Isolation and identification of Triticum aestivum L. em. Thell. cv Chinese Spring-T. peregrinum Hackel disomic chromosome addition lines

Analyses of RFLPs, isozymes, morphological markers and chromosome pairing were used to isolate 12 Triticum aestivum cv Chinese Spring (genomes A, B, and D)-T. peregrinum (genomes S^v and U^v) disomic chromosome addition lines. The evidence obtained indicates that each of the 12 lines contains an intact pair of T. peregrinum chromosomes. One monosomic addition line, believed to contain an intact 6S^v chromosome, was also isolated. A CS-7U^v chromosome addition line was not obtained. Syntenic relationships in common with the standard Triticeae arrangement were found for five of the seven S^v genome chromosomes. The exceptions were 4S^v and 7S^v. A reciprocal translocation exists between 4S¹ and 7S¹ in T. longissimum and evidence was obtained that the same translocation exists in T. peregrinum. In contrast, evidence for syntenic relationships in common with the standard Triticeae arrangements were found for only one U^v chromosome of T. peregrinum.; namely, chromosome 2U^v. All other U^v genome chromosomes are involved in at least one translocation, and the same translocations were found in the U genome of T. umbellulatum. Evidence was also obtained indicating that the centromeric regions of 4U and 4U^v are homoeologous to the centromeric regions of Triticeae homoeologous group-6 chromosomes, that the centromeric regions of 6U and 6U^v are homoeologous to the centromeric regions of group-4 chromosomes, and that 4U and 4U^v are more closely related overall to Triticeae homoeologous group-6 chromosomes than they are to group-4 chromosomes.     

Intraspecific karyotype divergence inTriticum araraticum (Poaceae)

Karyotypes of 185 accessions ofTriticum araraticum Jakubz. (2n = 28 = 4x = A^tA^tGG) from Iraq, Iran, Turkey, and Transcaucasia were analyzed using C-banding technique. All accessions showed a certain degree of C-banding polymorphism and further karyotypic diversity was generated by structural rearrangements, mainly translocations. Eighty-one accessions had the normal karyotype similar to that ofT. timopheevii (cultivation), i.e., they showed C-banding polymorphism but no chromosomal rearrangements based on the resolving power of the C-banding technique. One-hundred four accessions showed 34 karyotypic variants, 31 had reciprocal translocations with the breakpoints in the centromeric regions of chromosomes. Three showed reciprocal translocations with the breakpoints in intercalary regions of chromosomes. A paracentric inversion for 7A^t chromosome was observed in some accessions. The rearranged karyotypes differed from the normal by one translocation in 21 variants, by two in 9 variants, by three in 1 variant, and by four in 2 variants of karyotypes. Translocations occurred more frequenty in the chromosomes of G-genome than of A^t-genome. Individual chromosomes differed in the frequencies of their involvement in translocations. Each geographical region contained a unique spectrum of translocations. Karyotypic diversity was the highest in Iraq followed by Transcaucasia and Turkey. Iran showed little karyotypic variation. Based on karyotypic analysis, Iraq should be considered as a centre of origin and primary centre of diversity ofT. araraticum.     

Centromeric localization of an S-RNase gene in Petunia hybrida Vilm.

S-RNase has been identified to be an S-allele-specific stylar determinant contributing to the self-incompatibility response in Solanaceae. In order to examine the physical location of the S-RNase gene, multi-color fluorescence in situ hybridization (FISH) using the S ^B1 -RNase cDNA probe and ribosomal RNA gene (rDNA) probe was performed on an S ^B1 S ^B2 heterozygote of Petunia hybrida. The S ^B1 -RNase gene was detected as a doublet signal close to the centromere of chromosome III. Next, we performed FISH using a large genome probe prepared from a λSB1–311 clone (20 kb) which contains the S ^B1 -RNase gene and its 3′ flanking region. This probe hybridized to the centromeric regions of all P. hybrida chromosomes. Sequence analysis of the λSB1–311 clone revealed the presence of a repetitive sequence consisting of a novel 666 bp unit sequence. A subclone (pBS-SB1B5) containing this unit sequence also hybridized to all of the centromeric regions, confirming that this unit is the centromeric specific repetitive sequence. These data suggested that the S ^B1 -RNase gene is located very close to (within a distance of 12 kb from) the centromeric-specific repetitive sequence. Likewise, the pBS-SB1B5 probe hybridized to the centromeric regions of all chromosomes in P. littoralis, another Petunia species. However, the probe did not hybridize to the centromere of the chromosomes from other species in Solanaceae. These results suggested that this centromeric repetitive sequence might be a genus-specific one. Received: 3 December 1998 / Accepted: 8 December 1998<@head-com-p1a.lf>Communicated by F. Mechelke     

Giemsa banding,quinacrine fluorescence and DNA-replication in chromosomes of cattle (Bos taurus)

Almost all the 30 chromosome pairs of cattle can be identified by their banding patterns made be visible by a Giemsa staining technique described previously. The banding pattern of the X chromosome shows striking similarities with the banding pattern of the human X chromosome. — The centromeric region of the acrocentric autosomes contains a highly condensed DNA. This DNA is removed by the Giemsa staining procedure as can be shown by interference microscopic studies. If the chromosomes are stained with quinacrine dihydrochloride these centromeric regions are only slightly fluorescent. — Autoradiographic studies with ³H-thymidine show that the DNA at the centromeric regions starts and finishes its replication later than in the other parts of the chromosomes.     

Chromosomal evolution of the Chinese muntjac (Muntiacus reevesi)

The aim of this study was to test the validity of the hypothesis that the 2n=46 karyotype of the Chinese muntjac (Muntiacus reevesi) could have evolved through 12 tandem fusions from a 2n=70 hypothetical ancestral karyotype, which is still retained in Chinese water deer (Hydropotes inermis) and brown-brocket deer (Mazama gouazoubira). Combining fluorescence-activated chromosomal sorting and degenerate oligonucleotide-primed polymerase chain reaction, we generated chromosome-specific DNA paint probes for 13 M. gouazoubira chromosomes and most of the M. reevesi chromosomes with the exception of 18, 19 and X. These paint probes were used for fluorescence in situ hybridisation to chromosomal preparations of M. reevesi, H. inermis and M. gouazoubira. Chromosome-specific paint probes from M. reevesi chromosomes 1–5 and 11 each delineated more than one homologous pair (18 pairs in total) on the metaphases of H. inermis and M. gouazoubira. All the other probes from M. reevesi and probes from M. gouazoubira each hybridised to one pair of homologous chromosomes or regions. The C5 probe, derived from centromeric satellite sequences of M. reevesi, hybridised to the centromeric regions of all chromosomes of these three species. Most interestingly, several non-random interstitial signals, which are apparently localised to the putative fusion points, were found on chromosomes 1–5 and 11 of M. reevesi. Both the reciprocal painting patterns and localisation of the C5 probe demonstrate that M. reevesi chromosomes 1–5 and 11 could have evolved from 18 different ancestral chromosomes through 12 tandem fusions, thus providing direct molecular cytogenetic support for the tandem fusion hypothesis of karyotype evolution in M. reevesi. Received: 10 October 1996; in revised form: 18 December 1996 / Accepted: 27 December 1996     

Complex sex chromosome system in Eigenmannia sp. (Pisces,Gymnotiformes)

Chromosomes of Eigenmannia sp. (7 males and 15 females) collected from the Tietê River in Botucatu (SP, Brazil) were examined from gill, kidney and testicular cells. The diploid chromosome number in males was 2n=31 and in females, 2n=32. In both sexes the number of chromosomal arms was 40. The difference in diploid number was due to the fusion of two acrocentrics. Mitotic and meiotic studies suggested that one of the fused acrocentrics was the Y chromosome. The sex-determining mechanism in Eigenmannia sp. could therefore be XX, AA in the female and X, \-YA A in the males. One of the males presented 2n=30 chromosomes due to the occurrence of another fusion of acrocentrics. C-banding analysis of the mitotic chromosomes revealed constitutive heterochromatin in the centromeric regions of all acrocentrics. However, small metacentrics were C-band negative. The YA chromosome is C-band negative except for a small amount of heterochromatin in the centromeric region. The nucleolar organizer region as identified by Ag-staining is present in the interstitial region of chromosome pair No. 10.     

Locations of a Human Satellite DNA in Human Chromosomes

USING techniques for DNA/RNA or DNA/DNA hybridization in situ, Pardue and Gall¹ and Jones² made several significant discoveries on the chromosomal locations of the mouse satellite DNA: (1) this fraction of DNA is found in all chromosomes except the Y, (2) the cytological location of the satellite DNA is limited to the centromeric region of each chromosome and is probably absent in other regions and (3) the centromeric regions of all mouse chromosomes are hetero-chromatic.     

Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.     

Structure and replication of Phaseolus polytene chromosomes

The normal morphology of the polytene chromosomes of the embryo suspensor of Phaseolus coccineus is that of a tightly condensed cord with heavily Feulgen staining centromeric heterochromatic regions (α-heterochromatin) and other accessory heterochromatic regions (β-heterochromatin). The replication pattern of the chromosomes has been determined by autoradiographic analysis of material pulsed with ³H-thymidine for various lengths of time. The DNA replication cycle reqires 4–6 hours for completion. During replication chromosome structure becomes diffuse and the β-heterochromatic regions are indistinguishable from the euchromatic regions. The euchromatin is the first to replicate, and replication begins simultaneously at numerous sites in the euchromatin. The β-heterochromatin replicates next, and finally the centromeric heterochromatin. Replication is essentially complete in each of these parts of the chromosome before DNA synthesis begins in the next. The chromosomes are composed of numerous longitudinally running Feulgen positive strands, the equivalent portions of which replicate simultaneously. This indicates that there must be close control of the replication cycle in sister strands.     

Localisation of reiterated nucleotide sequences in Drosophila and mouse by in situ hybridisation of complementary RNA

The location of highly reiterated nucleotide sequences on the chromosomes has been studied by the technique of in situ hybridisation between the DNA of either Drosophila melanogaster salivary gland chromosomes or mouse chromosomes and tritium labelled complementary RNA (c-RNA) transcribed in vitro from appropriate templates with the aid of DNA dependent RNA polymerase extracted from Micrococcus lysodeikticus. The location of the hybrid material was identified by autoradiography after RNase treatment. — When Drosophila c-RNA, transcribed from whole DNA, was annealed with homologous salivary chromosomes in the presence of formamide the well defined labelling was confined to the chromocentre. With heat instead of formamide denaturation there was evidence of discontinuous labelling in various chromosome regions as well, apparently associated with banding. Xenopus ribosomal RNA showed no evidence of annealing to Drosophila chromosomes with the comparatively short exposure times used here. — When mouse satellite DNA was used as template the resulting c-RNA showed no hybridisation to Drosophila chromosomes but, when annealed with mouse chromosomes, the centromeric regions were intensely labelled. The interphase nuclei showed several distinct regions of high activity which suggested aggregation of centromeric regions of both homologous and non-homologous chromosomes. The results of annealing either c-RNA or labelled satellite DNA to homologous chromosomes were virtually indistinguishable. Incubation of Drosophila c-RNA with mouse chromosomes provided no evidence of localisation of grains. — It is inferred that both in mouse and Drosophila the centromeric regions of all chromosomes are enriched in highly reiterated sequences. This may be a general phenomenon and it might be tentatively suggested that the highly reiterated sequences play some role in promoting the close physical approximation of homologous and non-homologous chromosomes or chromosome regions to facilitate regulation of function.     

The chromosomal localization of a heavy satellite DNA in the testis of Plethodon c. cinereus

When DNA from blood or liver of Plethodon c. cinereus is centrifuged to equilibrium in cesium chloride it separates out into 2 components. The smaller or satellite component is relatively rich in G + C and is therefore heavy, and it amounts to about 2% of the total DNA. The heavy satellite does not include the ribosomal cistrons, and it is unrelated to the nucleolar organizer. When squash preparations of cells from the testis of P. c. cinereus are incubated in synthetic E³RNA complementary to the satellite DNA, the RNA anneals specifically to the centromeric heterochromatin of spermatogonia, spermatocytes, and spermatids, and to the centromeric regions of all discernible chromosomes. RNA/DNA hybrids were located by autoradiography. H³RNA complementary to the major component of the DNA anneals to all nuclei and to all parts of the chromosomes. H³RNA complementary to nucleolar DNA from Xenopus laevis anneals specifically to the chromatin associated with nucleoli in nuclei at various stages of the meiotic divisions. The nature of the centromeric heterochromatin and its role in the meiotic divisions are discussed.     

Molecular cytogenetics of the equidae. II. purification and cytological localization of a (G + C)-rich satellite DNA from Equus hemionus onager and cross-species hybridization to E. asinus chromosomes

A (G + C)-rich satellite DNA component (p = 1.716 g/ml) has been fractionated from the total DNA of the Iranian subspecies of the Asiatic wild ass, Equus hemionus onager, by successive dactinomycin-CsCl and netropsin sulfate-CsCl isopycnic gradients. Complementary 3H-RNA (cRNA) transcribed from the satellite DNA hybridized predominantly to the centromeric and telomeric constitutive heterochromatic regions of onager chromosomes. These studies have suggested that satellite DNA's with similar sequences are present in the centromeric, as well as telomeric, heterochromatic regions of some onager chromosomes. The centromeric region of the fusion metacentric t(23;24) of the onager is deficient in sequences homologous to the onager 1.716 g/ml satellite DNA, indicating a loss of satellite DNA during fusion or an amplification of the satellite DNA in the centromeric regions of the acrocentric chromosomes 23 and 24 subsequent to fission. Sequences complementary to onager 1.716 g/ml satellite DNA show extensive hybridization to the constitutive heterochromatin of the feral donkey (E. asinus) karyotype, consistent with a view of conservation and amplification of similar or identical sequences in the two species.     

Karyomorphology of six taxa in Chrysanthemum sensu lato (Anthemideae) in Egypt and their genetic relationships by Giemsa C‐banding

Abstract Giemsa C‐banding was applied to the chromosome complements of six diploid species belonging to six genera in Chrysanthemum sensu lato (Anthemideae) distributed in Egypt. Four types of C‐banding distribution were observed in the taxa as follows: (i) negative C‐banding in Anacyclus monanthos (L.) Thell.; (ii) all bands in terminal regions in Achillea fragrantissima (Forssk.) Sch. Bip, which showed 32 bands on 18 chromosomes; (iii) all eight bands at centromeric regions on eight chromosomes in Matricaria recutita L.; and (iv) bands at terminal and centromeric regions in Brocchia cinerea Vis. (12 terminal and six centromeric bands on 12 chromosomes), Cotula barbata DC. (four terminal, six centromeric, and eight short arm bands on 16 chromosomes), and Glebionis coronaria (L.) Cass. ex Spach. (eight terminal on the short arms and four large bands in centromeric regions on 12 chromosomes).     

New Technique for Distinguishing between Human Chromosomes

A GIEMSA staining procedure that preferentially stains centromeric heterochromatin in mouse chromosomes has been described¹. This specificity was observed when fixed preparations were treated with sodium hydroxide to denature the DNA and then incubated in warm saline to allow annealing, in the presence of ³H-labelled single stranded satellite DNA or its complementary RNA. In this way mouse satellite DNA was located in the centromeric heterochromatin^1,2. It is known to consist of highly repetitive sequences³ and to anneal much more rapidly than non-repetitive DNA⁴. It seems probable, therefore, that the darker staining with Giemsa of these regions, after denaturation and annealing, indicates the presence of highly repetitive DNA.     

Banding in Human Chromosomes treated with Trypsin

THE differential staining properties of the Giemsa stain were first observed by Pardue and Gall¹. They were studying in situ hybridization between mouse satellite DNA and mouse chromosomes and observed that following certain pretreatment the centromeric regions of mouse chromosomes were more densely stained by Giemsa stain than other regions. The darkly stained regions were considered to consist of constitutive heterochromatin. Similar observations were later made on human chromosomes by Arrighi and Hsu² and Gagné et al.³. Through modifications of the original methods used in the DNA hybridization work, techniques have been developed which make each chromosome identifiable^4–6.     

Centromere structures highlighted by the 100%-complete Cyanidioschyzon merolae genome

Centromere dynamics are largely unknown in lower plants (algae). We have recently identified the centromere-specific histone H3 variant (CENH3) and clarified the dynamic centromere rearrangement at mitosis in the primitive red alga Cyanidioschyzon merolae. We also showed that the CENH3-containing nucleosomes constituted the kinetochore closely interacting with the nuclear envelope. CENH3 visualization during the whole cell cycle suggests that C. merolae centromeres are monocentric and confined to specific loci. We completed 100% no-gap telomereto-telomere sequencing of the C. merolae genome. Interestingly, a single A+T-rich region has been identified on each fully sequenced chromosome. No centromere-like A+T-rich repetitive sequence have been found within these regions, implying that the C. merolae centromeres may be ‘point’ centromeres, or be comprised of nonrepetitive heterogeneous DNA sequences.Key words: centromere, chromosome structure, complete nuclear genome, Cyanidioschyzon, repetitive DNACentromere function is evolutionarily conserved in almost all eukaryotes. It is known that centromeric DNAs undergo rapid evolution and have no obvious constraints on their sequence conservation. However, several centromere proteins are conserved at the domain and motif level, suggesting that key protein-protein interactions, retained through centromere evolution, might allow for the functional conservation and DNA sequence diversity of the centromere. Most prominent are centromere-specific histone H3 (CENH3) family proteins, because the histone fold domain of this family is well conserved among all the eukaryotic lineages to assemble the centromeric nucleosomes with other conventional histones.¹We previously clarified the centromere movement and reconstitution during the cell cycle by tracing the CENH3 in the ultrasmall unicellular red alga Cyanidioschyzon merolae. On the relationship between the kinetochore and the nuclear envelope (NE), which had been poorly understood in red algae, we demonstrated using electron microscopy that they are closely associated at mitosis. Given the cellular characteristics that the chromosomes barely condense and the NE remains intact throughout the cell cycle in C. merolae, we postulate that this kinetochore-NE interaction might produce a ‘signal’ until the uncondensed and lagged chromosome arm regions have been completely segregated and the tension on the NE has been attenuated. Visualization of CENH3 proved that the C. merolae chromosomes are not holocentric (entire chromosomes serve as centromeres) but are likely to be monocentric (one ‘regional’ or ‘point’ centromere occurs on each chromosome).²Previous C. merolae genome sequencing showed that several chromosomes possess single A+T-rich regions, which are annotated as putative centromeric regions. However, there were many gaps in the whole genome assembly that had not been fully sequenced, and a full picture of the putative centromeric regions was unclear.³ Recently, we finished the 100% complete genome sequencing and obtained all the 20 chromosome assemblies as full telomere-to-telomere sequences.⁴ Although generally the rDNA regions are highly repetitive and structurally unstable in most eukaryotes, the C. merolae complete genome sequence included the complete set of three ‘static’ singlet rDNA clusters scattered across different chromosomal loci, which is one of the most distinguishing structural characteristics.⁵Figure 1 shows the overall G+C content distribution on the fully sequenced chromosomes. It is interesting to note that a single A+T-rich region is present on each chromosome. We postulate that the single A+T-rich regions are something more than just stochastic distribution, and are likely to play some role in the maintenance of chromosome structure, since these regions show essentially a one-on-one relationship with chromosomes. Although less clear, the genome sequence of the unicellular green alga Ostreococcus tauri (Prasinophyceae) similarly shows a biased A+T distribution pattern.⁶ To determine whether the A+T-rich regions are associated with repetitive sequences like most centromeric regions in other species, we employed Tandem repeats finder, a program used to search for repeat sequences.⁷ However, we have not found any repeat sequence, any common pattern or any particular rule concerning the size, A+T% or sequence of these regions.Open in a separate windowFigure 1G+C content and distribution on the Cyanidioschyzon merolae chromosomes. Arrowheads indicate the positions of the putative centromeric regions.Several pioneering works provide useful information on the centromere structures in lower eukaryotes. The centromeres of the human malaria parasite Plasmodium falciparum are composed of extremely A+T-rich repetitive elements.⁸ In the trypanosome parasites, the Trypanosoma brucei chromosomes possess A+T-rich repeats within the centromeric region as identified by etoposide-mediated topoisomerase-II cleavage analysis, while these A+T elements are not found in T. cruzi.⁹ It is also important to note that centromeres in Candida albicans are all comprised of different and unique DNA sequences, and are maintained by an epigenetic mechanism.^10,11We presume that monocentric C. merolae chromosomes are unlikely to possess ‘regional’ centromeres composed of A+T-rich centromeric repeats, but rather the centromere structure is similar to ‘point’ (approximately 120 bp) centromeres in Saccharomyces cerevisiae.¹ Alternatively, the functional C. merolae centromeres might be non-repetitive, heterogeneous DNA elements, lacking in inter-chromosomal sequence similarities. With the increasing numbers of lower plant genome sequences that are available, comparative analysis of centromeric sequences will help to elucidate the evolution of centromere DNA-protein interactions in the plant kingdom.     

In situ nick translation distinguishes between C-band positive regions on mouse chromosomes

In situ nick translation of mouse metaphase chromosomes by non-radioactive detection means and DNase I digestion followed by Giemsa staining were used to analyse the DNase I resistance of two different C-band positive regions. These were the centromeric heterochromatin of aero- and metacentric chromosomes and an interstitial C- band on chromosome 1 of wild mice, IS(HSR;1C5D)1Lub. Whereas the centromeric heterochromatin was clearly resistant to DNase I, the interstitial C-band showed very high DNase I sensitivity. Among centromeric C-bands, the heterochromatin in Robertsonian fusion biarmed chromosomes was more resistant to DNase I action than was the centromeric heterochromatin of the acrocentric chromosomes.     

Characterization of heterochromatin in Schistocerca gregaria by C- and N-banding methods

Mitotic and meiotic chromosomes of Schistocerca gregaria were C-, mild N- and strong N-banded. After C-banding, three out of eleven autosomes show, in addition to the centromeric C-bands, a second C-band. — The mild N-banding method produces a single N-band in each of only four chromosomes. With the exception of one N-band these mild N-bands correspond to the non-centromeric, second C-bands, indicating the heterochromatic character of at least three mild N-band regions. — The strong N-banding technique produces bands both at the C- and mild N-band positions and additionally a third band in one chromosome (M₈), not present after C- or mild N-banding. — The N-bands do not correspond to the nucleolus organizer regions. Because of the mechanisms of the N-banding methods, it is concluded that the centromeric heterochromatin, as well as the non-centromeric N-band regions, contain high quantities of non-histone proteins. Presumably a specific difference exists between the non-histone proteins in the centromeric and non-centromeric N-band regions because the centromeres are banded by the strong N-banding technique, but not after mild N-banding. It is concluded that the N-band regions (two exceptions) contain a heterochromatin type which has the following features in common with the -heterochromatin of Drosophila: C- as well as N-banding positive, high nonhistone protein content, repetitive and late replicating DNA. It is discussed whether the N-banded heterochromatin regions of Schistocerca contain that DNA fraction which is, like the Drosophila -heterochromatin, underreplicated in polyploid nuclei.