Two types of constitutive heterochromatin in the chromosomes of some Fritillaria species

A Giemsa banding technique has been used to study C-banding in mitotic chromosomes in root tips of Fritillaria graeca, F. crassifolia and F. rhodocanakis, all diploids (2n=24) belonging to the graeca group. In the first two the C-bands were of two types, diverging in respect of staining regularly and specifically within chromosomes. In one type it was weak, being intermediate between that of intensely stained ones, representing the other class, and the euchromatin. In F. graeca the pale bands were proximally localized and confined to 5 pairs, whereas in F. crassifolia they occurred only in the 4 M chromosomes, in each within the centromeric constriction as a large inclusion. The interphase nuclei of both species contained pale and heavily stained chromocentres. No pale ones occurred in such nuclei of F. rhodocanakis. The probability is discussed that the two classes of C-band represent distinct types of heterochromatin, differing both in respect of condensation throughout the whole mitotic cycle and in the repetitive DNA sequences they most likely contain. In all 3 species pairs of Giemsa-positive centromeric dots, representing the centromeres, were masked both by proximally or centromerically localized bands, irrespective of the class of heterochromatin they represented.     

部分柑桔属及其近缘属Giemsa C-带带型研究

本文应用Giemsa显带技术研究了枳属(Poncirus)、金柑属(Fortunella)和柑桔属(Citrus)16个分类群的染色体。枳属以末端带和着丝点带为主,金柑属与柑桔属主要以末端带为主;统计分析了各分类群每对染色体及全组染色体的异染色质含量,并列出其带型公式;探讨了金柑属的分类学地位;赞同把柚(C.grandis)作为柑桔属的基本种之一;根据异染色质含量的变化对柑桔属的带型演化进行了讨论。     

Differential staining of plant chromosomes with Giemsa

Simple Giemsa staining techniques for revealing banding patterns in somatic chromosomes of plants are described. The value of the methods in the recognition of heterochromatin was demonstrated using five monocotyledonous and two dicotyledonous species. In Trillium grandiflorum the stronger Giemsa stained chromosome segments were shown to be identical with the heterochromatic regions (H-segments) revealed by cold treatment. Preferential staining of H-segments was also observed in chromosomes from three species of Fritillaria and in Scilla sibirica. Under suitable conditions the chromosomes of Vicia faba displayed a characteristic banding pattern and the bands were identified as heterochromatin. The Giemsa techniques proved to be more sensitive than Quinacrine fluorescence in revealing a longitudinal differentiation of the chromosomes of Crepis capillaris, where plants with and without B-chromosomes were examined. Again all chromosome types had their characteristic bands but there was no difference in Giemsa staining properties between the B-chromosomes and those of the standard complement.     

Comparison of areas of quinacrine mustard fluorescence and modified Giemsa staining in human metaphase chromosomes

The methods of quinacrine mustard fluorescence and modified Giemsa staining were compared in view of the structural details revealed in human mitotic chromosomes derived from the peripheral blood of normal healthy humans. Over the chromatids both techniques produced a crossbanding pattern where larger segments of heavy staining in the latter technique and the fluorescing bands in the former occurred at similar locations. The centromeric heterochromatin, intensely stained with Giemsa was, however, negative in fluorescence, except for chromosome no. 3 and less often no. 6. The regularly occurring secondary constrictions in chromosomes 1, 9, and 16 behaved generally like areas of centromeric heterochromatin. The area of secondary constriction in the Y chromosome as also that of chromosome 9 in the ASG modification of the Giemsa technique was both non-fluorescent and non-staining.     

C-banded karyotypes of two Silene species with heteromorphic sex chromosomes.

Mitotic metaphase chromosomes of Silene latifolia (white campion) and Silene dioica (red campion) were studied and no substantial differences between the conventional karyotypes of these two species were detected. The classification of chromosomes into three distinct groups proposed for S. latifolia by Ciupercescu and colleagues was considered and discussed. Additionally, a new small satellite on the shorter arm of homobrachial chromosome 5 was found. Giemsa C-banded chromosomes of the two analysed species show many fixed and polymorphic heterochromatic bands, mainly distally and centromerically located. Our C-banding studies provided an opportunity to better characterize the sex chromosomes and some autosome types, and to detect differences between the two Silene karyotypes. It was shown that S. latifolia possesses a larger amount of polymorphic heterochromatin, especially of the centromeric type. The two Silene sex chromosomes are easily distinguishable not only by length or DNA amount differences but also by their Giemsa C-banding patterns. All Y chromosomes invariably show only one distally located band, and no other fixed or polymorphic bands on this chromosome were observed in either species. The X chromosomes possess two terminally located fixed bands, and some S. latifolia X chromosomes also have an extra-centric segment of variable length. The heterochromatin amount and distribution revealed by our Giemsa C-banding studies provide a clue to the problem of sex chromosome and karyotype evolution in these two closely related dioecious Silene species.     

Phylogenetic relationship in Fritillaria spp. of Iran inferred from ribosomal ITS and chloroplast trnL-trnF sequence data

The genus Fritillaria embraces up to 165 taxa in the family Liliaceae, most of which are of high medicinal and ornamental value and importance. In this study, 44 specimens of the genus representing 9 species were collected from their natural habitats located in 10 provinces of Iran. Phylogenetic analysis was performed based on DNA sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal cistron and the trnL-trnF regions. The phylogeny was constructed using the neighbor joining inference method. Results indicate that the examined samples were evidently diverged into 2 distinct clades. Members of the subgenera Fritillaria and Rhinopetalum formed one clade while the other clade contained the subgenera Theresia and Petilium. There can be seen a high degree of similarity between the only yellow-colored crown imperial specimen and the red-colored specimens. The endemic species of Fritillaria straussii, Fritillaria zagrica and Fritillaria kotschyana which their status within the subgenera known in the genus Fritillaria has been remained undefined, fell into the subgenus Fritillaria. The clades also had relatively reasonable distribution patterns based on the genetic structure, geographical conditions and climate specifications. This study revealed the feasibility of the ITS and trnL-trnF DNA sequence for phylogeny of the genus Fritillaria. This is the first phylogenetic analysis of Fritillaria spp. in Iran.     

Distribution of heterochromatin in a reconstructed karyotype of Vicia faba as identified by banding- and DNA-late replication patterns

1) The distribution pattern of heterochromatin characterized by Giemsa-banding, Quinacrine-banding and DNA-late replication has been studied in a reconstructed karyotype of Vicia faba with all chromosome pairs interdistinguishable. 2) By means of two Giemsa-banding methods both an interstitial and a centromeric Giemsa-banding pattern are described. The former one comprehends 14 marker and 18 additional bands of lower but characteristic visualization frequencies. The centromeric Giemsa-banding pattern consists of 7 bands, located in the centromeric and in the secondary constrictions of the metaphase chromosomes. Chromosomes with banding patterns intermediate between the interstitial and the centromeric Giemsa-banding have also been observed. 3) Quinacrine-banding revealed 10–12 brightly fluorescent bands and 1–2 regions of dim fluorescence. Most Q-bands occupy chromosomal positions also characterized by interstitial Giemsa bands. 4) The DNA-late replication pattern, analyzed both by autoradiography and by FPG-technique, revealed 9 late replicating chromosome regions; all of these correspond positionally to the sites of interstitial Giemsa bands. 5) The results are discussed with respect to (a) the relationships between the banding- and the DNA-late replication pattern; (b) banding and heterochromatin characteristics; (c) the correlations between the distribution of chromatid aberrations and special types of heterochromatin. — The patterns of heterochromatin distribution found are in basic conformity with the corresponding patterns reported for the standard karyotype of Vicia faba. The heterochromatin type characterized by both Giemsabanding and late replication is characteristic of all those chromosome regions which after mutagen treatments show up as aberration hot spots. Positional correlations between interstitial Giemsa marker bands and chemically induced isochromatid breaks are indicative of preferential aberration clustering in heterochromatin/euchromatin junctions.     

Nectary morphology in South West Asian Fritillaria (Liliaceae)

Nectaries of 3 1 taxa belonging to 4 subgenera of the genus Fritillaria are investigated by scanning electron and light microscopy. In most of the material investigated nectary cells were smaller and narrower, and less irregular in shape than those of the neighbouring tissue of the tepals. Species belonging to subgenus Rhinopetalum clearly differ from all other species. Their nectaries are deeply impressed, and the slit-like nectary orifice is bordered by two lobes, at least in the lower part densely hairy. In F. gibbosa, E karelinii and F. ariana, the flowers are ± zygomorphic as the nectary on the upper tepal is more deeply depressed than the others, and the nectary lobes are rather broad and fringed. In E stenanthera and E bucharica, nectaries are equally impressed in all tepals and the nectary orifice is bordered by narrow, unfringed ridges. The unique structure of nectaries in all species of this subgenus supports its separation from Fritillaria into a separate genus (Rhinopetalum Fisch. ex Alexand.). In the other subgenera, the nectaries are less impressed, often ± flattish, and usually linear to lanceolate or ovate, except in subgenus Petzlium where they are ± circular. One complex in subgenus Fritillaria is markedly distinguished from the rest of the subgenus: in the F. crassifolia group, the nectaries consist of a long and linear raised ridge with a median furrow. F. crassifolia ssp. poluninii is raised to specific level, E poluninii (fix) Bakhshi Khaniki & K. Persson, stat. nov. It is concluded that data on nectary morphology support the latest classification of the genus Fritillaria into subgenera and informal groups.     

Comparison of the Giemsa C-banded karyotypes of Hystrix coreana and H. patula (Poaceae, Triticeae)

The karyotypes of Hystrix coreana from eastern USSR and H. patula from USA were investigated by Giemsa C-banding. Both species are outbreeders and have 2n = 4x = 28. The karyotype of two plants of H. coreana has 10 metacentric, 6 submetacentric, 8 heterobrachial and 4 SAT chromosomes; two plants differed by having 12 metacentric, 4 submetacentric, 8 heterobrachial and 4 SAT-chromosomes, and 10 metacentric, 4 submetacentric, 9 heterobrachial and 5 SAT-chromosomes, respectively. The C-banding pattern had no or few inconspicuous intercalary bands, but conspicuous telomeric C-bands in one or both arms giving a high content of heterochromatin (16.3–18.2%). The chromosome complement of one plant of H. patula had 8 metacentric, 6 submetacentric, 8 heterobrachial and 6 SAT-chromosomes. The C-banding pattern had between 1 and 4 intercalary or centromeric bands and conspicuous telomeric bands on one or both arms giving a high content of constitutive heterochromatin (16.4%).     

GIEMSA C-BANDED KARYOTYPES OF SEVEN NORTH AMERICAN SPECIES OF ALLIUM

The karyotypes of seven North American Allium species were studied by Giemsa C-banding technique. Two species (A. shoenoprasum and A. tricoccum) were diploids with 2n = 16 chromosomes. Three species (A. cernuum, A. douglasii and A. geyeri) were also diploids but with 2n = 14 chromsomes. Diploid and tetraploid populations of A. textile (2n = 14, 28) were studied. The population of A. canadense studied here was a tetraploid (2n = 28). All these North American species, except A. geyeri, possessed centromeric bands in all their chromosomes and nucleolar constriction bands in their satellited chromosomes. Allium shoenoprasum contained telomeric bands in most of its chromosomes but the other species had them only in a small number of chromsomes. Only three species (A. shoenoprasum, A. textile and A. tricoccum) were found to have intercalary bands in some chromosomes. The heterochromatin distribution in B chromosomes of three species was also observed. In A. cernuum, the heterochromatin occupied most of the length of all its Bs, but in A. shoenoprasum, heterochromatin was concentrated in the centromeric region. One population of A. textile (CC1179) was found to have a B chromosome in which very little heterochromatin existed.     

Giemsa C-banded karyotypes in Serupius L. (Orchidaceae)

Giemsa C-banding is utilized for the first time to characterize eight taxa of the genus Serapias . Heterochromatin distribution indicated that the Serapias species form a very homogeneous group. All the species possess chromosome pairs with similar heterochromatin patterns. C-banding showed conspicuous bands located around the centromeres, with some het-erochromatic short arms. There was more heterochromatin in S. apulica and S. nurrika than in the other taxa. Extensive centromeric heterochromatin may indicate recent structural rearrangements in the chromosome complement. Taken altogether, karyomorphology indicates a rather recent origin for the genus Serapias , which might also account for the small amount of interspecific variation observed.     

THE DISTRIBUTION OF HETEROCHROMATIN IN THE GENUS NICOTIANA (SOLANACEAE)

A survey of the species of the genus Nicotiana was carried out to determine the distribution and the cytological characteristics of heterochromatin in this genus. All examined species of the genus possess knob-type heterochromatin, which is defined as spherical, densely staining regions of the pachytene chromosomes. These knobs are most frequently located near the centromere, the nucleolar organizer, and the ends of the chromosomes. Block-type heterochromatin, defined as any longer-than-broad heterochromatic segment seen at pachytene, was found in three species of the section Paniculatae, three species of the section Tomentosae, and two species of the section Noctiflorae. Three categories of the block-type heterochromatin, corresponding to the three subgenera, were found to differ with respect to overall size, staining properties, and location of the blocks. The distribution of these three types of block heterochromatin is discussed in the light of the latest taxonomic treatments of the genus.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

C-Banding Patterns and Quantitative Karyotype Characteristics of Bulgarian Species of Crepis (Asteraceae)

Abstract: Giemsa C-banded idiograms were established from eight Crepis species of Bulgarian origin ( Crepis viscidula, C. paludosa, C. conyzaefolia, C. bithynica var. bithynica and var. fodorii, C. pulchra, C. sancta, C. setosa, and C. zacintha ) that had been previously studied for genome size and karyotype shape using classical methods. All taxa are diploids with descending chromosome numbers, x = 6, 5, 4, and 3. Satellites were always heterochromatic. Small but distinct centromeric bands were of general occurrence (except perhaps in C. paludosa ) and terminal bands were present at least in some chromosomes of the karyotypes. Higher amounts of heterochromatin in the form of terminal and intercalary bands were found in C. bithynica and C. zacintha, but banding patterns were not so specifically similar as to indicate common origin. There are a number of particular karyotypic characters which may turn out to be phylogenetically significant when corresponding information on many related taxa becomes available. Correlations of karyotypic parameters were calculated. In heterochromatin only percent heterochromatin, but not absolute heterochromatin amount, and genome size were significantly correlated. This is the result of a strong euchromatin variation but independent heterochromatin variation.     

玉米花粉单倍体植株染色体上异染色质的变异

我们用Giemsa BSG C-带技术检查了玉米花药培养获得的花粉单倍体植株根尖细胞染色体上异染色质的变异,观察结果表明,有的植株所显示的C-带数目是与供体植株的相一致,有的植株所显示的C-带数目则发生了显著变化,其中有的增加,有的减少。并讨论了异染色质发生变异的可能原因。还相应地观察到间期核中染色中心的变化是与中期染色体上C-带数目的变化相一致。     

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study

Summary A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%–85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.     

Thoracic underreplication in Drosophila species estimates a minimum genome size and the dynamics of added DNA

Many cells in the thorax of Drosophila were found to stall during replication, a phenomenon known as underreplication. Unlike underreplication in nuclei of salivary and follicle cells, this stall occurs with less than one complete round of replication. This stall point allows precise estimations of early-replicating euchromatin and late-replicating heterochromatin regions, providing a powerful tool to investigate the dynamics of structural change across the genome. We measure underreplication in 132 species across the Drosophila genus and leverage these data to propose a model for estimating the rate at which additional DNA is accumulated as heterochromatin and euchromatin and also predict the minimum genome size for Drosophila. According to comparative phylogenetic approaches, the rates of change of heterochromatin differ strikingly between Drosophila subgenera. Although these subgenera differ in karyotype, there were no differences by chromosome number, suggesting other structural changes may influence accumulation of heterochromatin. Measurements were taken for both sexes, allowing the visualization of genome size and heterochromatin changes for the hypothetical path of XY sex chromosome differentiation. Additionally, the model presented here estimates a minimum genome size in Sophophora remarkably close to the smallest insect genome measured to date, in a species over 200 million years diverged from Drosophila.     

Karyomorphological analyses and heterochromatin characteristics disclose phyletic relationships among 2n = 32 and 2n = 36 species ofOrchis (Orchidaceae)

The karyotypes of eight taxa ofOrchis L. with 2n = 32 and 2n = 36 have been investigated using morphometrical measurements following staining with Feulgen, Giemsa (C-banding) and the DNA specific fluorochrome Hoechst 33258. The karyotypes ofO. coriophora subsp.fragrans, andO. papilionacea proved to be the most asymmetrical, whileO. morio andO. longicornu exhibited the most symmetrical karyotypes. Using C-banding and the fluorochrome H33258 only the taxa with high asymmetry indices showed the presence of differentially stained chromatin bands. In most chromosomes heterochromatin bands were present at the telomeric position. The present results seem to indicate that the analysed species do not form a homogeneous group and further subdivisions are possible, which, in turn, do not always correlate with divisions based on morphological characters. Both karyomorphology and heterochromatin distribution coincide in indicating a possible evolutionary pathway.     

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.     

Morphological diversity of centromere regions in polytene chromosomes of blackflies (Diptera, Simulidae)

Karyotypes of more than 120 species of 33 genera of the Palearctic blackflies (Simuliidae) were studied on squashed acetoorcein stained preparations of salivary gland polytene chromosomes in larvae. In the course of evolution of the family, a significant complication was noticed in the morphology of centromere regions of polytene chromosomes. In plesiomorphic species, centromeres are not pronounced morphologically and the general picture does not differ from that of other bands and interbands of the polytene chromosome. In species with apomorphic characters, a distinct precentromeric heterochromatin appears, whose manifestation is responsible for morphological diversity of centromere zones in polytene chromosomes. They are represented either by conspicuous slightly thickened heterochromatic bands or by large amplified blocks of heterochromatin or puff-like structure, being considerably extended as a result of despiralization of precentromeric heterochromatin. There are species, which more commonly lack chromocentre and their chromosomes are separated. Some other species have ectopic contacts between pricentromeric heterochromatin. In some species, this heterochromatin is organized as a compact chromocentre. This has been found only in representatives of southern latitudes, most frequently in evolutionarily young species with narrow specialization.