1s and MaSRS2s may co-compose 5S rDNA multigenes and NTSs in M. aculeatus. FISH analysis revealed that MaSRS1 and MaSRS2were the main components of the heterochromatic regions of the X and Y chromosomes. This finding contributes additional data about differentiation of heteromorphic sex chromosomes in lower vertebrates.     

Characterization of Japanese flounder karyotype by chromosome bandings and fluorescence in situ hybridization with DNA markers

The chromosomes of Japanese flounder, Paralichthys olivaceus, were examined by conventional differential staining methods including G-, Q-, C-, silver (Ag)-, fluorochrome, and replication R-bandings and by fluorescence in situ hybridization (FISH) with 5S and 18S rDNAs and telomeric DNA as probes. Replication R-banding substantially made it possible to identify 24 homologous pairs by their RBG-banding pattern and relative length. Both rDNA loci were mapped to chromosome 1, where 5S and 18S rDNA loci were located at the centromeric region and secondary constriction, respectively. C-banding revealed that both rDNA loci were heterochromatic, and 18S rDNA loci were positive for chromomycin A₃ but negative for 4′,6-diamidino-2-phenylindole (DAPI) staining. Telomeric FISH signals were observed at all chromosome ends and at the interstitial region of some chromosomes. The observed results were discussed in relation to the karyotype evolution in the order Pleuronectiformes.