Cytotaxonomy of Astyanax (Characiformes,Characidae) from the Upper Iguaçu River Basin: confirmation of the occurrence of distinct evolutionary units

Taxonomic studies of the genus Astyanax from the Iguaçu River (Brazil) indicate that they may be differentiated into 11 distinct species, some of which have not been formally described and named so for. This study focuses on three of these species, Astyanax sp. B, Astyanax sp. C and Astyanax sp. D from the Upper Iguaçu River Basin. Comparative cytogenetic analyses of C‐banding, Ag‐NORs (silver nitrate stained nucleolar organizer region) and 18S and 5S rDNA corroborate that they are distinct species. A diploid number of 50 chromosomes and similar karyotypic formulae were observed in the three taxa, with the exception of Astyanax sp. D that differs in the number of submetacentric and subtelocentric chromosomes. However, the NOR silver‐staining pattern, the heterochromatic bands (C‐bands) and the mapping of the 18S and 5S rDNA sites in the chromosomes showed divergences between all three species under study, supporting the occurrence of distinct evolutionary units.     

Physical mapping of rDNA genes establishes the karyotype of almond

The localisation of ribosomal RNA genes on chromosomes of almond (Prunus amygdalus, 2n = 16) was studied by fluorescence in situ hybridisation. Simultaneous double-colour hybridisation with both 18S–5.8S–25S and 5S rDNA probes demonstrated that all chromosomes can be identified. In spite of the small size, differences in length between chromosomes that hybridised with the same rDNA probe as well as between chromosomes without hybridisation signal are apparent. Chromosomes were ordered in the karyotype according to their length. The 18S-5.8S-25S rDNA genes were detected in subdistal positions of chromosomes 2, 3, and 8. Sites located on chromosomes 2 and 3 carry a higher number of repeats than the site of chromosome 8. The 5S rDNA genes were found proximally located on chromosomes 5 and 7, the signal on chromosome 5 showing higher intensity than the signal on chromosome 7. Chromosomes 1, 4, and 6 show no hybridisation signal.     

Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA(3) ) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO (3) staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.     

Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes,Prochilodontidae)

A single NOR-bearing chromosome pair was identified by silver nitrate staining in a previous study of the fish Prochilodus argenteus from the S ã o Francisco River (MG, Brazil), with a third metacentric chromosome sporadically bearing active NOR. The present study focused on an analysis of the chromosomal localization of both the major (45S) and the minor (5S) rRNA genes using FISH. The use of the 18S rDNA probe confirmed the previous Ag-NOR sites interstitially located in a large metacentric pair and also identified up to three other sites located in the telomeric regions of distinct chromosomes, characterizing an interindividual variation of these sites. In addition, the 5S rDNA site was revealed adjacent to the major NOR site, identified at the end of the large Ag-NOR bearing metacentric chromosome. In a few metaphases, an additional weak hybridization signal was observed in a third chromosome, possibly indicating the presence of another 5S rDNA cluster. Despite a lower karyotype diversification (2n=54 and FN=108) often observed among species of Prochilodontidae, variations involving both 45S and 5S rRNA genes could play an important role in their chromosome diversification.     

Sex chromosome system ZZ/ZW in Apareiodon hasemani Eigenmann, 1916 (Characiformes,Parodontidae) and a derived chromosomal region

Parodontidae fish show few morphological characteristics for the identification of their representatives and chromosomal analyses have provided reliable features for determining the interrelationships in this family. In this study, the chromosomes of Apareiodon hasemani from the São Francisco River basin, Brazil, were analyzed and showed a karyotype with 2n = 54 meta/submetacentric chromosomes, and a ZZ/ZW sex chromosome system. The study revealed active NORs located on pair 11 and additional 18S rDNA sites on pairs 7 and 22. The 5S rDNA locus was found in pair 14. It showed a pericentric inversion regarding the ancestral condition. The satellite DNA pPh2004 was absent in the chromosomes of A. hasemani, a shared condition with most members of Apareiodon. The WAp probe was able to detect the amplification region of the W chromosome, corroborating the common origin of the system within Parodontidae. These chromosomal data corroborate an origin for the ZW system of Parodontidae and aid in the understanding of the differentiation of sex chromosome systems in Neotropical fishes.     

Structure and variability of nuclear ribosomal genes in the genus Helianthus

Summary The restriction map of the rDNA unit of Helianthus annuus was constructed using EcoRI, BamHI, HindIII, KpnI and SacI restriction enzymes. Variations in this map among 61 ecotypes representing 39 species of the genus Helianthus were analyzed. The sizes of the rDNA unit ranged from 9.8 to 11.0 kbp, due to a length-repeat heterogeneity of the external non-transcribed spacer by increments of 200 base pair segments. Lengthrepeat heterogeneity and restriction polymorphism were found to be characteristic of populations or species of Helianthus. Restriction patterns and thermal melting with probes of a cloned H. annuus ENTS segment allowed us to differentiate species from each other. However, most lines of the cultivated sunflower were found to be identical on the basis of the physical properties of their ribosomal DNA.     

Chromosome characterization and biogeographic relations among three populations of the driftwood catfish Parauchenipterus galeatus (Linnaeus, 1766) (Siluriformes: Auchenipteridae) in Brazil

The present study employed basic and molecular cytogenetic methods to characterize three populations of Parauchenipterus galeatus from the basins of the Paraná and São Francisco Rivers, and a region of connection between the two basins. Although the diploid number was equal to 58 chromosomes, variations in karyotype formula were detected among the populations. B chromosomes were detected only in the population from the São Francisco River. Heterochromatin was located in the terminal position in almost all the chromosomes and in the pericentromeric position in some acrocentric chromosomes in the three populations. A single nucleolus organizer region was detected by silver nitrate and 18S rDNA‐fluorescent in situ hybridization in the short arm of one subtelocentric pair in the three populations, varying only in the chromosome pair bearing this site. The 5S rDNA sites were located in two submetacentric chromosome pairs in the three populations, varying only in the chromosome pairs bearing these sequences. Classic and molecular chromosome markers, along with the context of the natural history of the formation of hydrographic basins, ecological aspects, and the geographic isolation of populations between hydrographic basins and within the same basin, were important contributions to the discussion on possible biogeographic relations among the populations of Parauchenipterus galeatus. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 648–656.     

FISH在人类未受精卵染色体异常分析中的应用

分子细胞遗传学的主要技术代表———荧光原位杂交 (FISH)是用荧光标记的依靠探针杂交原理在细胞核中或染色体上显示某一特定核酸序列的位置 ,并可进行相对定量分析 .它广泛应用于遗传病的诊断、产前诊断、肿瘤遗传学、进化遗传学研究和基因定位等领域 ,随着辅助生殖技术的进展 ,将在植入前胚胎遗传学诊断 (PGD)、生殖细胞 (卵母细胞和精子 )染色体异常的研究方面发挥更大的用途 .它是联系分子遗传学和细胞遗传学之间的桥梁 .     

Chromosomal diversity in three species of electric fish (Apteronotidae,Gymnotiformes) from the Amazon Basin

Cytogenetic studies were carried out on samples of Parapteronotus hasemani, Sternarchogiton preto and Sternarchorhamphus muelleri (Apteronotidae, Gymnotiformes) from the Amazon basin. The first two species exhibited both a 2n = 52 karyotype, but differed in their karyotypic formulae, distribution of constitutive heterochromatin, and chromosomal location of the NOR. The third species, Sternarchorhamphus muelleri, was found to have a 2n = 32 karyotype. In all three species the DAPI and chromomycin A3 staining results were consistent with the C-banding results and nucleolar organizer region (NOR) localization. The 18S rDNA probe confirmed that there was only one pair of ribosomal DNA cistron bearers per species. The telomeric probe did not reveal interstitial telomeric sequences (ITS). The karyotypic differences among these species can be used for taxonomic identification. These data will be useful in future studies of these fishes and help understanding the phylogenetic relationships and chromosomal evolution of the Apteronotidae.     

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.     

Chromosome mapping of repetitive sequences in four Serrasalmidae species (Characiformes)

The Serrasalmidae family is composed of a number of commercially interesting species, mainly in the Amazon region where most of these fishes occur. In the present study, we investigated the genomic organization of the 18S and 5S rDNA and telomeric sequences in mitotic chromosomes of four species from the basal clade of the Serrasalmidae family: Colossoma macropomum, Mylossoma aureum, M. duriventre, and Piaractus mesopotamicus, in order to understand the chromosomal evolution in the family. All the species studied had diploid numbers 2n = 54 and exclusively biarmed chromosomes, but variations of the karyotypic formulas were observed. C-banding resulted in similar patterns among the analyzed species, with heterochromatic blocks mainly present in centromeric regions. The 18S rDNA mapping of C. macropomum and P. mesopotamicus revealed multiple sites of this gene; 5S rDNA sites were detected in two chromosome pairs in all species, although not all of them were homeologs. Hybridization with a telomeric probe revealed signals in the terminal portions of chromosomes in all the species and an interstitial signal was observed in one pair of C. macropomum.     

Insights into chromosomal evolution of Cicadomorpha using fluorochrome staining and mapping 18S rRNA and H3 histone genes

The infraorder Cicadomorpha (Hemiptera) is a cosmopolitan species‐rich lineage of phytophagous insects. They have holocentric chromosomes and vary greatly in diploid number across families, with X0 as the predominant sex male mechanism. Here, we advance the understanding of chromosome mapping of repetitive elements of four families of cicadomorphan insects, the spittlebugs (Cercopidae), leafhoppers (Cicadellidae), and treehoppers (Aetalionidae and Membracidae). Sampled individuals from 19 species show considerable variation in diploid number, which may have originated from fusions between autosomes or between autosomes and the ancient X. The distribution of CMA₃⁺ blocks, primarily observed in low numbers in autosomal regions, was a conserved trait. Likewise, fluorescence in situ hybridization (FISH) mapping revealed mainly one locus per haploid genome for the 18S rRNA gene and for H3, each of which is located on distinct chromosomes. Despite the extensive variation in the number of autosomes and sex systems, the number of loci of ribosomal and H3 genes remained stable and may reflect the ancestral genome organizations in these groups. These results shed light on the chromosomal‐level organization in Cicadomorpha and provide new insights into the evolutionary history of karyotypes and repetitive elements in this diverse insect lineage.     

FISH-mapping of 18S ribosomal RNA genes and telomeric sequences in the Japanese bitterlings Rhodeus ocellatus kurumeus and Tanakia limbata (Pisces,Cyprinidae) reveals significant cytogenetic differences in morphologically similar karyotypes

The Japanese rose bitterling, Rhodeus ocellatus kurumeus, and the oily bitterling, Tanakia limbata, were cytogenetically studied by silver (Ag)- and chromomycin A₃ (CMA₃)-staining, by C-banding and by mapping of the 18S ribosomal genes and of the (TTAGGG) _n telomeric sequence. These two representative species of related genera of the subfamily Acheilognathinae show very similar chromosome complements. Nevertheless, significant differences in the chromosomal distribution of nucleolus organizer regions (NORs) and interstitial telomeric sequences were observed. Whereas R. ocellatus kurumeus shows a single NOR-bearing chromosome pair, T. limbata is characterized by a higher number of variable NORs. Multiple telomeric sequence sites were found at the pericentromeric regions of several chromosomes in the rose bitterling. No telomeric sequence sites were detected near centromeres, but they were found to be scattered along the NORs in the oily bitterling. Two karyoevolutive trends might have been identified in the subfamily.     

Chromosome analysis and FISH mapping of ribosomal DNA (rDNA), telomeric (TTAGGG)n and (GATA)n repeats in the leech Haemopis sanguisuga (L.) (Annelida: Hirudinea)

In the present paper the chromosome complement (n = 13; 2n = 26) of the common leech Haemopis sanguisuga (L.) (Annelida: Hirudinea: Hirudinidae) was analyzed using banding techniques and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG) _n and (GATA) _n ]. FISH with the rDNA probe consistently mapped major ribosomal clusters (18S–28S rDNA) in the pericentromeric region of one large metacentric chromosome pair; this region, which consisted of heterochromatin rich in GC base pairs, was preferentially stained by silver nitrate (Ag-NOR). The (TTAGGG) _n telomeric probe was hybridized with the termini of nearly all chromosomes, whereas the (GATA) _n probe did not label any chromosome areas.     

Physical mapping of 35S rRNA genes and genome size variation in polyploid series of Sisyrinchium micranthum and S. rosulatum (Iridaceae: Iridoideae)

Sisyrinchium micranthum and S. rosulatum are part of a species complex in which S. micranthum displays considerable morphological variation. S. rosulatum is a tetraploid species, whereas S. micranthum plants may present three different ploidy levels (2x, 4x, and 6x), so that polyploidy might have an important role in the diversification of this group. Notwithstanding, most cytogenetic studies on these species are based on chromosome counting. Aiming to understand how polyploidy may have impacted the genomes of these species, the DNA content of 184 specimens was estimated; fluorochrome banding with chromomycin A₃ and fluorescent in situ hybridization using an 18S-5.8S-26S ribosomal DNA (rDNA) probe were also performed. The results showed a reduction in monoploid genome size (1Cx), as well as in the number of heterochromatin bands and rDNA sites per monoploid genome, from diploids to polyploids. Additionally, intraspecific and within-ploidy variations in genome size and number of rDNA sites were observed. The source of varying structure in genome organization of these plants may be the multiple independent formations of polyploids along with an ongoing diploidization process. However, the intraspecific and within-ploidy polymorphisms indicate genetic mechanisms other than genome duplication and diploidization to be important to the genome evolution of these taxa.     

Cytogenetic studies in six species of Scinax (Anura,Hylidae) clade Scinax ruber from northern and northeastern Brazil

Scinax species are still underrepresented in cytogenetic studies, mainly with respect to populations from northeastern and northern Brazil. In this study, we provide new chromosomal information on Scinax boesemani, S. camposseabrai, S. garbei, S. pachycrus, S. trilineatus and S. x-signatus, all belonging to clade S. ruber. They were collected at two locations in the Caatinga biome (northeastern Brazil) and at one in the Amazon (northern Brazil) biomes. Chromosomes were analyzed by conventional staining, C-banding, Ag-NOR staining, and fluorochrome staining. All species shared a modal diploid value of 2n = 24 and fundamental arm number (FN) of 48. Moreover, both chromosomal size and morphology were similar to other species in this Scinaxclade. C-banding revealed centromeric heterochromatin in all species, along with terminal species-specific C-bands in some species. Active nucleolar organizer regions (Ag-NORs) were identified at 11q in most species, except for S. boesemani and S. garbei (Ag-NORs at interstitial region of 8q). Differing from most anurans, GC-rich regions were not restricted to NORs, but also coincident with some centromeric and terminal C-bands. These data contribute to the cytotaxonomy of Scinax by providing chromosomal markers and demonstrating the occurrence of microstructural rearrangements and inversions on chromosomal evolution of Scinax.     

Physical mapping of the 18S-25S and 5S ribosomal RNA genes in diploid bananas

Fluorescent in situ hybridisation (FISH) was used to determine the number and distribution of the 18S-25S and 5S rDNA sites on mitotic chromosomes of 6 wild and 2 edible diploid (2n=22) accessions belonging to the two banana species, Musa acuminata and M. balbisiana. FISH with the 18S-25S probe resulted in signals on one pair of chromosomes, the position of signals corresponded to the secondary constriction at the end of a short arm. The intensity of labelling was different between the homologues and the larger site corresponded to a larger secondary constriction. This labelling pattern was observed consistently in all genotypes. On the other hand, differences in the number of 5S sites were observed between the accessions. While in some of the wild seeded species, the 5S rDNA was localised on two pairs of chromosomes, hybridisation signals appeared on three pairs of chromosomes in other wild accessions. Quite unexpectedly, only five sites of 5S rDNA were reproducibly observed in the two vegetatively propagated diploid edible cultivars, Pisang Mas and Niyarma Yik, evidence for structural heterozygosity. A dual colour FISH showed that in all accessions, the satellite chromosomes carrying the 18S-25S loci did not carry the 5S loci. The results demonstrate that molecular cytogenetics can be applied to Musa and that physical cytogenetic maps can be generated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Karyotypic conservatism in samples of Characidium cf. zebra (Teleostei, Characiformes, Crenuchidae): Physical mapping of ribosomal genes and natural triploidy

Basic and molecular cytogenetic analyses were performed in specimens of Characidium cf. zebra from five collection sites located throughout the Tietê, Paranapanema and Paraguay river basins. The diploid number in specimens from all samples was 2n = 50 with a karyotype composed of 32 metacentric and 18 submetacentric chromosomes in both males and females. Constitutive heterochromatin was present at the centromeric regions of all chromosomes and pair 23, had additional interstitial heterochromatic blocks on its long arms. The nucleolar organizer regions (NORs) were located on the long arms of pair 23, while the 5S rDNA sites were detected in different chromosomes among the studied samples. One specimen from the Alambari river was a natural triploid and had two extra chromosomes, resulting in 2n = 77. The remarkable karyotypic similarity among the specimens of C. cf. zebra suggests a close evolutionary relationship. On the other hand, the distinct patterns of 5S rDNA distribution may be the result of gene flow constraints during their evolutionary history.     

Chromosome rearrangements in Pectinidae (Bivalvia: Pteriomorphia) implied based on chromosomal localization of histone H3 gene in four scallops

Chromosomal structural rearrangement in four scallops, Chlamys farreri (n = 19), Patinopecten yessoensis (n = 19), Chlamys nobilis (n = 16) and Argopecten irradians (n = 16), was studied by fluorescence in situ hybridization using histone H3 gene probes. The results show that histone H3 gene sites differ strikingly with regard to number, location, and intensity among, or even within these species. For example, two histone H3 gene loci were detected on the metaphase chromosomes of P. yessoensis, while one locus was found in the others. In P. yessoensis, differing intensities of hybridization signals were detected between homologues 5 and 11, and within homologue 11. These data suggest that the histone H3 gene is a qualified chromosome marker for the preliminary understanding of the historical chromosomal reconstructing of the Pectinidae family. The variable distribution patterns of the histone H3 gene suggest that gene duplication/diminution as well as chromosome rearrangements by inversion and translocation may have played important roles in the genomic evolution of Pectinidae. We also compiled our present results with former published data regarding the chromosome mapping of rDNAs in species of the Pectinidae family. Such comparative chromosomal mapping should improve our understanding of historical chromosomal reconstructions of modern-day scallops.     

Cytogenetic characterization of the sole Solea senegalensis (Teleostei: Pleuronectiformes: Soleidae): Ag-NOR, (GATA) n , (TTAGGG) n and ribosomal genes by one-color and two-color FISH

A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for the nucleolus organizer region (Ag-NOR) identification, one-color FISH for chromosomal mapping of 45S and 5S ribosomal DNAs (rDNAs), (GATA) _n , and (TTAGGG) _n , and two-color FISH for co-localization of both rDNAs. The Ag-NORs and the 45S rDNA were mapped to a medium-sized submetacentric chromosomal pair. Hybridization with the 5S rDNA showed a major signal on the short arm of a medium-sized submetacentric chromosome pair and a minor signal on a centromeric site of a small acrocentric chromosome pair. Differences in the Ag-NOR and 45S and 5S rDNAs FISH signal sizes were observed between homologous chromosomes and among individuals. A two-color FISH co-localized 45S and 5S rDNAs to a medium-sized submetacentric chromosomal pair. The hybridization with the telomeric (TTAGGG) _n repeat displayed small signals at all chromosomal telomeres. Finally, the (GATA) _n probe produced dispersed and small hybridization signals on all chromosome spreads, showing its ubiquitous existence in the genome. These results were compared with those from other Pleuronectiformes and discussed in terms of karyotype evolution.