Fluorescent Chromosome Banding Research Articles

Comparative Cytogenetics and Fluorescent Chromosome Banding in Five Indian Species of Dipcadi Medik

The genus Dipcadi Medik. (Subfamily: Scilloideae) has a narrow distribution in India and several overlapping morphological traits make the genus taxonomically challenging at the species level. Cytogenetic characterization can provide additional taxonomic data and can be used to evaluate genetic diversity at the species level. We have accomplished comparative karyotype analysis and fluorescence banding patterns using 4′-6-Diamidino-2-phenylindole (DAPI) and Chromomycin A3 (CMA) in five Indian species for the first time. The karyotypes of D. concanense and D. goaense exhibited similar fluorochrome banding profiles. However, D. montanum, D. ursulae and D. erythraeum differ distinctly in their karyotypes. In all taxa, CMA+ve/DAPI−ve or DAPI0 (GC-rich) constitutive heterochromatin was located at the constriction region or terminal satellite of the nucleolar chromosome. DAPI+ve/CMA−ve or CMA0 (AT-rich) heterochromatin dominates in D. montanum, D. ursulae and D. erythraeum. However, D. erythraeum shows a distinct variation in fluorochrome banding pattern from all other species. The distribution of CMA and DAPI bands is a reflection of heterochromatin composition and variations acquired by different species. This characterization can be used to assess phylogenetic relationships in the understudied genus Dipcadi and may serve as a basis for other genomic analyses and evolutionary studies.

Incipient speciation in Calceolaria polyrhiza complex (Calceolariaceae): cytogenetic analysis supports molecular and morphological data

ABSTRACT Calceolaria polyrhiza is a perennial herb distributed in southern South America. The species is highly polymorphic and includes four described morphotypes that were previously considered different species. We performed a detailed study of the karyotype of each morphotype considering at least two localities for each one, with the aim of contributing to the taxonomic delimitation of the Calceolaria polyrhiza complex. In addition, a principal component analysis and a mapping of chromosome traits onto a phylogeny of nuclear DNA sequences were performed. This detailed karyotypic study has revealed specific markers, which have allowed to characterize each morphotype. ‘Prichardii’ morphotype was consistently polyploid. ‘Mendocina’ and ‘Lanceolata’ morphotypes had the highest total heterochromatin amount, with two pairs of chromosomes carrying NORs. ‘Mendocina’ samples showed the most asymmetrical karyotypes. ‘Polyrhiza’ morphotype had the most symmetrical karyotypes of the four morphotypes and the simplest fluorescent chromosome banding pattern. Thus, the four morphotypes could be characterized by chromosomal data and, together with the phylogenetic analysis of molecular data, could be considered taxonomic units in the process of diversification.

Biología reproductiva y citogenética de Distichia muscoides (Juncaceae).

El género Distichia Nees & Meyen (Juncaceae) está formado por tres especies nativas de Sudamérica, siendo Distichia muscoides Nees & Meyen la más ampliamente distribuida (Kirschner et al., 2002). Esta especie dioica es el componente principal de los bofedales, ecosistema muy importante de la región altoandina. En este trabajo se estudiaron distintos aspectos biológicos de D. muscoides como: 1) la viabilidad del polen por medio de métodos indirectos de coloración; 2) la receptividad estigmática a través de una prueba con peróxido de hidrógeno; 3) la viabilidad de las semillas mediante su germinación; y 4) sus datos citogenéticos, empleando la técnica de bandeo cromosómico fluorescente CMA/DA/DAPI. Los resultados muestran que las flores estaminadas presentan polen con un porcentaje alto de viabilidad (100%); las flores pistiladas alcanzan la receptividad estigmática una vez que las ramas del estigma desarrollan su máxima longitud (4 mm) y permanecen en este estado al menos durante dos días. El número promedio de óvulos por ovario es 34, y el número promedio de semillas por fruto es 25. Las semillas germinaron a partir del día 15, mostrando 92% de germinación y un tiempo promedio de germinación de 18,4 días. La especie posee 2n = 80-84 cromosomas y el bandeo de fluorescencia mostró dos pares de bandas terminales CMA+/DAPI-, asociadas a regiones organizadoras nucleolares (NORs). Se reportan por primera vez datos reproductivos y cariológicos para esta especie.

Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level.

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR-, C- and fluorescent chromosome banding, and fluorescent insitu hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the 'Paranense Sea', probably forced the ancestors of these species to find refuge in unflooded areas for about 2Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.

Fluorescent chromosome banding patterns of several species in the Cupressaceae <i>sensu stricto</i>

Fluorescent chromosome banding patterns of several species in the Cupressaceae <i>sensu stricto</i>

Karyological relationships in Indian species of Drimia based on fluorescent chromosome banding and nuclear DNA amount.

The genus Drimia (syn. Urginea), commonly called squill, represents a species complex, infrageneric delimitation being ill-defined due to morphological variability, population variation within species and polyploidy. In the present study, fluorescent chromosome banding and measurements of nuclear DNA content by flow cytometry were performed in five Indian species of Drimia: Drimia indica, Drimia polyantha, Drimia razii, Drimia wightii and Drimia coromandeliana to elucidate taxonomic relationship and obtain possible insights into the evolutionary processes within this group. All taxa analyzed exhibited similar karyomorphology with subtle differences accounted by nucleolar chromosomes. Nuclear DNA content ranged from 20.41pg/2C in D. polyantha to 40.80pg/2C in D. coromandeliana and was positively correlated with chromosome number (r = 0.67, P = 0.02) and total diploid chromatin length (r = 0.59, P = 0.06). Fluorescent chromosome banding revealed the presence of CMA(+ve)/DAPI(-ve) signals associated with nucleolar chromosomes presumably coincident with NOR in all species and unique CMA(+ve) signals in diploid populations of D. indica. Satellite polymorphism between homologous NOR-bearing chromosomes was observed which supports hybrid origin of the taxon. UPGMA dendrogram and scatter diagrams based on karyological parameters indicated a close relationship of D. indica, D. razii and D. polyantha while D. wightii and D. coromandeliana appeared distant. D. wightii appeared more close to D. indica than to all other species based on genome size and karyomorphology. As a whole, D. indica showed high intra-specific variability with populations exhibiting intergrading characters with other species. In conclusion, it is likely that hybridization followed by reproductive isolation of polymorphic forms arising by adaptation to different ecological niches resulted in species diversification of Drimia in India, probably from a common ancestor similar to D. indica.

Increasing nuclear ploidy enhances the capability of antioxidant defense and reduces chromotoxicity in Lathyrus sativus roots under cadmium stress

The modulation of antioxidant defense and cytogenetic parameters was studied in Lathyrus sativus L. roots at 3 different ploidies under a control treatment (0 µM) and 5, 10, 25, and 50 µM of CdCl2 in nutrient media. Root growth was inhibited in diploids from 10 µM and in triploid from 25 µM. Growth was normal in tetraploid roots. Although Cd accumulated in ascending order of ploidy, perturbation of antioxidant defense was the severest in diploid, moderate in triploids, and not observed in tetraploid roots even at 50 µM. Low antioxidant enzyme activities led to overaccumulation of H2O2, consequently resulting in oxidative damage through membrane lipid peroxidation in treated diploid roots. Cd-induced chromotoxicity was exhibited by low mitotic fidelity in diploid chromosomes and to some extent in triploids. DNA base-specific fluorescent chromosome banding revealed GC to AT transition and even perturbation of the GC-rich region in the nucleolar organizing chromosome in diploid, presumably facilitating chromosome breakage at a lower ploidy level due to Cd treatment. By contrast, the normal to enhanced GC region was associated with a low percentage of chromosome breakage and other anomalies at higher ploidies exposed to Cd. Results suggest better tolerance of grass pea genotypes to Cd at elevated ploidy levels.

Classical and molecular cytogenetics and DNA content in Maihuenia and Pereskia (Cactaceae)

We studied cacti species of the subfamilies Pereskioideae (five species of the southern clade) and both species of Maihuenioideae using molecular cytogenetic techniques and DNA content. Mitotic chromosomes were analyzed for Pereskia aculeata, P. bahiensis, P. grandifolia, P. nemorosa, P. sacharosa, Maihuenia poeppigii, and M. patagonica, using the Feulgen stain, CMA/DAPI fluorescent chromosome banding, fluorescence in situ hybridization (FISH, probes of 5S rDNA and pTa71 for 18-5.8-26S rDNA), and DNA content by flow cytometry technique. The karyotypes were highly symmetrical, most of the pairs being metacentric (m). CMA/DAPI banding revealed the presence of CMA+/DAPI− bands associated with NORs in the first m pair of all species. The co-localization of 18-5.8-26S rDNA loci with CMA+/DAPI−/NORs blocks allowed the identification of homeologous chromosome pairs between species of both subfamilies. FISH using probe 5S rDNA was applied for the first time in both subfamilies. Diploid species had always one m pair carrying 5S rDNA genes, with pericentromeric location in different chromosome pairs. In the tetraploid cytotype of M. patagonica, the 5S rDNA probe hybridized to two pairs. The 2C DNA content obtained by FC varied twofold (from 1.85 to 2.52 pg), with significant differences between species. Mean chromosome length, karyotype formula, percentage of heterochromatin position of 5S rDNA locus, and nuclear Cx DNA content vary among Maihuenia and Pereskia species and allowed to differentiate them. Both genera are closely related and that the differences found are not strong enough to separate Maihuenioideae from Pereskioideae.

Karyological relationships among some South American species of Solanum (Solanaceae) based on fluorochrome banding and nuclear DNA amount

Fluorescent chromosome banding and measurements of nuclear DNA content by image cytometry of Feulgen-stained cells were performed in one sample each of eight diploid (2n = 24) species of Solanum: S. endoadenium, S. argentinum, S. pseudocapsicum, S. atropurpureum, S. elaeagnifolium, S. sisymbriifolium, S. chenopodioides, and S. palustre. The species studied could be distinguished by heterochromatin amount, banding patterns, and genome size. They exhibited only GC-rich heterochromatin and showed a comparatively low heterochromatin amount (expressed as percentage of haplotype karyotype length), ranging from 2.10 in S. argentinum to 8.37 in S. chenopodioides. Genome size displayed significant variation between species, with 1C-values ranging from 0.75 pg (735 Mbp) in S. palustre to 1.79 pg (1,754 Mbp) in S. sisymbriifolium. No significant correlation between genome size and heterochromatin amount was observed, but intrachromosomal asymmetry index (A1) was negative and significantly correlated with heterochromatin amount. DNA content was positively and significantly correlated with karyotype length. DNA C-value distribution in the genus as well as karyotype affinities and relationships between species are discussed in relation to different infrageneric classifications of Solanum.

Cytological and molecular characterization of Vicia barbazitae Ten. & Guss.

Vicia barbazitae, a taxon belonging to section Vicia of subgenus Vicia, was recovered and analysed by cytological, karyological and molecular methods with the aim of both proposing a general characterisation of this species and studying the relationships among the species of section Vicia . Phylogenetic relationships among the species of the section Vicia and those of the sections Microcarinae, Wiggersia and Atossa were also analysed. Automated karyotype analysis has been determined after Feulgen's reaction; chromosome banding was performed by sequence-specific fluorochrome staining. Fluorescent chromosome banding showed CMA(+)/DAPI(-) NOR-associated heterochromatin in the satellite pair. Karyomorphological parameters, based on symmetry indices, the dendrogram of linkage distance constructed on 37 chromosome parameters, as well as the molecular data based on internal transcribed spacer sequences provided information about phylogenetic position of this species inside the section Vicia and among the species belonging to the sections Microcarinae, Wiggersia, Atossa and Vicia. From our karyological and molecular results, it emerges that V. barbazitae can be considered a natural member of section Vicia.

Giemsa staining and fluorescent chromosome banding in someVitisL. species

Giemsa staining technique and fl uorescent chromosome banding with CMA 3 and DAPI were applied to the mitotic chromosomes of Vitis champinii, V. cinerea, V. girdiana, V. labrusca, V. rotundifolia, V. rupestris and V. vinifera for the purpose of chromosome measurement and constitutive heterochromatin characterization at the cytochemical level, respectively. Both fl uorescent CMA and conventional Giemsa staining constituted a valuable tool for chromosome characterization. Karyomorphometric data obtained after Giemsa staining allowed for an average ideogram and karyotype formulae based on chromosome length for the species. V. champinii and V. girdiana distinguished from the other fi ve species by means of the total haploid chromosome length, by the longest chromosome length and by the average chromosome length. The seven grape species have moderate chromosome asymmetry values and were classifi ed on 2A Stebbins' category. Positive CMA bands were seen in all species. V. girdiana distinguished from the other species solely by the presence of two CMA + bands, while V. champinii, V. cinerea, V. labrusca, V. rotundifolia and V. rupestris had four bands. V. vinifera showed chromosome heteromorphism for CMA bands. No clearly visible DAPI + band was seen in the species. According to the present observations, it seems that the evolutionary process of speciation involving North American and European Euvitis species studied, resulted in some discrete changes in chromosome measurements and also in heterochromatin base composition of at least one species. These data enlarge the chromosomal information of the genus Vitis and make possible further comparative studies into the Vitaceae family.

Karyotypes and fluorescent chromosome banding patterns in southern AfricanLycium(Solanaceae)

Abstract Chromosomal analyses of several southern African Lycium species resulted in diploid (2n = 24: L. amoenum, L. bosciifolium, L. ferocissimum, L. oxycarpum, L. tenue), tetraploid (2n = 48: L. gariepense, L. strandveldense, L. hantamense), and hexaploid (2n = 72: L. tetrandrum) counts. Chromosomes in all species were short (mean length = 2.00 μm; mean haploid genome length = 23.77 μm). Further, all species shared a highly symmetrical karyotype formula with 10 m pairs and 2 sm pairs, except L. bosciifolium with only one sm pair. The first m pair had a terminal microsatellite on the short arms. Fluorescent chromosome banding patterns with CMA/ DAPI staining in the diploid species showed NOR-associated heterochromatin in the first satellited pair. The tetraploids L. gariepense and L. hantamense had two chromosome pairs with a CMA+/DAPI−terminal band. Phylogenetic studies have shown that the southern African species are included in a monophyletic group including all Old World Lycium; these species are like...

Karyotype differentiation of four Cestrum species (Solanaceae) revealed by fluorescent chromosome banding and FISH

The karyotypes of four South American species of Cestrum (C. capsulare,C. corymbosum,C. laevigatum and C. megalophylum) were studied using conventional staining, C-CMA/DAPI chromosome banding and FISH with 45S and 5S rDNA probes. The karyotypes showed a chromosome number of 2n = 2x = 16, with metacentric chromosomes, except for the eighth submeta- to acrocentric pair. Several types of heterochromatin were detected, which varied in size, number, distribution and base composition. The C-CMA+ bands and 45S rDNA were located predominantly in terminal regions. The C-CMA + /DAPI + bands appeared in interstitial and terminal regions, and the C-DAPI + bands were found in all chromosome regions. The 5S rDNA sites were observed on the long arm of pair 8 in all species except C. capsulare, where they were found in the paracentromeric region of the long arm of pair 4. The differences in band patterns among the species studied here, along with data from other nine species reported in the literature, suggest that the bands are dispersed in an equilocal and non-equilocal manner and that structural rearrangements can be responsible for internal karyotype diversification. However, it is important to point out that the structural changes involving repetitive segments did not culminate in substantial changes in the general karyotype structure concerning chromosome size and morphology.

Differential Fluorescent Chromosome Banding of Solanum nigrum L. and Solanum villosum L. from Bangladesh

Two species of the genus Solanum viz. S. nigrum and S. villosum found in Bangladesh were cytogenetically investigated to confirm their taxonomic status. S. nigrum and S. villosum were found to possess 2n=24 and 2n=48 chromosomes, respectively. The centromeric formula 22m+2sm was found in S. nigrum and 48m in S. villosum. No gradual decrease of chromosomal length was observed in both the species indicated their karyotypes as primitive type. The individual chromosomal length ranged from 1.66 to 2.34 μm in S. nigrum and 1.66 to 2.66 μm in S. villosum. The total chromatin length in S. villosum (97.8 μm) was almost double to that of S. nigrum (48.94 μm). The range of relative length of chromosomes was similar in these two species. Solanum nigrum and S. villosum possessed 18 and 17 CMA positive bands, respectively. Most of the CMA-bands were present at the terminal region in both the species. The percentage of CMA banded region in S. villosum (35.43) was almost double to that of S. nigrum (18.69). A pair of DAPI positive bands was found on both the end of all the chromosomes in these 2 species. Each band was 0.5 μm in length. The karyotype of S. nigrum studied here indicated that the specimen was not actually S. nigrum rather it has much simillarities with S. americanum. Solanum villosum showed regular bivalent formation at metaphase-I and segregation at anaphase-I. The overall karyotypic features suggested S. villosum as an ancient auto-tetraploid of S. americanum which in course of time has started regular meiosis.

Replication, C- and Fluorescent Chromosome Banding Patterns in European Whitefish, Coregonus Lavavetus L.

The chromosome complement of Curegunus luvaretus was investigated using C-banding, DAPI and CMA,3 fluorescent stainings, and replication banding techniques. Diploid chromosome number was 2n = 80, and the arm number was NF = 100. Karyotype consisted of 8 pairs of metacentrics, 2 pairs of submetacentrics, 2 pairs of subtelocentrics and 28 pairs of acrocentrics, gradually decreasing in size, and of 0–4 supernumerary chromosomes. Chromomycin A3 staining revealed the location of nucleolar organizer regions (NORs), showing that examined fish possessed two pairs of submetacentric-subtelocentric (sm-st) NOR-bearing chromosomes. Staining with DAPI revealed the distribution of AT-rich regions. The use of in vivo 5-bromodeoxyuridine (BrdU) incorporation technique induced highly reproducible replication bands. All banding patterns obtained in the study allowed identification of numerous chromosome pairs in the Coregonus lacaretus karyotype. Chromosome 1 showed individual variation in the amount of C-positive heterochromatin resulting in the length polymorphism of this pair. Replication banding pattern in European whitefish showed differences between three forms of the first chromosome pair.

Técnicas para a obtenção de preparações citológicas com alta freqüência de metáfases mitóticas em plantas: Passiflora (Passifloraceae) e Crotalaria (Leguminosae)

Foram conduzidos experimentos visando à otimização de procedimentos para preparações citológicas de pontas de raiz com alta freqüência de metáfases mitóticas, apresentando cromossomos com morfologia nítida, em espécies pertencentes a dois gêneros de plantas, Passiflora (Passifloraceae) e Crotalaria (Leguminosae). Para o acúmulo de metáfases, bem como de prófases e prometáfases com cromossomos bem condensados, foram avaliados pré-tratamentos de raízes, em que foram utilizadas diferentes combinações de 8-hidroxiquinolina, inibidor do fuso mitótico, com cicloheximida, inibidor da síntese protéica. As preparações foram coradas pelo método de Feulgen. Os melhores resultados foram obtidos com o tratamento com 8-hidroxiquinolina a 300ppm combinado com cicloheximida a 3,125ppm, por 1 hora e 30 minutos para as espécies de Passiflora, e a combinação de 8-hidroxiquinolina a 300ppm + cicloheximida a 6,25ppm também durante 1 hora e 30 minutos para as espécies de Crotalaria. Esta investigação ilustra a situação de dois gêneros de plantas em que a adequação de pré-tratamentos diferiu entre os grupos, e é fundamental para estudos envolvendo comparação do cariótipo das espécies, sobretudo para a detecção de marcadores cromossômicos evidenciados por meio de métodos diversos de citogenética (bandamento-C, bandamento com fluorocromos e hibridação in situ fluorescente).

Fluorescent banding pattern analysis of eight taxa of Phaseolus and Vigna in relation to their phylogenetic relationships

Phylogenetic relationships among eight taxa of seven species of Phaseolus and Vigna (Phaseolus angularis, P. aureus, P. calcaratus, P. coccineus, P. vulgaris, Vigna sesquipedalis and V. sinensis; 2n = 22 each) were studied by the fluorescent chromosome banding technique. Preparations of somatic metaphase chromosomes of each taxon were sequentially stained with Giemsa, GC-specific fluorochrome chromomycin A3 (CMA) and AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). On the basis of the fluorescent banding patterns of the 22 chromosomes of each taxon, P. angularis, P. coccineus (from China and Korea) and P. vulgaris were grouped into one group ("Phaseolus group"), P. aureus and two Vigna species were grouped into another ("Vigna group") and P. calcaratus was grouped in an independent group.

Fluorescent chromosome banding in inbred chicken: quinacrine bands, sequential chromomycin and Dapi bands

Highly inbred White Leghorn chickens were used for an investigation of the banding pattern of the macrochromosomes. A standard was set for the Q-bands. GC-specific fluorochrome chromomycin and the AT-specific Dapi were used in a sequential stain. The comparison of these two stains disclosed quantitative differences in the base distribution of the DNA. Factors responsibe for the binding mechanism and the appearance of the bands are discussed.

Visualization of chromatin distribution in living PTO cells by Hoechst 33342 fluorescent staining

Chromatin distribution was visualized in living cells with the selective DNA fluorochrome Hoechst 33342. This dye was shown to be non-toxic on the rat kangaroo PTO cell line by measuring the labelled cell growth rate. The aim of this work was firstly to visualize chromatin distribution without fixation or dehydration and secondly to demonstrate that quantitative determination of DNA content was possible under these non-toxic labelling conditions. During interphase, condensed, decondensed and thin network chromatin configurations were visualized. In nucleolar regions the fluorochrome revealed well-defined chromocentres. During mitosis, fluorescent chromosome banding was observed in vital conditions and chromocentres on fixed chromosomes. Chromatin segregation was visualized after micronucleation, which induced chromosomal set distribution in individual micronuclei. By this means, we demonstrated that the chromocentres observed in interphase nuclei were part of nuclear organizer region (NOR)-bearing chromosomes. This vital staining of chromatin was shown to be compatible with the quantitative determination of DNA content, both in living PTO cells and in isolated nuclei.

Reverse fluorescent chromosome banding with chromomycin and DAPI.

Two DNA binding guanine-specific antibiotics, chromomycin A3 (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI.--In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI.--Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The cma-banding pattern appears to be similar to the pattern found by R-banding procedures.