Comparative cytogenetic studies of Curimatidae (Pisces, Characiformes) from the middle Paraná River (Argentina)

Almost all species of the Curimatidae family have a stable karyotype, with a diploid number of 54 metacentric (M) and submetacentric (SM) chromosomes, and one sole nucleolus organizer pair. This family has considerable specific diversity in Argentinean fluvial basins; however, no cytogenetic data are available. Eight species from the Paraná River (Argentina): Cyphocharax voga, C. spilotus, C. platanus, Steindachnerina brevipinna, S. conspersa, Curimatella dorsalis, Psectrogaster curviventris, and Potamorhina squamoralevis were analyzed cytogenetically. Chromosome preparations were obtained from direct samples and through cell culture, and they were processed for conventional, C- and nucleolar organizer region-banding. Six of the species exhibited the standard family karyotype, with 2n = 54 M-SM and fundamental number of chromosomes (FN) = 108, as well as variations in the chromosome formula, and in heterochromatic and nucleolar organizer regions. Though nucleolar organizer regions were located on only one chromosome pair, they varied in both carrier chromosomes and pairs involved. On the other hand, C. platanus showed a complement of 2n = 58 M-SM and subtelocentric with FN = 116, and P. squamoralevis presented 2n = 102, with some M-SM and a large number of acrocentric chromosomes. Even though the karyotype macrostructure appears to be conserved, the speciation process within the family has been accompanied by micro-structural rearrangements, as evidenced by pattern diversity in the heterochromatin and nucleolar organizer regions. Some changes in chromosome macrostructure have also occurred in this group, primarily in C. platanus and P. squamoralevis, in which there have been centric dissociations and inversions.     

Pericentric satellite DNA sequences in Pipistrellus pipistrellus (Vespertilionidae; Chiroptera)

This paper reports the molecular and cytogenetic characterization of a HindIII family of satellite DNA in the bat species Pipistrellus pipistrellus. This satellite is organized in tandem repeats of 418 bp monomer units, and represents approximately 3% of the whole genome. The consensus sequence from five cloned monomer units has an A-T content of 62.20%. We have found differences in the ladder pattern of bands between two populations of the same species. These differences are probably because of the absence of the target sites for the HindIII enzyme in most monomer units of one population, but not in the other. Fluorescent in situ hybridization (FISH) localized the satellite DNA in the pericentromeric regions of all autosomes and the X chromosome, but it was absent from the Y chromosome. Digestion of genomic DNAs with HpaII and its isoschizomer MspI demonstrated that these repetitive DNA sequences are not methylated. Other bat species were tested for the presence of this repetitive DNA. It was absent in five Vespertilionidae and one Rhinolophidae species, indicating that it could be a species/genus specific, repetitive DNA family.     

Replication Banding in two Mediterranean Moray eels: Chromosomal Characterization and Comparison

Early and late replication bandings have been obtained by in vitro BrdU incorporation in the Mediterranean Muraenidae species Muraena helena and Gymnothorax unicolor, and used to characterize their karyotypes. A comparative analysis of the banding patterns allowed to point out high karyotype similarity as well as chromosome rearrangements that occurred in karyotype evolution between these species.     

Differentiation of repetitive DNA sites and sex chromosome systems reveal closely related group in Parodontidae (Actinopterygii: Characiformes)

Parodon and Apareiodon lack sufficiently consistent morphological traits to be considered a monophyletic group in Parodontidae. Species within this family are either sex-homomorphic or sex-heteromorphic (i.e., lacking a differentiated sex chromosome system, ZZ/ZW or ZZ/ZW(1)W(2)). In this study, a DNA fragment from the heterochromatin segment of the W chromosome of Apareiodon ibitiensis (named WAp) was microdissected and used for in situ mapping of nine Parodontidae species. The species were also characterized using a satellite DNA probe (pPh2004). The species were phylogenetically clustered according to 17 characters, which were examined by both classical and molecular cytogenetic techniques. Given the present results, the single ZZ/ZW sex chromosome system seems to have been derived from a paracentric inversion of a terminal WAp site onto the proximal regions of the short arms of a metacentric chromosome pair, followed by WAp site amplification. We reason that these events restrained recombination and favored differentiation of the W chromosome in some species. Moreover, co-hybridization experiments targeting the WAp and pPh2004 repetitive DNA sites of A. affinis suggest that the ZZ/ZW(1)W(2) sex chromosomes of this species may have arisen from a translocation between the proto-sex chromosome and an autosome. Our phylogenetic analysis corroborates the hypothesis of sex chromosome differentiation and establishes groups of closely related species. The phylogenetic reorganization in response to these new data supports the presence of internal monophyletic groups within Parodontidae.     

Cytogenetic analyses of two Curimatidae species (Pisces; Characiformes) from the Paranapanema and Tietê Rivers.

Cytogenetic analyses were performed in two Curimatidae species (Steindachnerina insculpta and Cyphocharax modesta) from the Paranapanema and Tietê Rivers (S?o Paulo State, Brazil), showing a karyotype composed of 54 meta-submetacentric chromosomes in both species. Silver- and chromomycyn-staining and fluorescent in situ hybridization (FISH) using a 18S rDNA probe indicated that the nucleolar organizer regions (NORs) of both species are localized in the terminal region of the long arm of two metacentric chromosomes. Although a single NOR system was evidenced in both analyzed species, S. insculpta and C. modesta presented the nucleolar organizer regions in distinct chromosome pairs, indicating that these cistrons can be considered cytogenetic markers. Variation on the amount and distribution of the constitutive heterochromatin (C-bands) could also be detected between the two species - while S. insculpta presented few heterochromatic blocks, intensely stained C-bands were evidenced in C. modesta specially in the terminal region of the long arm of the NOR-bearing chromosomes. Although most Curimatidae species have been characterized by homogeneous karyotypes, isolated populations could be established under different environmental conditions leading to karyotype micro-structure variations specially related to the NORs localization and C-banding distribution. The obtained data were useful for the cytogenetic characterization and differentiation of S. insculpta and C. modesta and could be used in evolutionary inferences in the Curimatidae group.     

Mapping five repetitive DNA classes in sympatric species of Hypostomus (Teleostei: Siluriformes: Loricariidae): analysis of chromosomal variability

Fish belonging to the genus Hypostomus are known for exhibiting a striking diversity in its karyotype structure, however the knowledge concerning the distribution patterns of heterochromatin and location of repetitive DNA sequences in the karyotypes is still limited. Aiming a better understanding of the chromosomal organization in this group, we analyzed three sympatric species of Hypostomus collected in the Hortelã stream, a component of the Paranapanema River basin, Botucatu/SP/Brazil. The analyses involved the cytogenetic characterization and chromosomal mapping of repetitive sequences and intra/interspecific comparisons using sequences of the cytochrome C oxidase subunit I. The results revealed that H. ancistroides presents a karyotype with 2n = 68 chromosomes, H. strigaticeps 2n = 72 chromosomes, and H. nigromaculatus 2n = 76 chromosomes. In addition to differences found in the diploid number, it was also observed variations in karyotypic formulae, amount of constitutive heterochromatin, and location of nucleolus organizer regions. The cytogenetic mapping of 5S and 18S rDNA, as well as of the H3 histone gene, disclosed a differential dispersion process among the three species. In some cases the Rex1 transposable element showed to be co-located with 5S rDNA sites. The molecular analyses support the cytogenetic data and represent an additional tool for the characterization of the analyzed species. The results evidenced that chromosomal variations are not restricted to differences in diploid number or karyotypic macrostructure in the genus Hypostomus, indicating that events such as transposition of heterochromatin and rDNA segments may participate in the differentiation process occurred in these species.     

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.     

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.     

Cytogenetic characterization of the Trinidad endemic, Arachnocoris trinitatus Bergroth: the first data for the tribe Arachnocorini (Heteroptera: Cimicomorpha: Nabidae)

As an extension of the ongoing cytogenetic studies of the bug family Nabidae (Heteroptera: Cimicomorpha), the first evidence for the tribe Arachnocorini (the subfamily Nabinae), with reference to the Trinidad endemic, Arachnocoris trinitatus Bergroth, is provided. This is an attempt to gain a better insight into the evolution, systematics and within-family relationships of the family Nabidae. The studies were conducted using a number of cytogenetic techniques. The male karyotype (chromosome number and size; sex chromosome system; NOR location; C-heterochromatin amount, distribution and characterization in terms of the presence of AT-rich and GC-rich DNA), and male meiosis with particular emphasis on the behavior of the sex chromosomes in metaphase II are described. Also investigated are the male and female internal reproductive organs with special reference to the number of follicles in a testis and the number of ovarioles in an ovary. A. trinitatus was found to display a number of characters differentiating it from all hitherto studied nabid species placed in the tribe Nabini of the subfamily Nabinae, and in the tribe Prostemmatini of the subfamily Prostemmatinae. Among these characters are chromosome number 2n = 12 (10 + XY), the lowest within the family, nucleolus organizer regions (NORs) situated on the autosomes rather than on the sex chromosomes as is the case in other nabid species, and testes composed of 3 follicles but not of 7 as in other nabids. All the data obtained suggest many transformations during the evolution ofA. trinitatus.     

Primate evolution of a dispersed human repetitive DNA sequence

A dispersed middle repetitive DNA sequence isolated originally from human chromosome 12 did not show homology with rodent DNA under standard conditions of Southern DNA blot analysis. The evolutionary relationship of this human repetitive DNA to that of other primates was investigated using three hybridization methods: DNA dot blot, Southern DNA blot analysis, and chromosome in situ hybridization. Homology with the human repetitive DNA was found throughout the suborder Anthropoidea, in fourteen ape and New and Old World monkey species. In addition, the human pattern of hybridization to noncentromeric regions of all chromosomes was seen. No hybridization by any of the three techniques was found in five species of the suborder Prosimii. The phenomenon of marked differences in sequence homology and copy number of dispersed repetitive DNA from closely related species has been observed in protozoans (Plasmodia), Drosophila, sea urchins, mice and the great apes (Hominoidea). We report here a similar phenomenon that may have occurred at an early stage in primate evolution.     

Random BAC FISH of monocot plants reveals differential distribution of repetitive DNA elements in small and large chromosome species

BAC FISH (fluorescence in situ hybridization using bacterial artificial chromosome probes) is a useful cytogenetic technique for physical mapping, chromosome marker screening, and comparative genomics. As a large genomic fragment with repetitive sequences is inserted in each BAC clone, random BAC FISH without adding competitive DNA can unveil complex chromosome organization of the repetitive elements in plants. Here we performed the comparative analysis of the random BAC FISH in monocot plants including species having small chromosomes (rice and asparagus) and those having large chromosomes (hexaploid wheat, onion, and spider lily) in order to understand a whole view of the repetitive element organization in Poales and Asparagales monocots. More unique and less dense dispersed signals of BAC FISH were observed in species with smaller chromosomes in both the Poales and Asparagales species. In the case of large-chromosome species, 75-85% of the BAC clones were detected as dispersed repetitive FISH signals along entire chromosomes. The BAC FISH of Lycoris did not even show localized repetitive patterns (e.g., centromeric localization) of signals.     

A germline restricted, highly repetitive DNA sequence in Paramyxineatami: an interspecifically conserved, but somatically eliminated, element

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese hagfish, Paramyxine atami. The repeat unit of this family, which is 83?bp long, has been designated “EEPa1”, for Eliminated Element of P. atami 1. DNA filter hybridization using EEPa1 as a probe revealed that this family is shared among several species and is conserved in the germline DNA. Although eliminated, repetitive DNA that is shared interspecifically has not been reported in hagfish species, cases of chromatin diminution and chromosome elimination processes have been described previously in other organisms.The patterns and intensities of hybridization signals suggest that members of the repetitive DNA family defined by EEPa1 have undergone concerted molecular evolution.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Molecular cytogenetic characterization of the Amazon River dolphin Inia geoffrensis

Classical and molecular cytogenetic (18S rDNA, telomeric sequence, and LINE-1 retrotransposon probes) studies were carried out to contribute to an understanding of the organization of repeated DNA elements in the Amazon River dolphin (boto, Inia geoffrensis). Twenty-seven specimens were examined, each presenting 2n?=?44 chromosomes, the karyotype formula?12m?+?14sm?+?6st?+?10t?+?XX/XY, and fundamental number (FN)?=?74. C-positive heterochromatin was observed in terminal and interstitial positions, with the occurrence of polymorphism. Interstitial telomeric sequences were not observed. The nucleolar organizer region (NOR) was located at a single site on a smallest autosomal pair. LINE-1 was preferentially distributed in the euchromatin regions, with the greatest accumulation on the X chromosome. Although the karyotype structure in cetaceans is considered to be conserved, the boto karyotype demonstrated significant variations in its formula, heterochromatin distribution, and the location of the NOR compared to other cetacean species. These results contribute to knowledge of the chromosome organization in boto and to a better understanding of karyoevolution in cetaceans.     

A high-resolution karyotype of cucumber (Cucumis sativus L. 'Winter Long') revealed by C-banding, pachytene analysis, and RAPD-aided fluorescence in situ hybridization.

Using molecular cytogenetic DNA markers, C-banding, pachytene analysis, and fluorescence in situ hybridization (FISH), a high-resolution karyotype was established in the cucumber. C-banding showed distinct hetero chromatic bands on the pericentromeric, telomeric, and intercalary regions of the chromosomes. The C-banding patterns were also consistent with the morphology of 4'-6-diamino-2-phenylindole dihydrochloride (DAPI)-stained pachytene chromosomes. Two repetitive DNA fragments, CsRP1 and CsRP2, were obtained by PCR and localized on the mitotic metaphase and meiotic pachytene chromosomes. CsRP1 was detected on the pericentromeric heterochromatic regions of all chromosomes, except chromosome 1. CsRP2 was detected on 5 (chromosomes 1, 2, 3, 4, and 7) of 7 chromosomes. All homologous chromosome pairs could be distinguished by FISH using 2 RAPD markers. This is the first report on molecular karyotyping of mitotic and meiotic spreads of cucumber.     

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis,Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41-42 bp CNM repeat and the XHOI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.     

Chromosomal polymorphism in <Emphasis Type="Italic">Steindachneridion</Emphasis><Emphasis Type="Italic">melanodermatum</Emphasis> Garavello, 2005 (Siluriformes,Pimelodidae): a reappraisal the existence of sex chromosome system in the species

Fifty-five specimens of Steindachneridion melanodermatum were analyzed using molecular and conventional cytogenetic tools. Two polymorphisms were found: one involving the length of nucleolar organizer regions and another involving two submetacentric chromosomes previously identified as sex chromosomes. The polymorphism was confirmed by homogeneity between male and female karyotypes. Nucleotide sequencing and physical chromosome mapping were also used to identify and characterize one class of repetitive DNA, named SmAluI-Rex3. Based on the results and literature the present study offers an update of the occurrence of sex chromosome system in this species.     

Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis

Two recombinant DNA clones that are localized to single human chromosomes were isolated from a human repetitive DNA library. Clone pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. Clone pHuR 195, a variant satellite 2 sequence, specifically hybridizes to chromosome position 16qh. These locations were determined by fluorescent in situ hybridization to metaphase chromosomes, and confirmed by DNA hybridizations to human chromosomes sorted by flow cytometry. Pulsed field gel electrophoresis analysis indicated that both sequences exist in the genome as large DNA blocks. In situ hybridization to intact interphase nuclei showed a well-defined, localized organization for both DNA sequences. The ability to tag specific human autosomal chromosomes, both at metaphase and in interphase nuclei, allows novel molecular cytogenetic analyses in numerous basic research and clinical studies.     

Variations in repetitive DNA and heterochromatin in the genus Artemia

The genus Artemia (Crustacea, Phyllopoda) is widely distributed all over the world as a result partly of natural colonization and partly of spread by birds and man. Artemia offers a very interesting model for speciation studies, since the genus comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, heteroploidy and polyploidy). The finding of the clustered repetitive AluI DNA family in the heterochromatin of A. franciscana can provide a useful tool for investigating the relationship between the members of the genus Artemia at the molecular level. Sixteen strains of Artemia, comprising sibling species and parthenogenetic populations, were analysed for the presence of AluI repetitive DNA by dot-blot hybridization. The observed variation in the content of repetitive DNA together with genetical, biological and geological data, support the hypothesis that Artemia living in the New World are derived from ancestral species that evolved in the Mediterranean area.