DNA replication,3H-cRNA in situ hybridization and C-band patterns in the polycentric chromosomes ofLuzula purpurea Link

DNA late-replication,³H-cRNA in situ hybridization, and C-band distribution patterns were studied inLuzula purpurea Link chromosomes (2n=6). With each technique it was possible to identify homologous chromosomes. DNA late-replicating regions were present at the ends and in the middle of one chromosome pair (pair 1), on both ends of another chromosome pair with one end having more late-replicating regions than the other end (pair 2), and all along the length of the final pair (pair 3). The distribution of label following in situ hybridization of³H-cRNA complementary to Cot 1-reassociated DNA was similar to the DNA late-replication patterns. One chromosome pair had grains concentrated at the ends and in the middle of the chromosomes; another pair had grains at both ends with a greater grain concentration at one end; the final chromosome pair had grains distributed all along the length. C-band distribution patterns were also similar to the DNA late-replication and³H-cRNA in situ-hybridized ones. The results demonstrate that the constitutive heterochromatin ofL. purpurea polycentric chromosomes is similar to the constitutive heterochromatin of monocentric animal chromosomes in that it consists of highly repeated DNA sequences which are replicated late in the S stage of interphase.     

Localization of chromosome regions in potoroo nuclei (<Emphasis Type="Italic"> Potorous tridactylus </Emphasis>Marsupialia: Potoroinae)

Chromosome paints of the rat kangaroo ( Aepyprymnus rufuscens, 2n =32) were used to define chromosome regions in the long nosed potoroo ( Potorous tridactylus, 2n =12 female, 13 male) karyotype and localize these regions in three-dimensionally preserved nuclei of the potoroo to test the hypothesis that marsupial chromosomes have a radial distribution. In human nuclei chromosomes are distributed in a proposed radial fashion. Gene-rich chromosomes in the human interphase nucleus are preferentially located in the central area while gene-poor chromosomes are found more at the periphery of the nucleus; this feature is conserved in primates and chicken. Chromosome ordering in nuclei of P. tridactylus is related to their size and centromere position. Its relationship with replication patterns in interphase nuclei and metaphase was studied. In addition it was observed that the nucleus was not a smooth entity but had projections occupied by specific chromosome regions. Edited by: R. Allshire     

The characterization of somatic chromosomes of Gymnothorax unicolor (Delaroche, 1809) by C-banding and NOR staining (Osteichthyes,Anguilliformes)

The diploid chromosome number of Gymnothorax unicolor (Delaroche, 1809) is 2n=42, the karyotype comprising six pairs of meta-submetacentric and fifteen pairs of acrocentric chromosomes. C-positive chromatin is present in the centromeres of all chromosomes as well as in the paracentromeric regions of some chromosomes. A nucleolar organizer region was identified on the long arm of chromosome 9, near the centromere. This region is also positive to C-banding.Cytotaxonomical relationships are evidenced between the described karyotype and that of the related species Muraena helena.     

Karyotype analysis,nucleolus organizer regions and C-banding pattern of Eisenia foetida (oligochaeta,lumbricidae)

The diploid number 2n=22 and haploid number n=11 found for Eisenia foetida from Palermo, Italy, confirm earlier data for this species from other localities. Analyses of silver-stained and C-banded mitotic and meiotic chromosomes suggest that a single chromosome pair has active NORs which correspond with C-positive regions. The occurrence of nucleolus activity during spermatogenesis of E. foetida is ascertained.     

Karyotype and genome size variation in Ajuga L. (Ajugoideae–Lamiaceae)

Chromosome number changes and karyotype evolution play an important role in plant genome diversification and eventually in speciation. The genus Ajuga L. (Lamiaceae) has approximately 50 species distributed in temperate to subtropical regions. Four of these species are currently recognized in Korea (A. decumbens Thunb., A. multiflora Bunge, A. nipponensis Makino and A. spectabilis Nakai). Understanding the karyotype evolution in Ajuga has been hampered by the small size of their chromosomes and symmetrical karyotypes. Here we used classic Feulgen staining to establish chromosome numbers and construct karyotypes of the four species of Ajuga recognized in Korea and flow cytometry was used to study their variation in genome. The chromosome number of all investigated plants was 2n = 32. Still, the 2C DNA content ranged from 2.18 pg (A. decumbens) to 4.53 pg (A. multiflora). While the chromosome numbers were the same for all investigated species, the genome size variation could potentially be used as a taxonomic marker.     

Comparative cytogenetic study of the forms of <Emphasis Type="Italic">Macleaya cordata</Emphasis> (Willd.) R. Br. From different localities

A comparative cytogenetic study of two introduced forms of Makleaya cordata (Willd.) R. Br. = syn. Bocconia cordata Willd. grown in different ecological and geographical regions (Moscow and Donetsk areas) was carried out. In the study, a complex of methods utilizing various chromosomal markers, i.e., C- and DAPI-banding technique, fluorescence in situ hybridization (FISH) with probes of 26S and 5S rDNA, as well as estimation of the total area of C-positive regions (C-HCH) in prophase nucleoli and meiosis analysis, was used. In the karyotypes (2n = 20), each chromosome was identified on the basis of C-banding and FISH patterns and the chromosome ideograms were built. Pericentrometric and telomeric C-positive bands in chromosomes of the Moscow form karyotype were found to be smaller and intercalary bands, larger than the corresponding bands in the M. cordata form grown in Donetsk. It was found that the content of C-HCH in prophase nucleoli in the form of M. cordata grown in Donetsk was higher than in the form grown in Moscow. In both forms sites of 26S rDNA and 5s rDNA were localized on satellite chromosome 1 and on chromosome 4 respectively but the signals were more intensive in the plant form grown in Donetsk. The results of this study enable selecting M. cordata forms for use in pharmacology and recommending them for cultivation in various ecological and geographical regions.     

Replication banding patterns in the spined loach,Cobitis taenia L. (Pisces,Cobitidae)

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.     

Comparative FISH analysis of C-positive regions of chromosomes of wood mice (Rodentia,Muridae, <Emphasis Type="Italic">Sylvaemus</Emphasis>)

The homology of DNA of C-positive centromeric regions of chromosomes in wood mice of the genus Sylvaemus (S. uralensis, S. fulvipectus, S. sylvaticus, S. flavicollis, and S. ponticus) was estimated for the first time. DNA probes were generated by microdissection from the centromeric regions of individual autosomes of each species, and their fluorescence in situ hybridization (FISH) with metaphase chromosomes of representatives of all studied wood mouse species was carried out. Unlike in the chromosomal forms and races of S. uralensis, changes in the DNA composition of the chromosomal centromeric regions in the wood mouse species of the genus Sylvaemus (including closely related S. flavicollis and S. ponticus) are both quantitative and qualitative. The patterns of FISH signals after in situ hybridization of the microdissection DNA probes with chromosomes of the species involved in the study demonstrate significant differences between C-positive regions of wood mouse chromosomes in the copy number and the level of homology of repetitive sequences as well as in the localization of homologous repetitive sequences. It was shown that C-positive regions of wood mouse chromosomes can contain both homologous and distinct sets of repetitive sequences. Regions enriched with homologous repeats were detected either directly in C-positive regions of individual chromosomes or only on the short arms of acrocentrics, or at the boundary of C-positive and C-negative regions.     

High-resolution flow karyotyping and chromosome sorting in Vicia faba lines with standard and reconstructed karyotypes

Flow cytometric analysis has been performed on chromosomes isolated from formaldehyde-fixed root tips in a Vicia faba (2n = 12) line with a standard (wild-type) karyotype and in six V. faba translocation lines with reconstructed karyotypes. The resolution of individual chromosome types on histograms of chromosome fluorescence intensity (flow karyotypes) depended on the type of fluorochrome used for chromosome staining. The highest degree of resolution was achieved with 4,6-diamidino-2-phenylindole (DAPI). The lower resolution obtained after staining with mithramycin A (MIT) and propidium iodide (PI) was probably due to the sensitivity of these stains to changes in chromatin structure induced by formaldehyde fixation. After the staining with DAPI, only 1 chromosome type could be discriminated in the line with a standard karyotype. In the translocation lines, the number of chromosome types resolved on flow karyotypes ranged from 2 in the G and the ACB lines to all (6) chromosome types in the EFK and EF lines. Refined flow karyotyping permitted the sorting of a total of 15 different chromosome types from five of the translocation lines. It is expected that flow sorting of chromosomes from reconstructed karyotypes will become a powerful tool in the study of nuclear genome organisation in V. faba.     

Comparative cytogenetics and chromosomal characteristics of ribosomal DNA in the fish genus Vimba (Cyprinidae)

Karyotypic and cytogenetic characteristics of Vimba vimba and V. elongata were investigated using differential staining techniques (sequential C-banding, Ag- and CMA₃-staining) and fluorescent in situ hybridization (FISH) with 28S rDNA probe. The diploid chromosome number in both species was 2n = 50 with 8 pairs of metacentrics, 14 pairs of submetacentrics to subtelocentrics and 3 pairs of subtelo- to acrocentrics. The largest chromosome pair of the complements was characteristically subtelo- to acrocentric. The nucleolar organizer regions (NORs) in both species were detected in the telomeres of a single, middle-sized subtelocentric chromosome pair, a pattern common in a number of other Leuciscinae. FISH with rDNA probe produced consistently positive hybridization signals detected in the same regions indicated by Ag-staining and CMA₃-fluorescence. The distribution of C-positive heterochromatin was identical in both species, including a conspicuous size polymorphism of heterochromatic blocks in the largest metacentric and subtelo- to acrocentric chromosomal pairs. No heteromorphic sex chromosomes were detected. A single analyzed individual of V. melanops possessed the same karyotype and NOR phenotype as V. vimba and V. elongata. The apparent karyotype homogeneity and chromosomal characteristics of ribosomal DNA in all three species of the genus Vimba is consistent to that found in most other representatives of the European leuciscine cyprinid fishes.     

C-banding and in-situ hybridization analyses of Agropyron intermedium,a partial wheat x Ag. intermedium amphiploid,and six derived chromosome addition lines

Summary C-banded karyotypes of Agropyron intermedium (2n=6x=42, E₁E₂X), a partial amphiploid Triticum aestivum —Ag. intermedium (2n=8x=56, TAF46), and six derived chromosome addition lines, were analyzed. In Ag. intermedium, diagnostic C-bands were present on 14 pairs of chromosomes, designated from A to N, while the remaining seven pairs, designated O to U, either lacked, or had only faint, C-bands and were not always identified unambiguously. All seven Ag. intermedium chromosome pairs of the partial amphiploid TAF46, and the added Ag. intermedium chromosomes present in the six derived addition lines, were identified by their characteristic C-banding patterns. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent Ag. intermedium accession, suggesting that these chromosomes were not structurally rearranged. In-situ hybridization, using a 18s.265s rDNA probe, showed that the Ag. intermedium chromosomes 1Ai-1 and 5Ai-l present in the addition lines L3 and L5 were carrying actively transcribed nucleolus organizer regions. The results are discussed with respect to the genomic relationships of these chromosomes.Contribution no. 91-561-J from the Wheat Genetics Resource Center and Kansas Agricultural Experiment Station, Kansas State University, Manhatten, USA     

The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba

The Giemsa banding patterns of the standard karyotype of Vicia faba and of four new karyotypes with easily interdistinguishable chromosomes due to interchanges and inversions are described and compared with the data of other authors on preferential Giemsa staining in Vicia faba. All karyotypes contain 14 easily reproducible marker bands which characterize chromosome segments known to be heterochromatic. It is shown that the preferential Giemsa staining of chromosome regions is a valuable tool for the localization of translocation and inversion points in the chromosomes of the reconstructed Vicia karyotypes. A close correlation exists between banding patterns, segment extension by incorporation into chromosomal DNA of azacytidine and mutagen-specific clustering of induced chromatid aberrations in the new karyotypes.     

Interspersed distribution patterns of C-bands and satellite DNA in the holocentric chromosomes of Luzula flaccida (Juncaceae)

C-banding studies revealed the presence of C-positive regions interspersed amongst the euchromatin of the prophase holocentric chromosomes of Luzula flaccida. In-situ hybridization showed a highly-repeated DNA sequence to be located in clusters throughout the extended chromosomes. The interspersed distribution pattern of prophase C-bands and satellite DNA in L. flaccida is discussed in relation to those in organisms with monocentric chromosomes.     

In vitro culture ofBellevalia romana (L.) Rchb.

Summary Bellevalia romana (L.) Rchb., a monocotyledonous plant characterized by few (2 n=2 x=8) and very large chromosomes, is a useful subject for studying developmental problemsin vitro. Cytological analysis of callus revealed that the majority of cells were diploid, but the remaining cells had aneuploid nuclei with a wide range of chromosome numbers, tetraploid and haploid nuclei. The frequency of aneuploid and polyploid cells was higher in callus grown in the presence of 2,4-D than in callus grown in NAA plus BAP. These nuclei seemed to increase with the duration of culture. The chromosome number distribution as determined by chromosome counts in calli at different culture times was confirmed by DNA cytophotometry. Chromosome number mosaicism (mixoploidy and aneusomaty) also occurred in all root apices of 9 out of 46 plantlets regenerated from callusvia adventitious shoots.     

Comparison of homologous polytene chromosomes in Phaseolus coccineus embryo suspensor cells: morphological,autoradiographic and cytophotometric analyses

The functional behaviour of unpaired homologous polytene chromosomes (2n=22), was investigated in nuclei of Phaseolus coccineus embryo suspensor cells. Observations were carried out on the morphological level and after ³H-thymidine and ³H-uridine autoradiography. Histone and total protein contents in the chromatin were also investigated. It was shown that corresponding regions of homologous chromosomes may show different functional structures. ³H-thymidine incorporation demonstrated differences between homologues in both DNA synthesis leading to chromosome endoreduplication (polytenization) and DNA amplification (extra DNA synthesis). ³H-uridine autoradiography showed that homologous regions in a given chromosome pair may display three labeling patterns: i) both regions labeled; ii) both regions unlabeled; iii) one region labeled and the other unlabeled. These three states are found to occur in different cells of one and the same embryo suspensor. Differences between homologous chromosome regions were also found in the ratios between DNA and protein contents in their chromatin. These results, which show that the functional activity of homologous chromosomes of the same complement may greatly differ, are discussed in relation to the characteristics of the system investigated.     

Chromosomal evolution withinPiperaceae

Chromosome numbers for 26 different species of the generaPiper, Peperomia andPothomorphe (Piperaceae) are reported. The basic chromosome numbers are 2n = 26, x = 13 (Piper, Pothomorphe) and 2n = 22, x = 11 (Peperomia), polyploid series are characteristic forPiper andPeperomia. Piper has the smallest chromosomes and prochromosomal interphase nuclei,Peperomia the largest ones and mostly reticulate to euchromatic nuclei.Pothomorphe is intermediate in both characters. The karyomorphological differences betweenPothomorphe andPiper underline their generic separation. Interspecific size variation of chromosomes occurs inPiper andPeperomia. Infraspecific polyploidy was observed inPiper betle. C-banding reveals different patterns of heterochromatin (hc) distribution between the genera investigated. The genome evolution is discussed.     

Molecular cytogenetics and characterization of a ZZ/ZW sex chromosome system in Triportheus nematurus (Characiformes, Characidae)

Chromosomes of Triportheus nematurus, a fish species from family Characidae, were analyzed in order to establish the conventional karyotype, location of C-band positive heterochromatin, Ag-NORs, GC- and AT-rich sites, and mapping of 18S and 5S rDNA with fluorescence in situ hybridization (FISH). The diploid number found was 2n = 52 chromosomes in both males and females. However, the females presented a pair of differentiated heteromorphic chromosomes, characterizing a ZZ/ZW sex chromosome system. The Z chromosome was metacentric and the largest one in the karyotype, bearing C-positive heterochromatin at pericentromeric and telomeric regions. The W chromosome was middle-sized submetacentric, appearing mostly heterochromatic after C-banding and presenting heterogeneous heterochromatin composed of GC- and AT-rich regions revealed by fluorochrome staining. Ag-NORs were also GC-rich and surrounded by heterochromatic regions, being located at the secondary constriction on the short arms of the second chromosome pair, in agreement with 18S rDNA sites detected with FISH. The 18S and 5S rDNA were aligned in tandem, representing an uncommon situation in fishes. The results obtained reinforce the basal condition of the ZZ/ZW sex system in the genus Triportheus, probably arisen prior to speciation in the group.     

Cytological localization of fast renaturing and satellite DNA sequences inVicia faba

Summary DNA sequences reassociating within a Cot value of 1.8×10^–1 and those producing a light satellite in a CsCl density gradient were isolated fromVicia faba DNA and hybridizedin situ on squashes of roots of the same species. Silver grains were seen to be scattered over both the interphase nuclei and the metaphase chromosomes after hybridization with fast renaturing DNA sequences, indicating these are fairly regularly interspersed in theV. faba genome. Clustered labeling occurred after hybridization with satellite DNA sequences, indicating these are clustered in the genome. The localization of satellite DNA in chromosomes appeared to correspond closely to the position of the bright bands detectable after staining with quinacrine mustard. After hybridization with both DNA probes, labeling intensity over the nuclei of meristematic cells was higher than that over the nuclei of differentiating and/or differentiated cells. These results are discussed in relation to the structure of the cell nucleus, the mechanism of quinacrine banding and to previous data suggesting underrepresentation of nuclear repeated DNA sequences in differentiatingV. faba root cells.     

Asynchrony of DNA replication in the chromosomes of Luzula

Seedlings of Luzula purpurea (2n=6) were placed in contact with H³-thymidine for 30 minutes. After removal of the isotope the roots and leaf primordia were fixed at intervals between 0 and 14 hours. The percentage of labelled mitoses follows a very close curve in roots and leaf primordia. In both tissues the value of G₂ is approximately 3 to 4 hours and of S circa 8 hours. DNA replication in the chromosomes of L. purpurea is asynchronous. The discontinuous DNA synthesis discloses that Luzula chromosomes are composed of many segments replicating independently of each other. The results support a polycentric rather than a completely diffuse kinetochore system in this species.     

TheTy1-copia group retrotransposons inVicia species: copy number, sequence heterogeneity and chromosomal localisation

We present an in-depth study of theTy1-copia group of retrotransposons within the plant genusVicia, which contains species with widely differing genome sizes. We have compared the numbers and sequence heterogeneities of these genetic elements in three diploidVicia species chosen to represent large (V. faba, 1C=13.3 pg), medium (V. melanops, 1C=11.5 pg) and small (V. sativa, 1C=2.3 pg) genomes within the genus. The copy numbers of the retrotransposons are all high but vary greatly, withV. faba containing approximately 10⁶ copies,V. melanops about 1000 copies andV. sativa 5000 copies. The degree of sequence heterogeneity ofTy1-copia group elements correlates with their copy number within each genome, but neither heterogeneity nor copy number are related to the genome size of the host. In situ hybridization to metaphase chromosomes shows that the retrotransposons inV. faba are distributed throughout all chromosomes but are much less abundant in certain heterochromatic regions. These results are discussed in the context of plant retrotransposon evolution.