Interstitial telomeric repeats as markers of evolutionary changes in the mammalian karyotype: Human chromosome 2

The presence of conserved telomeric repeats represented by the hexamer (TTAGGG)_n at the chromosomal termini is necessary for the correct functioning and stability of chromosomes. A number of the genomes of mammals, including human, are known to contain interstitial telomeric sequences located far from the chromosomal termini. It is assumed that these repeats mark the regions of fusions or other rear-rangements of ancestral chromosomes. Exact localization of all interstitial telomeric sequences in the genome could significantly advance the understanding of the mechanisms of karyotype evolution and speciation. In this context, software was developed to search for degenerate interstitial telomeric repeats in complete sequences of mammalian chromosomes. The evolutionary significance of such repeats was demonstrated by the example of human chromosome 2. The results are available at http://www.bionet.nsc.ru/labs/theorylabmain/orlov/telomere/.     

Cytogenetic characterization and nuclear DNA content of three North African endemic Centaurea species

Three endemic Centaurea species from North Africa are investigated for the first time by chromomycin fluorochrome banding for GC-rich DNA distribution, fluorescence in situ hybridization for physical mapping of rRNA genes, and flow cytometry for genome-size assessment. Investigated species belong to three different sections and possess three basic chromosome numbers: C. tougourensis subsp. tougourensis 2n = 4x = 36 (x = 9), C. musimonum 2n = 2x = 20 (x = 10), and C. maroccana 2n = 2x = 24 (x = 12). The number and distribution of chromomycin positive bands (CMA⁺) and 18S-5.8S-26S (35S) rDNA loci were different among investigated species and ranged from 6 to 80 chromomycin bands and from 2 to 6 35S rDNA loci. The three species have just one 5S rDNA locus at intercalary position on a separate chromosome pairs, except in the case of C. musimonum in which both rDNA loci were localized on the same chromosome. All rDNA loci were co-localized with CMA⁺ bands, except three 35S in C. musimonum. Genome size ranged from 2C = 1.66 to 2C = 2.86 pg in diploid species (C. musimonum and C. maroccana, respectively) and to 2C = 4.51 pg in tetraploid C. tougourensis subsp. tougourensis.     

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

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

Molecular cytogenetics of Xeranthemum L. and related genera (Asteraceae, Cardueae)

Abstract: Seven representatives of the genera Amphoricarpus, Chardinia, Siebera, and Xeranthemum, all of them closely related as demonstraded by molecular phylogeny, have been studied from a cytogenetic perspective. Morphometrical karyotype parameters were calculated and idiograms obtained. Fluorochrome banding was performed with chromomycin A₃ to identify GC-rich regions in the chromosomes. Fluorescence in situ hybridization allowed us to locate the sites of 18S-5.8S-26S and 5S rDNA. Silver nitrate staining was used to count the number of nucleoli and to detect the active nucleolar organizing regions. Systematic and evolutionary issues are addressed in the light of these data.     

苜蓿核糖体基因物理定位及染色体荧光分带

利用核糖体基因为探针对,二倍体和四倍体苜蓿(Medicago sativa)进行原位杂交,结果表明,45s在四倍体、二倍体种中总是以单位点位于核仁组织区,5s则有2～3个位点;以二倍体种的基因组DNA为探针的原位杂交表明,蓝花苜蓿(M.coerulea)和黄花苜蓿(M.falcata)均能与四倍体染色体进行杂交,仅杂交信号强弱的染色体数目有差别;荧光染料DAPI使苜蓿的染色体显示带纹,蓝花苜蓿的DAPI带与C-带基本一致.文章对四倍体苜蓿的可能来源进行了讨论.     

Optimization of PRINS and C-PRINS for detection of telomeric sequences in Vicia faba

Primed in situ labelling (PRINS) of nucleic acids was developed as an alternative to traditionally used fluorescence in situ hybridization (FISH). Compared to FISH, PRINS is faster and does not require preparation of labelled probes. Nevertheless, the number of applications for physical mapping of DNA sequences on plant chromosomes remains low. This is due to the fact that there are a number of factors which influence the specificity and sensitivity of the reaction. The purpose of this work was to analyse the effect of some of them, including the age of slides, type of Taq DNA polymerase, number and concentration of primers, the presence and concentration of bovine serum albumine and MgCl₂ in the reaction mixture. Furthermore, the effect of various pre-treatments on signal intensity and non-specific fluorescence was studied. A consensus Arabidopsis-type telomeric sequence and Vicia faba mitotic chromosomes were used as a model system. We have found that the age of slides was critical and that under optimal conditions it was possible to achieve relatively high signal to noise ratio. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosomal distribution of interstitial telomeric sequences as signs of evolution through chromosome fusion in six species of the giant water bugs (Hemiptera,Belostoma)

Tandem arrays of TTAGG repeats show a highly conserved location at the telomeres across the phylogenetic tree of arthropods. In giant water bugs Belostoma, the chromosome number changed during speciation by fragmentation of the single ancestral X chromosome, resulting in a multiple sex chromosome system. Several autosome–autosome fusions and a fusion between the sex chromosome pair and an autosome pair resulted in the reduced number in several species. We mapped the distribution of telomeric sequences and interstitial telomeric sequences (ITSs) in Belostoma candidulum (2n = 12 + XY/XX; male/female), B. dentatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elegans (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elongatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. micantulum (2n = 14 + XY/XX), and B. oxyurum (2n = 6 + XY/XX) by FISH with the (TTAGG)_n probes. Hybridization signals confirmed the presence of TTAGG repeats in the telomeres of all species examined. The three species with reduced chromosome numbers showed additional hybridization signals in interstitial positions, indicating the occurrence of ITS. From the comparison of all species here analyzed, we observed inverse relationships between chromosome number and chromosome size, and between presence/absence of ITS and chromosome number. The ITS distribution between these closely related species supports the hypothesis that several telomere–telomere fusions of the chromosomes from an ancestral diploid chromosome number 2n = 26 + XY/XX played a major role in the karyotype evolution of Belostoma. Consequently, our study provide valuable features that can be used to understand the karyotype evolution, may contribute to a better understanding of taxonomic relationships, and also elucidate the high plasticity of nuclear genomes at the chromosomal level during the speciation processes.     

Telomere sequence localization and karyotype evolution in higher plants

Data for chromosomal localization of theArabidopsis-type of telomeric sequence repeats (TTTAGGG)_n are compiled for 44 species belonging to 14 families of angiosperms, gymnosperms and bryophytes. For 23 species and seven families this is the first report. Species of all families, except theAlliaceae, revealed these sequences at their chromosome termini. This indicates thatArabidopsis-type telomeric repeats are highly conserved. It is inferred that they represent the basic telomere sequence of higher plant phyla. In theAlliaceae, a deviating sequence (and mechanism?) for the stabilization of chromosome termini has possibly evolved secondarily. Nine species revealed interstitial telomeric sequences in addition to the terminal ones, in three species (Vicia faba, Pinus elliottii, P. sylvestris) also at centromeric positions. Interstitial telomeric sequences may indicate karyotype reconstructions, in particular alterations of chromosome numbers by chromosome fusion — or inversions with one breakpoint within the terminal array of repeats. They may contribute to stabilization of chromosome breaks, especially centric fissions, and increase the frequency of meiotic and illegitimate recombination.     

Conventional and ultrastructural analyses of the chromosomes of Discocyrtus pectinifemur (Opiliones, Laniatores, Gonyleptidae)

Among the Opiliones, species of the suborders Cyphophthalmi, Eupnoi, Dyspnoi and Laniatores have shown very diverse diploid chromosome numbers. However, only certain Eupnoi species exhibit XY/XX and ZZ/ZW sex chromosome systems. Considering the scarcity of karyotypical information and the absence of structurally identifiable sex chromosomes in the suborder Laniatores, we decided to analyse the chromosomes and bivalents of Discocyrtus pectinifemur (Gonyleptidae) to identify possible sex differences. Testicular cells examined under light microscopy showed a high diploid number, 2 n  = 88, meta/submetacentric chromosome morphology and a nucleolar organizer region on pair 35. Prophase I microspreading observed in transmission electron microscopy exhibited 44 synaptonemal complexes with similar electron density and thickness. The total and regular synapsis between the chromosomes of the bivalents was also noted in pachytene nuclei. Male mitotic and meiotic chromosomes revealed no distinct characteristic that could be related to the occurrence of heteromorphic sex chromosomes. Evolutionary trends of chromosome differentiation in the four suborders of Opiliones are discussed here.     

Cytogenetic and molecular characterization of the Abies alba genome and its relationship with other members of the Pinaceae

Genome size, karyotype structure, heterochromatin distribution, position and number of ribosomal genes, as well as the ITS2 sequence of the internal transcribed spacer (ITS) were analysed in silver fir (Abies alba Mill.). The analysis also included characterization of the Arabidopsis-type of telomeric repeats in silver fir and in related species. The results were compared with results from other species of the Pinaceae, to evaluate phylogeny and chromosomal and molecular evolution in the Pinaceae. Integrated chromosomal data provided insights into chromosome and karyotype evolution in the Pinaceae. The evolutionary trend for GC-rich heterochromatic blocks seems to involve loss of blocks that are not associated with rDNA. Similarly, numerous large blocks of interstitial plant telomeric repeats that are typical for all analysed species of the genus Pinus were not observed in the evolutionarily younger genera, such as Abies, Picea and Larix. On the contrary, the majority of telomeric sequences in these three genera appeared confined to the chromosome ends. We confirmed the current position of Abies and Tsuga in subfamily Abietoideae and the position of Pinus in the subfamily Pinoideae based on ITS2 sequences. Pseudotsuga is placed together with Larix into the subfamily Laricoideae. We conclude that the current position of the genus Picea in the subfamily Abietoideae should be reconsidered and, possibly, the genus Picea should be reclassified as a separate subfamily, Piceoideae, as recently proposed.     

Chromosomes of Theridiidae spiders (Entelegynae): Interspecific karyotype diversity in Argyrodes and diploid number intraspecific variability in Nesticodes rufipes

Theridiidae is a derived family within the Araneoidea clade. In contrast to closely related groups, the 2n(male) = 20+X(1) X (2) with acro/telocentric chromosomes is the most widespread karyotype among the theridiid spiders. In this work, the cytogenetic analysis of Argyrodes elevatus revealed original chromosome features different from those previously registered for Theridiidae, including the presence of 2n(male) = 20+X with meta/submetacentric chromosomes. Most individuals of Nesticodes rufipes showed family conserved karyotype characteristics. However, one individual had a 2n(male) = 24 due to the presence of an extra chromosome pair, which exhibited regular behavior and reductional segregation during meiosis. After silver staining, mitotic cells exhibited NORs localized on the terminal regions of the short arms of pairs 2, 3, and 4 of A. elevatus and on the terminal regions of long arms of pair 4 of N. rufipes. The comparative analysis with data from phylogenetically related species allowed the clarification of the origin of the interspecific and intraspecific chromosome variability observed in Argyrodes and in N. rufipes, respectively.     

Chromosomal localization of ribosomal DNA sequences in an apple rootstock using a digoxygenin detection system

A 6kb rDNA probe comprising the 18S coding plus spacer sequences has been hybridized to the metaphase chromosomes of apple rootstock cultivar MM106 demonstrating the localization of ribosomal gene arrays in the vicinity of the telomeric regions of the short arms of chromosomes 6 and 14.The in situ results using digoxygenin labelling coupled to an alkaline phosphatase immunoassay were confirmed by silver staining for NORs and nucleoli.This study demonstrates the feasibility of molecular cytogenetic analysis of very small chromosomes(1.0-2.7μm） of apple.     

Number and distribution of silver-stained nucleolar organizer regions and evolutionary relationships in domestic pigs

Variation in the numbers of silver-stained nucleolar organizer regions (Ag-NORS) were examined in 36 breeds of domestic pig of different geographic origins and five subspecies of wild boar. The relationship between Ag-NORs and evolution of domestic pigs was investigated. In all pigs observed, Ag-NORs were localized on the secondary constriction of chromosomes 10 and 8. The mean Ag-NOR numbers varied from 2.0–4.0, and decreased gradually with the different geographical distribution from south to north in China and from east to west in Europe. This regular change was caused mainly by the differences of frequency in chromosome 8 Ag-NOR type and was closely related to the evolution of domestic pig breeds.     

Karyotype analysis of four Vicia species using in situ hybridization with repetitive sequences

Mitotic chromosomes of four Vicia species (V. sativa, V. grandiflora, V. pannonica and V. narbonensis) were subjected to in situ hybridization with probes derived from conserved plant repetitive DNA sequences (18S-25S and 5S rDNA, telomeres) and genus-specific satellite repeats (VicTR-A and VicTR-B). Numbers and positions of hybridization signals provided cytogenetic landmarks suitable for unambiguous identification of all chromosomes, and establishment of the karyotypes. The VicTR-A and -B sequences, in particular, produced highly informative banding patterns that alone were sufficient for discrimination of all chromosomes. However, these patterns were not conserved among species and thus could not be employed for identification of homologous chromosomes. This fact, together with observed variations in positions and numbers of rDNA loci, suggests considerable divergence between karyotypes of the species studied.     

Genome size shifts: karyotype evolution in Crepis section Neglectoides (Asteraceae)

Plant genome size evolution is a very dynamic process: the ancestral genome of angiosperms was initially most likely small, which led to a tendency towards genome increase during evolution. However, findings in several angiosperm lineages demonstrate mechanisms that also led to genome size contraction. Recent molecular investigations on the Asteraceae genus Crepis suggest that several genomic reduction events have occurred during the evolution of the genus. This study focuses on the Mediterranean Crepis sect. Neglectoides, which includes three species with some of the smallest genomes within the whole genus. Crepis neglecta has the largest genome in sect. Neglectoides, approximately twice the size of the two species Crepis cretica and Crepis hellenica. Whereas C. cretica and C. hellencia are more closely related to each other than to C. neglecta the karyotypes of the latter species and C. cretica are similar, while that of C. hellenica differs considerably. Here, the karyotypic organisation of the three species is investigated with fluorescence in‐situ hybridisation and studied in a molecular phylogenetic framework based on the nuclear markers Actin, CHR12, CPN60B, GPCR1 and XTH23. Our findings further corroborate the occurrence of genome size contraction in Crepis, and suggest that the difference in genome size between C. neglecta and C. cretica is mostly due to elimination of dispersed repetitive elements, whereas chromosomal reorganisation was involved in the karyotype formation of C. hellenica.     

Uniqueness of the Gossypium mustelinum Genome Revealed by GISH and 45S rDNA FISH

Gossypium mustelinum ((AD)₄) is one of five disomic species in Gossypium. Three 45S ribosomal DNA (rDNA) loci were detected in (AD)₄ with 45S rDNA as probe, and three pairs of brighter signals were detected with genomic DNA (gDNA) of Gossypium D genome species as probes. The size and the location of these brighter signals were the same as those detected with 45S rDNA as probe, and were named GISH-NOR. One of them was super-major, which accounted for the fact that about one-half of its chromosome at metaphase was located at chromosome 3, and other two were minor and located at chromosomes 5 and 9, respectively. All GISH-NORs were located in A sub-genome chromosomes, separate from the other four allopolyploid cotton species. GISH-NOR were detected with D genome species as probe, but not A. The greatly abnormal sizes and sites of (AD)₄ NORs or GISH-NORs indicate a possible mechanism for 45S rDNA diversification following (AD)₄ speciation. Comparisons of GISH intensities and GISH-NOR production with gDNA probes between A and D genomes show that the better relationship of (AD)₄ is with A genome. The shortest two chromosomes of A sub-genome of G. mustelinum were shorter than the longest chromosome of D sub-genome chromosomes. Therefore, the longest 13 chromosomes of tetraploid cotton being classified as A sub-genome, while the shorter 13 chromosomes being classified as D sub-genome in traditional cytogenetic and karyotype analyses may not be entirely correct.     

Karyotyping and cytogenetic mapping of Atlantic cod (Gadus morhua Linnaeus, 1758)

The Atlantic cod (Gadus morhua) is an important natural resource for northern societies and is now also considered to be a promising candidate for aquaculture. In recent years, much effort has been directed towards the development of genomic tools, and genome initiatives for Atlantic cod have been established. Despite the growing attention devoted to the Atlantic cod genomics, basic aspects of its genome structure and organization remain unknown. Thus, the present work aims to study cytogenetic features of the Atlantic cod as a contribution to the knowledge of this species' genome. The Atlantic cod displays a diploid number of 46 chromosomes, with a karyotypic formula 16 m/sm + 30 st/t. Conventional karyotyping was improved by chromosomal mapping of two classes of repetitive sequences. 18S rDNA clusters were assigned to pairs 2 and 4; small amounts of 18S rDNA clusters were occasionally detected on pair 5. These findings could not be related to the geographical origin of the specimens, but were consistent with the variability of these repeated genes in fish in general. 5S ribosomal gene clusters, apparently corresponding to a single 5S rDNA class, were detected on twelve chromosomes (pairs 11, 12, 14, 17, 20 and 21). The present update of the existing but meagre information on the karyotype of Atlantic cod, plus the first physical mapping of repetitive genes in this species herein, opens the way for an integrated approach that combines genetic and physical mapping with the assembly of the genome of this commercially important species.     

Numerical variation of nucleolar organizer regions after silver staining in domestic and wild Suidae (Mammalia)

Selective silver staining was used to investigate the cellular distribution of numbers of nucleolar organizer regions (NORs) in domestic pigs (Sus scrofa) of eight different breeds, the European wild boar (S. scrofa scrofa), Indonesian wild boar (S. scrofa vittatus), Javan warty pig (S. verrucosus), Sulawesi warty pig (S. celebensis), and pigmy hog (S. salvanius). In the domestic pig as well as in the wild (sub)species of Sus, actively transcribing ribosomal RNA genes were found to be present in the secondary constrictions of chromosome pairs 10 and 8. Chromosomes 10 were consistently Ag-positive. Chromosomes 8 less frequently showed Ag-NORs, resulting in different mean numbers of Ag-NORs per individual animal. Mean Ag-NOR numbers per breed or (sub)species were generally higher in the wild representatives of Sus than in the domestic breeds. The highest mean numbers of Ag-NORs were observed in the Meishan breed and in S. celebensis and S. salvanius. The Meishan breed appears to be conservative in Ag-NOR staining pattern, being more comparable to the Asian wild Suidae than to the European breeds.     

Comparative cytogenetic analysis of Cestrum (Solanaceae) reveals different trends in heterochromatin and rDNA sites distribution

Species of Cestrum L. (Solanaceae) exhibit large variability in the accumulation of repetitive DNA, although their species possess a stable diploid number with 2n = 16. In this study, we used chromosome banding and fluorescence in situ hybridization (FISH) to characterize the karyotypes and populations of two species, Cestrum nocturnum L. and C. mariquitense Kunth. We also performed a karyotype comparison using 16 idiograms, of which 4 were developed in this study and 12 were obtained from the literature. Cestrum nocturnum displayed more bands than C. mariquitense, but the latter exhibited greater interpopulational variation in the band patterns. There was a tendency for large bands to be located at intercalary/terminal regions and for small bands to be located at intermediate/proximal regions. The idiogram comparison revealed a large variation in the amount, distribution, and size of heterochromatic bands. FISH with rDNA probes revealed stability in the number and location of 5S sites, while 45S was more variable in size and number of sites. Although 45S rDNA always appeared in the subterminal regions, this DNA family exhibited a mobility among chromosome pairs. These data highlight the dynamic of repetitive DNA families in these genomes, as well as the contribution for intra- and interspecific karyotype differentiation in Cestrum.