C-banding Procedure Research Articles

Metaphase Cells Enrichment for Efficient Use in the Dicentric Chromosome Assay.

The dicentric chromosome assay (DCA), is considered the 'gold standard' for radiation biodosimetry. Yet, DCA, as currently implemented, may be impractical for emergency response applications, especially when time is of the essence, owing to its labor-intensive and time-consuming nature. The growth of a primary lymphocyte culture for 48 h in vitro is required for DCA, and manual scoring of dicentric chromosomes (DCs) requires an additional 24-48 h, resulting in an overall processing time of 72-96 h for dose estimation. In order to improve this timing. we introduce a protocol that will detect the metaphase cells in a population of cells, and then will harvest only those metaphase cells. Our metaphase enrichment approach is based on fixed human lymphocytes incubated with monoclonal, anti-phosphorylated H3 histone (ser 10). Antibodies against this histone have been shown to be specific for mitotic cells. Colcemid is used to arrest the mitotic cells in metaphase. Following that, a flow-cytometric sorting apparatus isolates the mitotic fraction from a large population of cells, in a few minutes. These mitotic cells are then spread onto a slide and treated with our C-Banding procedure [Gonen et al. 2022], to visualize the centromeres with DAPI. This reduces the chemical processing time to ~2 h. This reduces the time required for the DCA and makes it practical for a much wider set of applications, such as emergency response following exposure of a large population to ionizing radiation.

Hybrid breeding boosted molecular genetics in rye

The history of rye (Secale cereale L.) breeding began from the first targeted selections made in Germany by the Probsteier Seed Cooperative around 1850, and over 150 years of breeding yielded a tremendous amount of results. Rye has also long been used as cytological subject due to its low number of chromosomes and their size. However, genetic findings in rye up to the early 1980s were rather scant. About 120 genes could be assigned to seven linkage groups. However, since then development of new methods such as C-banding procedures, in situ DNA hybridization, enzymology and molecular marker techniques achieved an enormous progress. Particularly, the latter of these was driven by the agronomic success of hybrid breeding in rye. The basic genetic knowledge resulting from intra- and interspecies genetic diversity assay and phylogenetic studies in the Secale genus used nuclear and cytoplasmic molecular markers to facilitate successful selection of parents for hybrid breeding and hence contribute to improvement of heterosis effect. The main achievements in rye hybrid breeding and genetic mechanisms exploited for different hybrid breeding strategies are discussed. The exploitation of landraces or wild rye species gene pools via advanced backcross QTL analysis or introgression libraries to increase the heterosis effect is a prospective strategy of marker-assisted hybrid breeding in rye. Prediction of hybrid performance can also be improved significantly by marker-assisted selection and genomic selection based on genome-wide marker data. The findings in rye genetics (including phylogenetic, mapping and population studies) are reviewed in their relation with the hybrid breeding purposes and demands. Overall, about 450 morphological and biochemical traits are mapped throughout the the genome plus about 5,000 DNA markers. They are not only associated with individual chromosome or segments but also efficiently used for comparative mapping (evolutionary studies), introgression monitoring, QTL tagging, and marker-assisted selection.

C-banded karyotype of Thinopyrum bessarabicum and identification of its chromosomes in wheat background

C-banded pattern in two accessions of Thinopyrum bessarabicum (Save ex Rayss) A. Love (2n = 2x = 14, EbEb) and their idiogram was established. C-banding analysis was further used to identify the chromosomes of Tritipyrum amphiploid (2n = 6x = 42, AABBEbEb) and a BC1F2 genotype from wheat and Tritipyrum. Two 18S-26S rDNA loci were detected on Th. bessarabicum chromosomes by in situ hybridization using an 18S-26S rDNA probe. Eb chromosomes in Tritipyrum generally were identified by their distinctive C-banding patterns which reflected heterochromatin regions. C-banding procedure resulted in sharp and distinct bands in one or both ends of Eb chromosomes without interval bands. Observed C-bands in Eb genome mainly reflected the telomeric and subtelomeric sequences which also showed more strong signals in genomic in situ hybridization. Results showed the importance of the C-banding technique as a screening tool in identification of addition and substitution lines in the progenies of wheat and Tritipyrum crosses during segregating generations.

The meaning of DAPI bands observed after C-banding and FISH procedures

Under specific technical conditions chromosome staining with 4',6-diamidino-2-phenylindole (DAPI) permits characterization of heterochromatic regions as AT-rich (DAPI(+)) or AT-poor (DAPI(-)), especially when the chromosomes are counterstained with chromomycin A(3) (CMA), which preferentially binds to GC-rich DNA. DAPI(+) bands also often have been observed after C-banding or FISH. In these cases, however, it is not clear whether only AT-rich regions stain positively with DAPI or other heterochromatins with different base compositions also are stained. We evaluated the meaning of DAPI bands observed after C-banding and FISH using three plant species bearing different types of heterochromatin: DAPI(+)/CMA(-), DAP(-)/CMA(+) and DAPI(0)/CMA(0) (neutral bands). Additional tests were performed using propidium iodide, a fluorochrome without preferential affinity for AT or GC. Our results indicate that AT-rich heterochromatin stains as DAPI(+) bands after C-banding or FISH, but other kinds of heterochromatin also may be stained by DAPI.

Comparative study of the grasshopperZoniopoda tarsata(Romaleide) andPaulinia acuminata(Pauliniidae) based on chromosome and C-banding analysis.

Abstract In most Acridoidea the basic karyotype is composed by 11 telocentric chromosomes and a telocentric X (2n=23, X0) in males and 12 telocentric chromosomes in females (2n=24, XX). In this paper we comparatively analyzed meiosis of Z. tarsata (Acrididae, Romaleinae) and P. acuminata (Pauliniidae) after C-banding and DAPI staining in order to investigate differences due to heterochromatin amount and localization. Z. tarsata and P. acuminata showed a karyotype composed by 11 somatic telocentric chromosomes and a telocentric X, corresponding to 2n=23, X0 diploid karyotype in males. After C-banding procedure Z. tarsata pachytene bivalents showed 12 densely stained spots including the X chromosome, 4 of these were observed in interstitial locations and the remaining in pericentric position of the telocentric bivalents. In P. acuminata 4 C-banded spots were observed, 2 of these in interstitial locations and the remaining in pericentric position. After pachytene DAPI staining, bivalents showed specific bright stained areas corresponding to those detected after C-banding in Z. tarsata, the same was observed in P. acuminata. These differences demonstrate that despite chromosome similarity between these taxa heterochromatin evolution seems to follow a different path, allowing a clear differentiation between these two karyotypes.

Genome size and Giemsa C-banded karyotype of tetraploid Bromus ciliatus L.

Tetraploid Bromus ciliatus L. is a North American bromegrass that has been placed in the Pnigma section of Bromus. The objective of this study was to characterize the genome of tetraploid B. ciliatus by cytogenetic methods and compare it to the genomes of other species included in the section Pnigma. All the plants of the accession (USDA PI 232214) selected for chromosome counting were tetraploids (2n = 28). The mean 2C nuclear DNA content for tetraploid B. ciliatus was 19.13± 0.07 pg as determined by flow cytometry which is significantly greater than the tetraploid DNA content of B. inermis Leyss. (11.74± 0.16 pg). C-banding procedures were used to identify individual mitotic chromosomes and to develop a karyotype for B. ciliatus. The genome of the tetraploid B. ciliatus consisted of 16 median chromosomes, eight submedian chromosomes, and four chromosomes with satellites which included one pair with a large satellite and one pair with a small satellite. The general pattern of the distribution of constitutive heterochromatin in B. ciliatus was quite different than the other bromegrasses that have been analyzed to date. Except for two pairs of chromosomes, all chromosomes in tetraploid B. ciliatus had telomeric bands on one or both arms. Some of the chromosomes with telomeric bands had centromeric bands that were located at one or both sides of the centromere and intercalary bands which were generally absent in the other bromegrass species. It was possible to identify all chromosomes of tetraploid B. ciliatus and to match the pairs of homologous chromosomes by using chromosome lengths, arm length ratios and C-banding patterns. The results of this study indicate that tetraploid B. ciliatus has different genomes than the European species evaluated to date in the section Pnigma.

C-Banding Analyses of Genomes

Smooth bromegrass (Bromus inermis Leyss.) has both tetraploid (2n = 28) and octaploid (2n = 56) ploidy levels that have been difficult to characterize cytogenetically because of similar chromosome morphology. Objectives of this study were to identify individual chromosomes of tetraploid and octaploid B inermis with C-banding procedures along with chromosome length and arm length ratios, develop more detailed karyotypes than those previously available, and use the karyotypes to examine the genomic relationship of tetraploid and octaploid B inermis Root tips of the plants from four tetraploid and three octaploid accessions were used to produce chromosome squash preparations for cytogenetic analysis. The tetraploid B inermis genome consisted of 12 chromosomes with a telomeric band on each arm and sixteen chromosomes with only one telomeric band on one arm. All of the chromosomes of the tetraploid form, except for four chromosomes, were identified by C-banding patterns, chromosome length, and arm length ratio. The octaploid B inermis genome consisted of four chromosomes with no C-bands, ≈14 chromosomes with two telomeric bands, and ≈38 chromosomes with only one telomeric band on either the short or long arm. The combined use of C-banding, chromosome size, and arm length ratio only enabled groups of 2, 4, 6, or 8 similar chromosomes to be identified because of similarities in chromosome morphology of the octaploids. Results indicate that tetraploid B inermis is an allotetraploid since all chromosomes except four could be separated into identifiable pairs. Because of differences between expected and actual numbers of satellite chromosomes and chromosomes with specific C-banding patterns, octaploid B inermis is probably not a doubled form of the tetraploid B inermis

Cytogenetics and cytotaxonomy ofVelloziaceae

Chromosome number and other cytological features are reported from 35 species ofVelloziaceae, including several African and Brazilian populations. All analyzed species show areticulate interphase nuclei and prophase/prometaphase chromosomes with proximal early condensation. Most heteropycnotic blocks do not seem to correspond to heterochromatin since, at least inVellozia patens, they do not stain differentially after C-banding procedures. Regarding the chromosome number, three main groups could be identified. The first comprised diploid species of the generaNanuza, Vellozia and the Brazilian species ofXerophyta with 2n = 14 or 16; the second comprised tetraploid species with 2n = 34, and included all Brazilian species of subfam.Barbacenioideae; the third group, of hexaploid species, comprised the African representatives of the genusXerophyta. A single population ofVellozia, possibly of hybrid origin, had 2n ≅ 32. A basic number of x = 8 is proposed for the family. The karyological information supports the hypothesis that theVelloziaceae originated on the South American, rather than on the African continent.

Sequential chromosome banding and in situ hybridization analysis

Different combinations of chromosome N- or C-banding with in situ hybridization (ISH) or genomic in situ hybridization (GISH) were sequentially performed on metaphase chromosomes of wheat. A modified N-banding-ISH/GISH sequential procedure gave best results. Similarly, a modified C-banding - ISH/GISH procedure also gave satisfactory results. The variation of the hot acid treatment in the standard chromosome N- or C-banding procedures was the major factor affecting the resolution of the subsequent ISH and GISH. By the sequential chromosome banding - ISH/GISH analysis, multicopy DNA sequences and the breakpoints of wheat-alien translocations were directly allocated to specific chromosomes of wheat. The sequential chromosome banding- ISH/GISH technique should be widely applicable in genome mapping, especially in cytogenetic and molecular mapping of heterochromatic and euchromatic regions of plant and animal chromosomes.

Biological dosimetry of absorbed radiation by C-banding of interphase chromosomes in peripheral blood lymphocytes.

Induction of premature chromosome condensation enables direct observation of radiation-induced cytogenetic damage in non-stimulated, interphase, human peripheral blood lymphocytes. This phenomenon can be explored in radiation protection for biological dosimetry in instances of accidental exposure to ionizing radiation. Quantification of an exposure by means of this approach has been limited so far mainly to the analysis of chromosome fragments. This limitation is due to the fact that conventional Giemsa staining of prematurely condensed chromosomes (PCCs) does not allow visualization of the centromeric regions and, as a result, the identification of dicentrics, centric rings and acentric fragments. In the present report a C-banding procedure, refined to avoid swelling and chromosome distortion of freshly prepared PCCs spreads, is used to identify such aberrations in non-stimulated human lymphocytes. The method allows immediate banding of the centromeric regions and enables scoring of aberrations within a time interval (3-4 h after blood sample withdrawal) that is only a fraction of that normally required when cells stimulated to proliferate are analysed at metaphase. The dose-response for dicentrics and centric rings measured in interphase lymphocytes was found to be similar to that obtained at metaphase. Measurement of dicentrics and centric rings in prematurely condensed chromosomes of human lymphocytes would provide valuable information on radiation dose estimates, especially in cases of extreme urgency.

Ag-NOR and C-banding analysis of spermatocyte chromosomes ofClavelina lepadiformis(Ascidiacea, Aplousobranchiata)

SUMMARYChromosome number n = 9 and 2n = 18 for Clavelina lepadiformis (Ascidiacea, Aplousobranchiata) from the Gulf of Palermo have been determined. Silver staining analysis of testicular cells reveals that within-individual variability in NOR banding patterns is present. Using the C-banding procedure, a very impressive heterochromatin amount seems to characterize the chromosome set of this species.

Genome size and «C-heterochromatic-DNA» in man and the african apes

The genome sizes and the amounts of DNA after C-banding pretreatments (C-heterochromatic DNA) were measured by quantitative cytochemical methods in man and the African apes,Gorilla gorilla andPan troglodytes. As evaluated by flow cytometry on propidium-iodide-stained lymphocytes, gorilla and chimpanzee have genome sizes larger than man. On the basis of the different resistance of metaphase chromosome DNA to the C-banding procedure, two genome compartments were defined, i.e.,C-heterochromatic-DNA andeuchromatic-DNA. The latter proved to be fairly constant in man and the African apes (as well as in two hylobatid species), whereas the variable amounts ofC-heterochromatic-DNA account well for the interspecific differences of genome size among the hominoid species studied so far. During karyotype diversification, quantitative changes (with either gains or losses) ofC-heterochromatic-DNA seem to have taken place independently in the hylobatid and the man/African ape lineages.

C-banding of meiotic chromosomes in male grasshopper, Poekilocerus pictus Fabr.

Testicular cells of grasshopper, Poekilocerus pictus Fabr., were subjected to standard C-banding procedure. Number of bands per genome during pachytene, diplotene and diakinesis varied from 15-20. Chromosomes were well spread and countable at metaphase-I and anaphase-I; however, no C-bands were observed at these stages, which might be due to high condensation of chromosomes. The 2n number was found to be 19, (9II+X) and the sex determining mechanism to be of XO type. The mean chiasma frequency per cell was 16.40±0.41 at metaphase-I.

C-banding as a simple tool to discriminate between micronuclei induced by clastogens and aneugens.

If mouse bone marrow preparations are treated with a classical C-banding procedure, it may be possible to distinguish between micronuclei with or without centromeres. This allows discrimination between micronuclei originating from chromosome breakage and those originating from chromosome loss. Thus, using C-banding, the micronucleus test can be used not only for the detection of clastogens but also aneugens. In this way, more exhaustive methods such as immunological staining using antikinetochore antibodies may not be necessary.

Topographical changes in rye chromosome ultrastructure caused by the C-banding procedure

The changes in the surface features of rye chromosome 1 induced by the C-banding procedure was investigated using the scanning electron microscope. Each step in the procedure produced significant changes in the surface topography of the chromosome and minor differences in its general morphology. The most noticeable alteration, chromosomes flattened and smooth, occurred when they were incubated in phosphate buffer without Leishman's stain. Since chromosomes not treated with barium hydroxide were uniformly stained, it seems probable that this alkali removes chromatin unselectively and that the C-bands produced here are due to a differential affinity of the heterochromatin to the stain. Key words: rye, heterochromatin, C-banding.

C-Banding Analyses and the Evolution of Heterochromatin among Arvicolid Rodents

Development of the C-banding procedure has enabled identification of the amount and location of constitutive heterochromatin (C-bands) in the karyotypes of many species. One group of mammals that has received considerable attention in this regard is the rodents. C-band data are currently available for species belonging to a number of rodent groups including the following familiar ones: sciurids, geomyids, heteromyids, New World cricetids, Old World cricetids, Eurasian murids, Australian murids and octodontids. Patton and Sherwood (1983) summarized chromosomal evolution among rodents and indicated that the following four categories of C-band variation have been observed: (a) differences in the amount of centromeric heterochromatin, (b) variability in the number of autosomal heterochromatic short arms, (c) presence or absence of telomeric or interstitial autosomal C-bands, and (d) variation in sex chromosome heterochromatin. The family Arvicolidae (voles, lemmings, and muskrats) is taxonomically diverse, containing more than 120 species. This group occurs in the temperate regions of North America and Eurasia, includes species that occupy a wide array of habitats, and has attracted considerable interest from classical taxonomists, population biologists, and behaviorists. However, the approximately 40 species of New World arvicolids have received little attention from comparative cytogeneticists utilizing differential staining procedures. Detailed banding data have been reported for fewer than 15 species (Judd, 1980; Judd et al., 1980; Modi, 1986; Nadler et al., 1976; Rausch and Rausch, 1975; Zenzes and Voiculescu, 1975) and no comprehensive statements regarding chromosomal evolution have been made. This report presents C-banded karyotypes for 17 North American species. These data are tabulated along with information from the literature concerning additional New World and Old World species, and are discussed with respect to the chromosomal distribution of constitutive heterochromatin, euchromatic rearrangements, and interspecific C-band differences.

Analysis of a centric shift in the S11 chromosome of Aiolopus strepens (Orthoptera: Acrididae)

A centric shift in the S11 chromosome of Aiolopus strepens, previously described as a pericentric inversion (Cabrero & Camacho, 1982), was analyzed by using the C-banding procedure. The location of breakage points as well as the posibilities of pericentric inversion and three-break transposition are discussed. The relationship between C-bands and NOR location as revealed by both silver staining and a double procedure Ag-NOR C-banding is also discussed.

Genome-specific control of meiotic pairing evidenced in mutant Aegilops ventricosa-Secale cereale amphidiploids

The Ae. ventricosa and S. cereale genomes were distinguished at meiosis by the C-banding procedure. Only two plants of the amphiploid Ae. ventricosa-S. cereale were found to exhibit the high degree of asynapsis limited to Aegilops ventricosa genomes. In addition, these genomes showed higher homoeologous pairing than homologous pairing frequencies. These results can be explained by the existence of separate genome-specific control of meiotic pairing between the chromosomes of both species in these synaptic mutant plants.

C-banding at meiosis as a means of analyzing cytogenetic structure in wheat

The characterization of reciprocal interchanges between chromosomes of wheat in the progeny of the backcross ((T. aestivum H-53 × S. cereale) × T. aestivum H-53) using Feulgen and C-banding staining techniques has been attempted. The translocated chromosomes were studied in detail in three lines using monosomic analysis. In the F1 of the crosses between these lines and the 21 monosomic lines of 'Chinese Spring' a total of five interchanges were identified. Using a Giemsa C-banding procedure it was possible to confirm the identity of the interchange chromosome pairs that were previously identified by monosomic analysis. Moreover, a total of 12 new interchanges were characterized or identified. C-banding also facilitated the identification of the arms involved in translocations and permitted the quantitative analysis of each multivalent in multiple interchange heterozygotes. The comparative availability of Feulgen and C-banding in studies of cytogenetic structure in wheat is discussed.Key words: C-banding, meiosis, heterochromatin, translocations, wheat, Triticum.

Meiotic behaviour of Un, D and R genomes in the amphiploid Aegilops ventricosa -Secale cereale and the parental species

Meiotic pairing frequencies of the Un and D genomes of Ae. ventricosa and the R of S. cereale could be easily established at metaphase I in Aegilops ventricosa - Secale cereale amphiploid plants as well as in its parental species by using the C-banding technique procedure. The results show a high diminution of chromosome pairing for all genomes in the amphiploid with respect to its parental species probably due to C-heterochromatin content and/or genotypic or cryptic interactions between the three genomes.