Variability of satellite DNA in 2 groups of field mice (Rodentia, Microtinae)

Nuclear DNAs of three forms of Microtus juldashi--M. carruthersi (group 1) and of three subspecies of polytypic species Alticola macrotis (group 2) were compared. Intensive interpopulational karyotype differentiation was observed in both groups, particularly, for centromeric heterochromatin quantity and localization. As satellite DNAs (satDNAs) were present in C-heterochromatin of the Rodents groups studied, the latter were used as a model for comparison of the satDNAs in the species in statu nascendi. With this view, the nuclear DNAs were studied by means of the neutral CsCl equilibrium centrifugation. In both groups examined, no correlation was found between the presence, the characteristics of the satDNAs and morphological as well as cytogenetic features of the animals. These results are an indirect confirmation of the idea, according to which satDNA does not possibly play significant role in the development of the reproductive isolation and species formation.     

The extensive amplification of heterochromatin in Melipona bees revealed by high throughput genomic and chromosomal analysis

Satellite DNAs (satDNAs) and transposable elements (TEs) are among the main components of constitutive heterochromatin (c-heterochromatin) and are related to their functionality, dynamics, and evolution. A peculiar case regarding the quantity and distribution of c-heterochromatin is observed in the genus of bees, Melipona, with species having a low amount of heterochromatin and species with high amount occupying almost all chromosomes. By combining low-pass genome sequencing and chromosomal analysis, we characterized the satDNAs and TEs of Melipona quadrifasciata (low c-heterochromatin) and Melipona scutellaris (high low c-heterochromatin) to understand c-heterochromatin composition and evolution. We identified 15 satDNA families and 20 TEs for both species. Significant variations in the repeat landscapes were observed between the species. In M. quadrifasciata, the repetitive fraction corresponded to only 3.78% of the genome library studied, whereas in M. scutellaris, it represented 54.95%. Massive quantitative and qualitative changes contributed to the differential amplification of c-heterochromatin, mainly due to the amplification of exclusive repetitions in M. scutellaris, as the satDNA MscuSat01-195 and the TE LTR/Gypsy_1 that represent 38.20 and 14.4% of its genome, respectively. The amplification of these two repeats is evident at the chromosomal level, with observation of their occurrence on most c-heterochromatin. Moreover, we detected repeats shared between species, revealing that they experienced mainly quantitative variations and varied in the organization on chromosomes and evolutionary patterns. Together, our data allow the discussion of patterns of evolution of repetitive DNAs and c-heterochromatin that occurred in a short period of time, after separation of the Michmelia and Melipona subgenera.

     

Satellite DNA content illuminates the ancestry of a supernumerary (B) chromosome

B chromosomes are supernumerary genomic elements most likely derived from the standard (A) chromosomes, whose dispensability has freed their DNA sequences to evolve fast, thus making it difficult to uncover their ancestry. Here, we show the ancestry of a B chromosome in the grasshopper Eumigus monticola by means of the high-throughput analysis of the satellitome, i.e., the whole collection of satellite DNA (satDNA). The satellitome found in this species consists of 27 satDNA families, with monomer length between 5 and 325 nt and A + T content between 42.9 and 83.3 %. Two out of the 20 clustered satDNA families (EmoSat26–41 and EmoSat27–102) were observed only on the B chromosome. The A chromosome carrying the highest number of satDNA families was the megameric S8 (13 families), six of which were also present in the B chromosome, and three of these were exclusive of the S8 and B chromosomes. The absence in the B chromosome of the H3 histone gene cluster (located interstitially on S8) and three satDNA families (located distally on S8) allowed delimiting the possible origin of the B chromosome to the proximal third of the S8 autosome, through a breakpoint between EmoSat11–122 and the H3 cluster. Interestingly, bioinformatic analysis revealed the presence of seeds for the two B-specific satDNAs in the A chromosomes, suggesting their massive amplification in the B chromosome after its origin. Therefore, intraspecifically arisen B chromosomes can harbor DNA sequences apparently being B-specific.     

Heterochromatin and rDNA sites distribution in the holocentric chromosomes of Cuscuta approximata Bab. (Convolvulaceae).

Cuscuta is a widely distributed genus of holoparasitic plants. Holocentric chromosomes have been reported only in species of one of its subgenera (Cuscuta subg. Cuscuta). In this work, a representative of this subgenus, Cuscuta approximata, was investigated looking for its mitotic and meiotic chromosome behaviour and the heterochromatin distribution. The mitotic chromosomes showed neither primary constriction nor Rabl orientation whereas the meiotic ones exhibited the typical quadripartite structure characteristic of holocentrics, supporting the assumption of holocentric chromosomes as a synapomorphy of Cuscuta subg. Cuscuta. Chromosomes and interphase nuclei displayed many heterochromatic blocks that stained deeply with hematoxylin, 4',6-diamidino-2-phenylindole (DAPI), or after C banding. The banded karyotype showed terminal or subterminal bands in all chromosomes and central bands in some of them. The single pair of 45S rDNA sites was observed at the end of the largest chromosome pair, close to a DAPI band and a 5S rDNA site. Two other 5S rDNA site pairs were found, both closely associated with DAPI bands. The noteworthy giant nuclei of glandular cells of petals and ovary wall exhibited large chromocentres typical of polytenic nuclei. The chromosomal location of heterochromatin and rDNA sites and the structure of the endoreplicated nuclei of C. approximata seemed to be similar to those known in monocentric nuclei, suggesting that centromeric organization has little or no effect on chromatin organization.     

Conserved DNA Motifs,Including the CENP-B Box-like,Are Possible Promoters of Satellite DNA Array Rearrangements in Nematodes

Tandemly arrayed non-coding sequences or satellite DNAs (satDNAs) are rapidly evolving segments of eukaryotic genomes, including the centromere, and may raise a genetic barrier that leads to speciation. However, determinants and mechanisms of satDNA sequence dynamics are only partially understood. Sequence analyses of a library of five satDNAs common to the root-knot nematodes Meloidogyne chitwoodi and M. fallax together with a satDNA, which is specific for M. chitwoodi only revealed low sequence identity (32–64%) among them. However, despite sequence differences, two conserved motifs were recovered. One of them turned out to be highly similar to the CENP-B box of human alpha satDNA, identical in 10–12 out of 17 nucleotides. In addition, organization of nematode satDNAs was comparable to that found in alpha satDNA of human and primates, characterized by monomers concurrently arranged in simple and higher-order repeat (HOR) arrays. In contrast to alpha satDNA, phylogenetic clustering of nematode satDNA monomers extracted either from simple or from HOR array indicated frequent shuffling between these two organizational forms. Comparison of homogeneous simple arrays and complex HORs composed of different satDNAs, enabled, for the first time, the identification of conserved motifs as obligatory components of monomer junctions. This observation highlights the role of short motifs in rearrangements, even among highly divergent sequences. Two mechanisms are proposed to be involved in this process, i.e., putative transposition-related cut-and-paste insertions and/or illegitimate recombination. Possibility for involvement of the nematode CENP-B box-like sequence in the transposition-related mechanism and together with previously established similarity of the human CENP-B protein and pogo-like transposases implicate a novel role of the CENP-B box and related sequence motifs in addition to the known function in centromere protein binding.     

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.     

Karyotype analysis of Lilium longiflorum and Lilium rubellum by chromosome banding and fluorescence in situ hybridisation.

Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red- and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.     

Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.     

Identification and chromosomal location of tandemly repeated DNA sequences in Allium cepa

A 314-bp tandemly repeated DNA sequence, named pAc074, was characterized in Allium cepa by fluorescence in situ hybridization (FISH) analyses using random amplified fragment as probe. The nucleotide sequences of the clone pAc074 is partially homologous to the satellite DNA sequences, ACSAT1, ACSAT2, and ACSAT3, of A. cepa with 81%, 81% and 78% similarity, respectively. Our sequential C-banding and FISH with pAc074 probe also clearly showed a close relation between Cheterochromatin at telomeric region and pAc074 sequences on all the chromosomes except on chromosome 6. On the long arm of chromosome 7, pAc074 sequences appeared as interstitial band which did not correspond to C-heterochromatin bands. Instead, the C-heterochromatin bands corresponded with the 5S rDNA signals. This is the first evidence of simultaneous banding of the 5S rDNA and C-band in A. cepa.     

Karyotype analysis of Nicotiana kawakamii Y. Ohashi using DAPI banding and rDNA FISH

Prometaphase cells were used to analyze the karyotype of Nicotiana kawakamii Y. Ohashi by means of sequential Giemsa/CMA/DAPI staining and multicolor fluorescence in situ hybridization with 5S and 18S rDNA. Observation of the DAPI-stained prometaphase spreads indicated that N. kawakamii had six pairs of large chromosomes, one pair of medium-sized chromosomes and five pairs of small chromosomes. The six pairs of large chromosomes possessed remarkable DAPI bands, and each could be identified from both the DAPI banding pattern and the length of the short arm. The DAPI banding pattern was approximately identical to the CMA and Giemsa banding patterns. Hybridization signals of the 18S rDNA probe were detected on two pairs of large chromosomes. In addition, two pairs of small chromosomes were identified based on the position of the 5S rDNA signals. An idiogram of N. kawakamii chromosomes was produced based on DAPI bands and rDNA loci. Received: 17 July 2000 / Accepted: 4 September 2000     

Accumulation of Y-specific satellite DNAs during the evolution of <Emphasis Type="Italic">Rumex acetosa</Emphasis> sex chromosomes

The study of the molecular structure of young heteromorphic sex chromosomes of plants has shed light on the evolutionary forces that control the differentiation of the X and Y during the earlier stages of their evolution. We have used the model plant Rumex acetosa, a dioecious species with multiple sex chromosomes, 2n = 12 + XX female and 2n = 12 + XY₁Y₂ male, to analyse the significance of repetitive DNA accumulation during the differentiation of the Y. A bulk segregant analysis (BSA) approach allowed us to identify and isolate random amplified polymorphic DNA (RAPD) markers linked to the sex chromosomes. From a total of 86 RAPD markers in the parents, 6 markers were found to be linked to the Ys and 1 to the X. Two of the Y-linked markers represent two AT-rich satellite DNAs (satDNAs), named RAYSII and RAYSIII, that share about 80% homology, as well as with RAYSI, another satDNA of R. acetosa. Fluorescent in situ hybridisation demonstrated that RAYSII is specific for Y₁, whilst RAYSIII is located in different clusters along Y₁ and Y₂. The two satDNAs were only detected in the genome of the dioecious species with XX/XY₁Y₂ multiple sex chromosome systems in the subgenus Acetosa, but were absent from other dioecious species with an XX/XY system of the subgenera Acetosa or Acetosella, as well as in gynodioecious or hermaphrodite species of the subgenera Acetosa, Rumex and Platypodium. Phylogenetic analysis with different cloned monomers of RAYSII and RAYSIII from both R. acetosa and R. papillaris indicate that these two satDNAs are completely separated from each other, and from RAYSI, in both species. The three Y-specific satDNAs, however, evolved from an ancestral satDNA with repeating units of 120 bp, through intermediate satDNAs of 360 bp. The data therefore support the idea that Y-chromosome differentiation and heterochromatinisation in the Rumex species having a multiple sex chromosome system have occurred by different amplification events from a common ancestral satDNA. Since dioecious species with multiple XX/XY₁Y₂ sex chromosome systems of the section Acetosa appear to have evolved from dioecious species with an XX/XY system, the amplification of tandemly repetitive elements in the Ys of the section Acetosa is a recent evolutionary process that has contributed to an increase in the size and differentiation of the already non-recombining Y chromosomes.     

Chromosomal organization of repetitive DNA sequences in Astyanax bockmanni (Teleostei, Characiformes): dispersive location, association and co-localization in the genome

Repetitive DNA sequences constitute a great portion of the genome of eukaryotes and are considered key components to comprehend evolutionary mechanisms and karyotypic differentiation. Aiming to contribute to the knowledge of chromosome structure and organization of some repetitive DNA classes in the fish genome, chromosomes of two allopatric populations of Astyanax bockmanni were analyzed using classic cytogenetics techniques and fluorescent in situ hybridization, with probes for ribosomal DNA sequences, histone DNA and transposable elements. These Astyanax populations showed the same diploid number (2n = 50), however with differences in chromosome morphology, distribution of constitutive heterochromatin, and location of 18S rDNA and retroelement Rex3 sites. In contrast, sites for 5S rDNA and H1, H3 and H4 histones showed to be co-located and highly conserved. Our results indicate that dispersion and variability of 18S rDNA and heterochromatin sites are not associated with macro rearrangements in the chromosome structure of these populations. Similarly, distinct evolutionary mechanisms would act upon histone genes and 5S rDNA, contributing to chromosomal association and co-location of these sequences. Data obtained indicate that distinct mechanisms drive the spreading of repetitive DNAs in the genome of A. bockmanni. Also, mobile elements may account for the polymorphism of the major rDNA sites and heterochromatin in this genus.     

Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa

We describe the morphology and molecular organization of heterochromatin domains in the interphase nuclei, and mitotic and meiotic chromosomes, of Brassica rapa, using DAPI staining and fluorescence in situ hybridization (FISH) of rDNA and pericentromere tandem repeats. We have developed a simple method to distinguish the centromeric regions of mitotic metaphase chromosomes by prolonged irradiation with UV light at the DAPI excitation wavelength. Application of this bleached DAPI band (BDB) karyotyping method to the 45S and 5S rDNAs and 176 bp centromere satellite repeats distinguished the 10 B. rapa chromosomes. We further characterized the centromeric repeat sequences in BAC end sequences. These fell into two classes, CentBr1 and CentBr2, occupying the centromeres of eight and two chromosomes, respectively. The centromere satellites encompassed about 30% of the total chromosomes, particularly in the core centromere blocks of all the chromosomes. Interestingly, centromere length was inversely correlated with chromosome length. The morphology and molecular organization of heterochromatin domains in interphase nuclei, and in mitotic and meiotic chromosomes, were further characterized by DAPI staining and FISH of rDNA and CentBr. The DAPI fluorescence of interphase nuclei revealed ten to twenty conspicuous chromocenters, each composed of the heterochromatin of up to four chromosomes and/or nucleolar organizing regions.     

Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis

The karyotype of the Mediterranean species Anemone hortensis L. (Ranunculaceae) was characterized with emphasis on heterochromatin distribution and localization of ribosomal (18S−5.8S−26S and 5S rDNA) and telomeric repeats (TTTAGGG). Diploid chromosome complement, 2 n  = 2 x  = 16, common to all investigated populations, consisted of three acrocentric, one meta-submetacentric and four metacentric chromosomes ranging in size from 6.34 to 10.47 µm. Fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes revealed two 18S−5.8S−26S rDNA loci on a satellite and secondary constriction of acrocentric chromosome pair 2 and terminally on acrocentric chromosome pair 3, and two 5S rDNA loci in the pericentromeric region of meta-submetacentric chromosome pair 4 and in the proximity of the 18S−5.8S−26S rDNA locus on chromosome pair 2. The only GC-rich heterochromatin, as revealed by fluorochrome Chromomycin A3 staining, was that associated with nucleolar organizer regions, whereas AT-rich heterochromatin, stained with 4,6-diamino-2-phenylindole (DAPI), was distributed intercalarly and terminally on the long arm of all three acrocentric chromosomes, and terminally on chromosomes 4 and 5. FISH with Arabidopsis -type telomeric repeats (TTTAGGG) as a probe revealed two classes of signals, small dot-like and large bands, at chromosome termini exclusively, where they corresponded to terminal DAPI-stained heterochromatin. Heteromorphism of chromosome pair 4, which refers to terminal DAPI bands and FISH signals, was observed in populations of Anemone hortensis . Chromosome pairing during meiosis was regular with formation of localized chiasmata proximal to the centromere.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 177–186.     

X-linked heterochromatin distribution in the holocentric chromosomes of the green apple aphid <Emphasis Type="Italic">Aphis pomi</Emphasis>

Chromatin organization in the holocentric chromosomes of the green apple aphid Aphis pomi has been investigated at a cytological level after C-banding, NOR, Giemsa, fluorochrome staining and fluorescent in situ hybridization (FISH). C-banding technique showed that heterochromatic bands are exclusively located on X chromosomes. This data represents a peculiar feature that clearly contradicts the equilocal distribution of heterochromatin typical of monocentric chromosomes. Moreover, silver staining and FISH carried out with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; CMA₃ staining reveals that these silver positive telomeres are the only GC-rich regions among A. pomi heterochromatin, whereas all other C-positive bands are DAPI positive thus containing AT-rich DNA.     

Comparative cytogenetics between the species Passiflora edulis and Passiflora cacaoensis

Passiflora edulis Sims is the most economically important species of the genus Passiflora. A new species was described recently, Passiflora cacaoensis Bernacci & Souza, which displayed morphologic characteristics very similar to P. edulis. Due to the need for delimitation of the two species, karyomorphological and banding analyses were carried out. Both species have 2n = 18, with the same karyotype formula 16 m + 2sm. There was variation between the species regarding the location of satellites and the width of chromosome pairs 2, 4 and 8. C banding revealed the presence of constitutive heterochromatin in the centromeric and telomeric regions of all chromosomes in both species. However, only in P. cacaoensis did chromosomes 3 and 9 have a large quantity of heterochromatin. Fluorochrome banding revealed CMA⁺ bands only in the satellites, but no DAPI⁺ bands. Fluorescence in situ hybridisation (FISH) showed that in P. cacaoensis the rDNA 5S probe is located in a single site in the subterminal position of the long arm of chromosome 5. However, for the rDNA 45S probe, two sites were detected in terminal positions of the long arms of chromosome 7, with a bigger and stronger signal, and of chromosome 9. According to the asymmetry index and the quantity of heterochromatin, P. cacaoensis is a more basal species than P. edulis. The cytogenetic data indicate that P. cacaoensis is closely related to P. edulis, but is a different species.     

C banding in polytene chromosomes of Simulium ornatipes and S. melatum (Diptera: Simuliidae)

Polytene and mitotic chromosomes of Simulium ornatipes and S. melatum were subjected to C banding procedures. In both species polytene chromosomes consistently show C banding of centromere regions, telomeres, nucleolar organiser and, unexpectedly, numerous interstitial sites. The interstitial C banding sites correspond to morphologically single polytene bands. Their response is graded and independent of band size. Interstitial C bands in S. ornatipes are scattered throughout the complement, whereas in S. melatum they are clustered. Supernumerary heterochromatic segments in S. ornatipes also exhibit strong C banding and inverted segments can differ from standard in C banding pattern. — Mitotic chromosomes of both species show a single centric C band with indications of two weak interstitial bands in S. ornatipes, suggesting that many C band regions, detectable in polytene chromosomes, are not resolved by present techniques in mitotic chromosomes. — Contrary to current opinion that C banding is diagnostic for constitutive heterochromatin, the interstitial C band sites of polytene chromosomes are regarded as euchromatic. Conversely, the heterochromatic pericentric regions of S. ornatipes are not C banded. — It appears that polytene chromosomes offer a promising system for the elucidation of C banding mechanisms.