5S rDNA Probes Research Articles

Chromosome Studies of Astyanax jacuhiensis Cope, 1894 (Characidae) from the Tramandai River Basin, Brazil, Using in situ Hybridization with the 18S rDNA Probe, DAPI and CMA3 Staining

The genus Astyanax comprises 86 species of fish distributed in Brazilian river basins and is considered of the Incertae sedis group within the family Characidae. This study presents an analysis of 12 specimens of Astyanax jacuhiensis from the Tramandai River Basin, RS Brazil: 6 from the Maquiné River and 6 from the Quadros Lagoon. All specimens showed a diploid number equal to 50 chromosomes with different karyotypic formula between the two localities. The population from the Maquiné River showed 10m+26sm+6st+8a (FN=92). Fish from the Quadros Lagoon showed 12m+20sm+6st+12a (FN=88). AgNORs were evidenced in the short arm of one acrocentric chromosome pair in both populations, confirmed by FISH with the 18S rDNA probe. CMA3 fluorochrome corresponded with the AgNOR sites, while DAPI staining was negative in these regions. C banding revealed that heterochromatin was weakly distributed, mainly in the pericentromeric and terminal regions in most chromosomes. Analyses of male gonadal tissue were conducted with the objective of characterizing the meiotic chromosome behavior in A. jacuhiensis. The following stages were evidenced: spermatogonial with 50 chromosomes, pachytene and metaphase I with 25 bivalents, and metaphase II with 25 chromosomes, thus confirming the diploid number of the species. Chromosomal abnormalities were not observed. This study shows preliminary data on A. jacuhiensis from the Tramandai River Basin, contributing with more chromosomal information for this group of fish.

The Unique Karyotype of Henochilus wheatlandii, a Critically Endangered Fish Living in a Fast-Developing Region in Minas Gerais State, Brazil

Henochilus wheatlandii, the only species of this genus, is critically endangered and was considered extinct for over a century. The rediscovery of this fish in 1996 made it possible to study its phylogenetic relationships with other species in the subfamily Bryconinae. The aim of this study was to characterise the karyotype of H. wheatlandii. Standard staining, C-positive heterochromatin and nucleolar organiser region (NOR) banding, chromomycin A3 staining, and fluorescent in situ hybridisation (FISH) using 5S rDNA and 18S rDNA probes were conducted on nineteen specimens collected in the Santo Antonio River, a sub-basin of the Doce River in Ferros municipality, Minas Gerais State, Brazil. Henochilus wheatlandii shared the same diploid number and chromosome morphology as other species of Bryconinae. However, its heterochromatin distribution patterns, NOR localisation, and FISH patterns revealed a cytogenetic profile unique among Neotropical Bryconinae, emphasizing the evolutionary uniqueness of this threatened species.

Investigation of genome polymorphism and seed coat anatomy of species of section Adenolinum from the genus Linum

Twelve species of section Adenolinum from genus Linum have been studied using cytogenetic methods (DAPI/C-banding, Ag-NOR staining, FISH with 5S and 26S rDNA probes), RAPD analysis, and seed surface ultrastructure and seed coat anatomy investigation. Karyotype analysis revealed that these species have a chromosome number of 2n = 18 or 2n = 36. The DAPI/C-banding patterns enabled the identification of all chromosomes and revealed the similarities in chromosome patterns in all of the studied species. It was found that 18-chromosome plants are diploids, whereas 36-chromosome forms are tetraploids. In diploid species, 5S and 26S rRNA genes are located in similar positions on a single pair of chromosomes or on two chromosome pairs in tetraploids. Ag-NOR staining showed NOR activity in both genomes of tetraploids. RAPD analysis and seed coat anatomy investigation revealed some interspecies differences. Species from nearby areas have more similar RAPD characteristics compared with more geographically distant species. The results of this study may serve as an additional argument in favour of a monophyletic origination of the Adenolinum species and its strong isolation from other Linum species.

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n=12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.

Cytogenetics of Synbranchiformes: a comparative analysis of two Synbranchus Bloch, 1795 species from the Amazon

Synbranchidae belongs to the Synbranchiformes and occurs in Africa, Asia, Australia, Mexico, and Central and South America. This family comprises four genera: Synbranchus, Ophisternon, Monopterus, and Macrotrema. Only two are known from the neotropical region, Ophisternon and Synbranchus. According to current classification, Synbranchus has three valid species: S. marmoratus (Bloch 1795), S. madeirae (Rosen and Rumney 1972), and S. lampreia (Favorito, Zanata and Assumpção 2005). Thus the present research is aimed to cytogenetically characterize (by classical and molecular methods) two syntopic species-S. aff. lampreia and S. madeirae-from the central Amazon basin to validate the taxonomy of both species and provide a revisionary discussion on the cytogenetics of Synbranchiformes. Synbranchus aff. lampreia was found to possess 2n=44 chromosomes (6m+2st+36a, NF=50), while S. madeirae had 2n=46 chromosomes (6m+2st+38a, NF=52). Constitutive heterochromatin was dominant in the centromeric and terminal regions of most of the chromosomes in both species, although the precise distribution patterns were species-specific. The nucleolar organizing region was single in S. aff. lampreia and multiple in S. madeirae, as indicated by both AgNO(3) and hybridization using 18S rDNA probes. The 5S rDNA sites were located interstitially on the long arms of an acrocentric pair in both species, and the telomeric probe did not show any interstitial sites in either species. These data indicate the occurrence of interspecific karyotypic variability in Synbranchus and suggest that taxonomic review for this genus is necessary.

Karyotypic variation in Rhinophylla pumilio Peters, 1865 and comparative analysis with representatives of two subfamilies of Phyllostomidae (Chiroptera)

The family Phyllostomidae belongs to the most abundant and diverse group of bats in the Neotropics with more morphological traits variation at the family level than any other group within mammals. In this work, we present data of chromosome banding (G, C and Ag-NOR) and Fluorescence In Situ Hybridization (FISH) for representatives of Rhinophylla pumilio Peters, 1865 collected in four states of Brazil (Amazonas, Bahia, Mato Grosso and Pará). Two karyomorphs were found in this species: 2n=34, FN=64 in populations from western Pará and Mato Grosso states and 2n=34, FN=62 from Amazonas, Bahia, and northeastern Pará and Marajó Island (northern). Difference in the Fundamental Number is determined by variation in the size of the Nucleolar Organizer Region (NOR) accompanied with heterochromatin on chromosomes of pair 16 or, alternatively, a pericentric inversion. The C-banding technique detected constitutive heterochromatin in the centromeric regions of all chromosomes and on the distal part of the long arm of pair 15 of specimens from all localities. FISH with a DNA telomeric probe did not show any interstitial sequence, and an 18S rDNA probe and silver staining revealed the presence of NOR in the long arm of the pair 15, associated with heterochromatin, and in the short arm of the pair 16 for all specimens. The intra-specific analysis using chromosome banding did not show any significant difference between the samples. The comparative analyses using G-banding have shown that nearly all chromosomes of Rhinophylla pumilio were conserved in the chromosome complements of Glossophaga soricina Pallas, 1766, Phyllostomus hastatus Pallas, 1767, Phyllostomus discolor Wagner, 1843 and Mimon crenulatum Geoffroy, 1801, with a single chromosomal pair unique to Rhinophylla pumilio (pair 15). However, two chromosomes of Mimon crenulatum are polymorphic for two independent pericentric inversions. The karyotype with 2n=34, NF=62 is probably the ancestral one for the other karyotypes described for Rhinophylla pumilio.

Comet‐FISH with rDNA probes for the analysis of mutagen‐induced DNA damage in plant cells

We used comet-fluorescence in situ hybridization (FISH) in the model plant species Crepis capillaris following exposure of seedlings to maleic hydrazide (MH). FISH with 5S and 25S rDNA probes was applied to comets obtained under alkaline conditions to establish whether these DNA regions were preferentially involved in comet tail formation. MH treatment induced significant fragmentation of nuclear DNA and of rDNA loci. A 24-h post-treatment recovery period allowed a partial reversibility of MH-induced damage on nuclear and rDNA regions. Analyses of FISH signals demonstrated that rDNA sequences were always involved in tail formation and that 5S rDNA was more frequently present in the tail than 25S rDNA, regardless of treatment. The involvement of 25S rDNA in nucleolus formation and differences in chromatin structure between the two loci may explain the different susceptibility of the 25S and 5S rDNA regions to migrate into the tail. This work is the first report on the application of FISH to comet preparations from plants to analyze the distribution and repair of DNA damage within specific genomic regions after mutagenic treatment. Moreover, our work suggests that comet-FISH in plants may be a useful tool for environmental monitoring assessment.

Cytogenetic characterisation of the razor shells Ensis directus (Conrad, 1843) and E. minor (Chenu, 1843) (Mollusca: Bivalvia)

The European razor shell Ensis minor (Chenu 1843) and the American E. directus (Conrad 1843) have a diploid chromosome number of 38 and remarkable differences in their karyotypes: E. minor has four metacentric, one metacentric–submetacentric, five submetacentric, one subtelocentric and eight telocentric chromosome pairs, whereas E. directus has three metacentric, two metacentric–submetacentric, six submetacentric, six subtelocentric and two telocentric pairs. Fluorescent in situ hybridisation (FISH) using a major ribosomal DNA probe located the major ribosomal genes on one submetacentric chromosome pair in both species; FISH with a 5S ribosomal DNA (5S rDNA) probe rendered one chromosomal (weak) signal for E. minor and no signal for E. directus, supporting a more dispersed organisation of 5S rDNA compared to the major ribosomal genes. The vertebrate telomeric sequence (TTAGGG) n was located on both ends of each chromosome, and no interstitial signals were detected. In this work, a comparative karyological analysis was also performed between the four Ensis species analysed revealing that the three European species studied so far, namely E. minor, E. siliqua (Linne 1758) and E. magnus Schumacher 1817 show more similarities among them than compared to the American species E. directus. In addition, clear karyotype differences were found between the morphologically similar species E. minor and E. siliqua.

First karyotype description of Hypostomus iheringii (Regan, 1908): a case of heterochromatic polymorphism

In this study, which is the first karyotype analysis of Hypostomus iheringii, nine specimens collected in Córrego da Lapa (tributary of the Passa-Cinco River) showed a diploid number of 80 chromosomes. Silver nitrate staining and fluorescence in situ hybridization (FISH) with an 18S rDNA probe revealed the presence of multiple nucleolus organizer regions (NORs) (chromosome pairs 13, 20, and 34). FISH with a 5S rDNA probe showed that this cistron was only present in chromosome pair 2. When the karyotypes of individual animals were compared, unique heterochromatic polymorphisms were detected on chromosome pairs 1 and 5. Specifically, specimens had heterochromatic blocks (h+h+) on both chromosomes, one chromosome with heterochromatic blocks (h+h-) or chromosomes that lacked heterochromatic blocks (h-h-). Considering that heteromorphic pattern is not correlated with variation in size, the process of heterochromatinization might act on the long arms of these chromosomes. In summary, all chromosomal markers indicate that the karyotype of Hypostomus iheringii is highly differentiated and that the heterochromatinization of chromosomal segments may have contributed to its karyotypic differentiation.

Banding and FISH in three species of Vernonia, subsection Macrocephalae (Asteraceae, Vernonieae)

To complement our knowledge about the karyotypes of the genus Vernonia Schreb., different techniques of chromosome banding, including AgNOR, triple staining with fluorochromes CMA/DA/DAPI (CDD), and fluorescence in situ hybridization (FISH) for the 45S rDNA probe, were applied to three species of subsection Macrocephalae. Vernonia bardanoides was collected from an area of cerrado (savanna) vegetation in Itirapina, Sao Paulo State, Brazil, and V. linearifolia and V. tomentella were collected from areas of rocky, open altitudinal vegetation in Joaquim Felicio and Diamantina, respectively, in Minas Gerais State. All species showed two terminal CMA+ and NOR bands. FISH indicated two terminal 45S rDNA sites in V. linearifolia and V. tomentella, and six in V. bardanoides.

Comparative cytogenetic study on two species of the genus Entedon Dalman, 1820 (Hymenoptera, Eulophidae) using DNA-binding fluorochromes and molecular and immunofluorescent markers.

Karyotypes of Entedon cionobius Thomson, 1878 and Entedon cioni Thomson, 1878 (Hymenoptera: Eulophidae) were studied using DNA-binding ligands with different base specificity (propidium iodide, chromomycin A3, methyl green and DAPI; all these ligands, except for the last one, were used for the first time in parasitic wasps), C-banding, fluorescence in situ hybridization (FISH) with a 45S rDNA probe and 5-methylcytosine immunodetection. Female karyotypes of both species contain five pairs of relatively large metacentric chromosomes and a pair of smaller acrocentric chromosomes (2n = 12). As in many other Hymenoptera, males of both Entedon Dalman, 1820 species have haploid chromosome sets (n = 6). Fluorochrome staining revealed chromosome-specific banding patterns that were similar between the different fluorochromes, except for the CMA3- and PI-positive and DAPI-negative band in the pericentromeric regions of the long arms of both acrocentric chromosomes. The obtained banding patterns were virtually identical in both species and allowed for the identification of each individual chromosome. C-banding revealed a pattern similar to DAPI staining, although centromeric and telomeric regions were stained more intensively using the former technique. FISH detected a single rDNA site in the same position on the acrocentric chromosomes as the bright CMA3-positive band. Immunodetection of 5-methylcytosine that was performed for the first time in the order Hymenoptera revealed 5-methylcytosine-rich sites in the telomeric, centromeric and certain interstitial regions of most of the chromosomes.

Karyoevolutionary aspects of Atlantic hogfishes (Labridae–Bodianinae), with evidence of an atypical decondensed argentophilic heterochromatin

Fish from the family Labridae elicit considerable ecological interest, especially due to their complex interactions with the reef environment. Different karyoevolutionary tendencies have been identified in the subfamilies Bodianinae, Corinae and Cheilinae. Chromosomal analyses conducted in the Atlantic species Bodianus rufus (2n=48; 6m+12sm+14st+16a, FN=80), Bodianus pulchellus (2n=48; 4m+12sm+14st+18a, FN=78) and Bodianus insularis (2n=48; 4m+12sm+14st+18a, FN=78) identified Ag-NOR/18SrDNA sites located only in the terminal region of the short arm (p) of the largest subtelocentric pair. The 5S rDNA genes were mapped in the terminal region of the long arm (q) of the largest acrocentric pair and the p arm of chromosome 19 in B. insularis. The karyotype of the three species shows an extensive heterochromatic and argentophilic region, exceptionally decondensed, located in the p arm of the second subtelocentric pair. This region does not correspond to a NOR site, since it is not hybridized with 18S rDNA probes, and is not GC-rich, as generally occurs with nucleolus organizer regions of lower invertebrates. Heterochromatin in the three species is reduced and distributed over the centromeric and pericentromeric regions of chromosomes. The elevated number of two-armed chromosomes in species of Bodianus, in relation to other Labridae, shows karyotype diversification based on pericentric inversions, differentiating them markedly in terms of evolutionary tendencies that occur in subfamilies Corinae and Cheilininae. Structural cytogenetic similarities between B. pulchellus and B. insularis, in addition to the conserved chromosomal location pattern of ribosomal multigenic families, indicate phylogenetic proximity of these species.

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

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

Cytogenetic studies of Atlantic mojarras (Perciformes - Gerreidae): chromosomal mapping of 5S and 18S ribosomal genes using double FISH

Fish of the family Gerreidae, mainly species of the genera Diapterus and Eugerres, have high potential for cultivation, because of their saline tolerance. A detailed cytogenetic analysis of Diapterus auratus, Diapterus rhombeus and Eugerres brasilianus was conducted using conventional staining, C-banding, Ag-NOR, AT/GC-specific fluorochrome staining and mapping of ribosomal sequences with 5S and 18S rDNA probes. All the species exhibited symmetrical karyotype, 2n = 48 acrocentric chromosomes. Ag-NORs and 18S rDNA are present in the interstitial position on pair 1 (genus Diapterus) and pair 6 in Eugerres brasilianus. The 5S rDNA sites, located in the interstitial position (pair 11), are conserved in the three species. Heterochromatic regions are similar in the Diapterus species, showing a pattern of reduced and centromeric bands, differing from E. brasilianus, where, in addition to these, more prominent interstitial bands were observed. GC-rich regions are located at ribosomal sites. Karyotypic comparison between Diapterus and Eugerres reveals similarity in chromosomal macrostructure, differing in C-positive heterochromatin distribution and position of 18S sites, indicating the occurrence of structural microrearrangements. Although complementary analyses are needed, the similarities observed for these and other species suggest the possibility of breaking postzygotic barriers and their potential use, through induced interspecific or intergeneric hybridizations.

Dual-color FISH karyotype and rDNA distribution analyses on four Cucurbitaceae species

Karyotype and ribosomal DNA distribution on four Cucurbitaceae species was analyzed through dual-color fluorescence in situ hybridization (FISH) using 5S and 45S rDNA probes. Chromosome sizes varied slightly among the species with Cucumis sativus relatively the largest (∼2.5 μm) and Momordica charantia the smallest (∼1 μm). In Cucumis sativus L. (2n = 14), the 45S rDNA hybridized on the pericentromeric area of five chromosomes (metacentric a, b, c, g, and submetacentric d) and the 5S rDNA on the region proximal to the centromere of the short arm of chromosome e. In Luffa cylindrica (L.) Roem. (2n = 26), the 45S rDNA hybridized on the distal regions of the short arms of five chromosomes (metacentric a, b, c, f, and submetacentric d) and the 5S rDNA on the region proximal to the centromere of the short arm of chromosome e. In Lagenaria siceraria (Molina) Standl. (2n = 22), the 45S rDNA hybridized on the distal regions of the short arms of two chromosomes (submetacentric b and metacentric e) and the 5S rDNA juxtaposed with the 45S rDNA signal in a region proximal to the centromere on chromosome e. In Momordica charantia L. (2n = 22), the 45S rDNA hybridized on the majority of the short arms of two metacentric chromosomes (d and k) and the 5S rDNA on the proximal region of the short arm of chromosome e. The interphase and metaphase rDNA distribution and FISH karyotype analyses of the four species showed the possible fate of rDNAs through the process that lead to the interspecific variations among the cucurbits. These results will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species.

Intraspecific polymorphism of ribosomal DNA loci number and morphology in Brachypodium pinnatum and Brachypodium sylvaticum

The genus Brachypodium has become the target of extensive cytomolecular studies since one of its representatives, B. distachyon, has been accepted as a model plant for temperate cereals and forage grasses. Recent preliminary studies suggested that intraspecific rDNA polymorphism can occur in at least two members of the genus, B. sylvaticum and B. pinnatum, so the aim of this study was to further analyse this phenomenon. FISH with 25S rDNA and 5S rDNA probes was performed on somatic metaphase chromosomes, supplemented by the silver staining technique which distinguishes transcriptionally active from inactive 18S-5.8S-25S rDNA loci. The number, size and chromosomal distribution of 5S rDNA loci were very constant: two loci were invariably observed in all studied diploid accessions of both species, while four 5S rDNA loci were present in the tetraploid B. pinnatum. In contrast to 5S rDNA loci, those of the 35S rDNA were more variable. Two or three loci were observed in the diploid B. pinnatum and four in tetraploid accessions. In chromosome complements of B. sylvaticum accessions from two to six 35S rDNA sites were detected. Regardless of total rDNA locus number, only two were transcriptionally active in diploid accessions of both species, while two or four were active in the tetraploid B. pinnatum. Additionally, the fluorescent CMA/DAPI banding method was used to identify the relation between rDNA sites and CMA+ bands. It was revealed that the number and chromosomal distribution of CMA+ bands are in congruence only with 35S rDNA loci which gave strong FISH signals.

Population divergence and peculiar karyoevolutionary trends in the loricariid fish Hypostomus aff. unae from northeastern Brazil

Loricariidae (Siluriformes, Hypostominae) is one of the most diverse catfish families. In spite of the wide distribution of loricariids in South America, cytogenetic reports are available for only a few species, mostly from southern and southeastern Brazil. We made the first chromosomal analysis of Hypostomus aff. unae from the Contas River basin in northeastern Brazil. Four populations isolated by short distances but from distinct landscapes were studied based on conventional staining, C-banding, argyrophilic nucleolar organizer regions (Ag-NOR), CMA(3)/DAPI fluorochrome staining, and fluorescent in situ hybridization with 18S rDNA probes. Although sharing the same diploid number (2n = 76) and NOR locations, each population presented exclusive karyotype formulae and specific patterns of heterochromatic and AT-rich regions. The derived karyotypes of H. aff. unae (2n >54; high number of acrocentrics bearing AT-rich interstitial heterochromatin) indicated a divergent karyoevolution, mostly driven by centric fissions, pericentric inversions and particular heterochromatin dispersion models. This finding of distinct evolutionary units in H. aff. unae will be useful for understanding the natural history of loricariids from relatively unexplored coastal basins in South America.

Karyotypic Variation and Karyomorphology in Iranian Endemic Ecotypes of <i>Plantago ovata</i> Forsk.

Isabgol is an annual herb cultivated as a medicinal plant. In the present study, karyotypic analysis of 12 Iranian endemic Isabgol (Plantago ovata Forsk.) ecotypes was carried out. The chromosome number was 2n=2x=8 with 4m+4st karyotype formula. The mean value of total chromosome length (TL) was 2.86 μm, ranging from 2.44 μm (E4) to 3.26 μm (E12). All ecotypes had 2 pairs of satellites (3S, 4S), varying in size from 0.34 to 0.36 μm. Most of the chromosomal parameters were significantly different among the various ecotypes. In general, karyotypes were symmetrical and fell in the Stebbins 2A category. The scatter diagram of A1 and A2 asymmetry indicated 4 groups of ecotypes while PCA analysis together with cluster analysis and scatter diagram of TL and CI, showed 3 groups, containing 9, 2 and 1 ecotypes, respectively. C-Banding patterns and fluorescence in situ hybridization (FISH) with 45S rDNA probe provided landmarks for chromosome identification in somatic preparations.

Karyotype characterization of two populations of Vernonia geminata (Asteraceae, Vernonieae) using banding and FISH techniques

In order to extend our knowledge concerning karyotypes of the genus Vernonia, we applied various techniques of chromosome banding, including AgNOR and triple staining with the fluorochromes CMA/DA/DAPI (CDD), and of fluorescent in situ hybridization (FISH) for the 45S rDNA probe to specimens of two populations of Vernonia geminata collected from an open-pasture area, in southern Brazil. B chromosomes were observed in one of the populations. Both populations of V. geminata presented a pair of CMA(3)(+) terminal bands and one pair of chromosomes with terminal AgNOR banding. The FISH evidenced, in one population, two pairs of small sites of 45S rDNA; these being two small terminal sites and two centromeric sites. In the other population, there was only one pair of small terminal sites and two sites in two B chromosomes, one in each chromosome. There was coincidence of localization between CMA(+) and NOR bands with one of the small terminal sites of 45S rDNA of one chromosome of the normal complement, but not in B chromosomes.

Karyotype analysis of three Solanum plants using combined PI-DAPI staining and double fluorescence in situ hybridization with 45S and 5S rDNA probes

In this study, mitotic metaphase chromosomes of Solanum surattense Burm ., Solanum lyratum Thunb. and Solanum photeinocarpum Nakam. were well prepared using an advanced chromosome preparation method. The chromosomes were distinguished by combined PI-DAPI (CPD) staining and double fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes and their molecular cytogenetic karyotypes were established. Although, the karyotype of S. surattense Burm. and S. photeinocarpum Nakam was first established, the karyotype formulas of the three Solanum plants ( S. surattense Burm., S. lyratum Thunb. and S. photeinocarpum Nakam) can be described as follows: 2n=24=18m +4sm+2st (2SAT), 2n=24=18m +4sm+2st (2SAT) and 2n=24=18m +6sm (2SAT). Moreover, the karyotype asymmetry of the three species belongs to 2A type. After CPD staining, the centromeres of all chromosomes in the three species were shown as red CPD bands, indicating the presence of GC-rich DNA sequences in the chromosomes. Subsequential double FISH shows that S. surattense Burm., S. lyratum Thunb. and S. photeinocarpum Nakam, all have a pair of 45S rDNA sites located on chromosomes, and all of these 45S rDNA sites correspond to the respective prominent CPD banded regions. S. surattense Burm., S. lyratum Thunb. and S. photeinocarpum Nakam correspond to the CPD banded regions at the ends of chromosomes 8, 10 and 12, respectively. The three Solanum plants all have a pair of 5S rDNA sites. In S. surattense Burm., the 5S rDNA sites are located on the long arm of the 6th chromosome pair, while the 5S rDNA sites of S. lyratum Thunb. and S. photeinocarpum Nakam are all located on the short arms of the 4th chromosome pair. Our study shows that the CPD bands and rDNA FISH signals provide effective chromosome markers allowing us to establish accurate molecular cytogenetic karyotypes of the three species tested. Key words: Solanum surattense Burm. Solanum lyratum Thunb., Solanum photeinocarpum Nakam , karyotype, CPD staining, fluorescence in situ hybridization (FISH).