CMA Bands Research Articles

Chromosomal Characterization of Five Medicinally Significant Phyllanthus Species in Bangladesh by DNA Base-Specific Fluorochrome Banding Technique

ABSTRACT Five Phyllanthus species were characterized using a fluorochrome chromosome banding technique with CMA and DAPI. This genus displayed a range of somatic chromosomal counts, including 2n = 2x = 26 (diploid) in P. acidus, P. niruri, and P. reticulatus; 2n = 6x = 48 (hexaploid) in P. urinaria; and 2n = 10x = 100 (decaploid) in P. emblica (wild and cultivated varieties), which exhibited a multi-basic chromosome number. The centromeric and terminal regions of the chromosomes contained most of the CMA and DAPI bands, indicating that GC- and AT-rich repeats had accumulated in these locations. The diversity based on the heterochromatin distribution patterns made it possible to use the CMA and DAPI banding approaches to analyze and characterize these five Phyllanthus species.

Chromosome numbers and heterochromatin variation in introgressed and non-introgressed populations ofEpidendrum(Orchidaceae: Epidendroideae): interspecific transfers of heterochromatin lead to divergent variable karyotypes in the parental populations

AbstractSome groups in Epidendrum have stable chromosome numbers, whereas the species of Epidendrum subgenus Amphiglottium exhibit high levels of chromosome number variability and other genomic alterations. Polyploidy and dysploidy have both been invoked to explain chromosome number variation in the subgenus, but little is known about what is driving the high frequency of chromosome modification within some species in this group. To test whether hybridization and introgression are involved, we use fluorochrome staining to evaluate the chromosome numbers and heterochromatin bands in E. subgenus Amphiglottium, including individual parental genotypes from introgressed and non-introgressed populations. Two main types of heterochromatin were observed forming CMA and DAPI bands in Epidendrum spp. B chromosomes were identified in some individuals. In non-introgressed populations, E. secundum has three cytotypes, 2n = 28, 56 and 84, found in different parts of its geographical range and a small number of DAPI bands. In introgressed populations of E. secundum, there was dysploid variation among individuals that were morphologically typical parental plants, and in one introgressed population a high number of DAPI bands was observed in what appeared on morphological grounds to be pure parental individuals. Our data corroborate the hypothesis that hybridization and introgression play a role in generating chromosome variation (number and form) and heterochromatin diversification in E. subgenus Amphiglottium.

Cytomolecular diversity of the subtribe Laeliinae (Epidendroidae, Orchidaceae) suggests no relationship between genome size and heterochromatin abundance

The subtribe Laeliinae (Orchidaceae) comprises approximately 2000 species and 38 genera, with numerically stable karyotypes of 2n = 40 (x = 20) in most species. Cytomolecular and chromosome banding analyses have not yet been combined with phylogenetic studies, making the understanding of chromosomal evolution in the group more difficult. The present work sought to perform comparative cytogenetic analyses of representatives of the subtribe Laeliinae, followed by the interpretation of that data within an evolutive context. We analyzed 23 species from seven genera for their chromosome numbers and morphology, the distribution of CMA and DAPI bands, fluorescent in situ hybridization (FISH) with 5S and 35S rDNA and DNA quantification by flow cytometry. All of the species showed 2n = 40, suggesting an apparent macrostructural karyotypic stability, except the polyploids Encyclia alboxanthina, E. jenischiana, E. seidelii, Laelia gouldiana and Prosthechea faresiana (2n = 80). At least two CMA+/DAPI− terminal bands were observed in all analyzed species. Additionally, CMA+/DAPI− blocks were observed in the pericentromeric region of species of the genera Brassavola, Cattleya (except C. trianae) and Laelia. Terminal CMA−/DAPI+ bands were observed in representatives of Brassavola, Laelia and Guarianthe. The clade Brassavola + Cattleya + Laelia, that presented high number of heterochromatic bands, also showed expansions of the numbers of 5S rDNA sites. DNA contents varied from 2C = 2.85 pg (Prosthechea fragrans) to 2C = 5.76 pg (Laelia gouldiana) without relation with number of CMA/DAPI bands or 5S rDNA sites. The relationship between the amplification of repetitive DNAs (viewed here as rDNA sites or heterochromatic bands) and genome size variation is discussed and compared with other plant groups.

Heterochromatin Bands and rDNA Sites Evolution in Polyploidization Events in Cynodon Rich. (Poaceae)

Cynodon is a genus with a wide distribution in tropical and subtropical areas. Ploidy levels in Cynodon range from diploid to hexaploid; hence, polyploidy is the most important driver of chromosome number variation in this genus. After polyploidization, structural rearrangements such as deletions, insertion, or duplications of DNA sequences frequently occur, allowing variation of chromosome morphology, size, and number. These events might result in a wide diversity of karyotypes, contributing to reproductive isolation, and consequently to speciation. In this study, we investigate the karyotype variation of Cynodon based on comparative cytogenetic analyses. We conducted chromosome counts, DNA quantification, CMA/DAPI double staining, and FISH mapping of 5S and 35S rDNA sites. Cytomolecular data were analyzed in a phylogenetic framework, in order to trace the evolutionary history of the karyotype variation considering polyploidy events in this group. Our results indicate that the most recent common ancestor of Cynodon had two 35S and 5S rDNA sites, two CMA bands, nine DAPI+ bands on the long arm, five DAPI+ bands on the short arm, and three DAPI+ bands in the pericentromeric region. During polyploidization events, there were losses and gains of heterochromatic sequences mainly on the short arms and centromeric regions.

Karyological traits related to phylogenetic signal and environmental conditions within the Hymenaea clade (Leguminosae, Detarioideae)

The Hymenaea clade is a lineage of the subfamily Detarioideae of Leguminosae. The clade is currently composed of three genera: Guibourtia, Hymenaea, and Peltogyne. The genera Guibourtia and Hymenaea present an Amphi-Atlantic distribution, whereas Peltogyne is exclusively found in the Neotropics. Previous studies have focused on the phylogeny of the group but there are no studies that demonstrate mechanisms that could be involved in the evolution of this Amphi-Atlantic clade. The present work is the largest cytogenetic analysis for the group published so far. We aimed to provide a study of karyotypic characteristics of the Hymenaea clade, interpreted using the plastidial trnK phylogenetic reconstruction as background. We analyzed karyological characteristics such as chromosome number, chromosome size, centromeric position, CMA/DAPI patterns, and banding for 5S and 45S rDNA. With the present data available, multivariate analyses (PCA) and Phylogenetic Eigenvector Regression (PVR) demonstrated that centromeric asymmetry and number of 5S rDNA sites may present a strong phylogenetic signal, whereas number of CMA+ sites is associated with geography/climate characteristics. The association of CMA bands and geography has been reported for other legumes of the Caesalpinia group. This study demonstrates that the use of statistical tools for analyzing karyotypic characteristics can result in more precise interpretation of evolutionary processes related to structural changes in chromosomes and how these changes can also be related to environmental conditions.

Characterization of aneuploidy in interspecific hybrid between Urochloa ruziziensis (R. Germ. & Evrard) Crins and Urochloa decumbens (Stapf) R. D. Webster.

The aim of the study was to characterize the type of aneuploidy present in the hybrid Urochloa ruziziensis×Urochloa decumbens and to confirm the origin of the additional chromosomes through comparative analysis of the hybrid and parental karyotypes. C andCMA bandingtechniques were used for chromosome differentiation. The parental genotypes showed 36 chromosomes. The hybrid presented plants with 36 + 2 chromosomes and plants with 36 + 1 chromosomes. Urochloa ruziziensis (4x) presented four chromosomes with CMA and C bands co-located in the terminal position. In U. decumbens, four chromosomes presented terminal CMA bands, eight chromosomes were distinguished by Cbanding with pericentromeric and terminal bands, one chromosome with terminal band at both ends and one chromosome presented one Cterminal band. For the hybrid, CMA bands were found on five chromosomes and C bands on seven chromosomes, all in terminal position. Aneuploidy was identified in pairs 3' and 4' in the hybrid plants with 36 + 2 chromosomes, characterizing it as double trisomy. The karyotype of hybrid plants with 36 + 1 chromosomes indicated elimination of the additional chromosome identified in pair 4' and maintenance of trisomy on pair 3'. The comparative analysis of karyotypes indicates that the additional chromosomes that characterize the trisomy were inherited from U. ruziziensis (artificial tetraploid).

B-Chromosomes in Gymnosperms

Abstract B-chromosomes show a non-random distribution in different taxa of gymnosperms. They are generally smaller and show similar centromeric position as the normal complement and are heterochromatic in nature and may show DAPI or CMA banding. They are generally devoid of any gene content, do not pair at meiosis and show accumulation through female line. They show incremental effect on genome size and some adaptive value in particular ecological conditions.

Chromosomal variability in Brazilian species of Anthurium Schott (Araceae): Heterochromatin, polyploidy, and B chromosomes.

The genus Anthurium has a Neotropical distribution, with karyotype predominance of x = 15, although some species show disploidy or polyploid variations. The karyotypes of seven species and different populations of Anthurium were analyzed using fluorochrome CMA and DAPI staining. The karyotypes were composed of meta- and submetacentric chromosomes, with numbers varying from 2n = 30 to 2n = 60. Supernumerary euchromatic chromosomes were observed in A. affine, and supernumerary heterochromatic chromosomes were observed in A. gladiifolium and A. petrophilum. Polyploidy was recurrent in the Anthurium species analyzed, with records of 2n = 30 and 60 in different A. pentaphyllum populations. Fluorochrome staining revealed different CMA+ banding distributions between diploid and polyploid cytotypes of A. pentaphyllum, suggesting structural alteration events. Anthurium petrophilum, on the other hand, showed a more consistent banding profile, with 10 to 12 proximal CMA bands in the three populations analyzed. DAPI+/CMA0 regions occurred exclusively in populations of A. gracile and A. pentaphyllum. The heterochromatic fraction in Anthurium was found to be quantitatively variable among species and populations, which may be related with adaptive aspects, different environmental conditions, or phylogenetic position.

Cytogenetic phenogram with high resolution chromosome configurations of some Korean landrace citrus by CMA banding and rDNA loci

Abstract Metaphase chromosomes of five Korean landrace citrus were analyzed to understand the phylogenetic relationship among them and to compare these characteristics with those of other Citrus species including previously reported six Korean landrace mandarins at a cytogenetic level using CMA banding patterns and rDNA loci. The CMA banding patterns of the five Korean landrace citrus were 1 A+2B+2C+6D+7E in dongjeongkyul (C. erythrosa), 3B+1C+7D+5E+2 F in hongkyul (C. tachibana), 2 A+1B+3C+4D+8E in sadoogam (C. pseudogulgul), 1 A+3B+1C+7D+6E in dangyooza (C. grandis), 1 A+1B+1C+9D+6E in jigak (C. aurantium). All types of chromosome bands were present in all accessions except in hongkyul, in which type A chromosome was absent but two of type F chromosomes were observed. The numbers of type A, B, and C chromosomes were lower in all accessions. In contrast, the type D and E chromosomes were remarkably constant and predominantly observed in all accession. The distributions of 5S and 45S rDNA loci by FISH were heterogeneous among them. All accessions possessed one 5S rDNA locus except for hongkyul, which displayed two 5S rDNA loci. And they always co-localized with 45S rDNA locus. All 45S rDNA loci were homotopic to CMA-positive regions. Every type A and B chromosomes possessed one 45S rDNA locus in the proximal region of the chromosomes. There was no type C and E chromosome with rDNA observed. Hierarchical cluster analysis and UPGMA phenogram based on CMA banding pattern combined with 5S and 45S rDNA loci of 11 Korean landrace citrus showed the strong karyotype dissimilarity in the invested taxa.

Heterochromatic and cytomolecular diversification in the Caesalpinia group (Leguminosae): Relationships between phylogenetic and cytogeographical data

The pantropical Caesalpinia group comprises 27 genera and approximately 205 species. The Neotropical species of the group are distributed in three main centers of diversity: the Andes, Mesoamerica (including Mexico, Central America, the southern USA and the Caribbean), and Northeastern Brazil. Our study investigates patterns of phylogenetic, environmental and geographical variation in an attempt to explain the karyotypic diversity which occurs within the Caesalpinia group. Cytogenetic analyses were based on chromosome number and morphology, CMA and DAPI heterochromatic bands, distribution of rDNA sites, and their molecular phylogenetic relationships based on the plastid markers matK, rps16, trnDT, trnL and ycf6 and the nuclear ribosomal marker ITS. Ecological differentiation among cytotypes was tested by comparing sets of climatic variables. In total, twenty species from ten genera of the Caesalpinia group were analysed, and all of them were found to have the same chromosome number (2n=24). CMA+/DAPI− bands were observed on the short arms of the acrocentric chromosomes in all of the species. Additionally, three different patterns were observed on the metacentric chromosomes: proximal CMA+/DAPI− bands, proximal CMA0/DAPI− bands and terminal CMA+/DAPI− bands. The 45S rDNA sites varied from eight to 14, always co-localizing with CMA+/DAPI− bands of acrocentrics, while 5S rDNA was localized in only a single chromosome pair in most species. A correlation between heterochromatic patterns and the geographic distributions/ecological niche of species was observed. Our analyses support some shared effects of the environment, when integrated with phylogeny and geography, on the CMA/DAPI variability. Thus, CMA+ heterochromatin is highly dynamic and its amplification/deamplification might be non-random, generating similar karyotypes in species which are not sister taxa in the molecular phylogeny but which co-occur in similar environments.

Karyotype diversity of Korean landrace mandarins by CMA banding pattern and rDNA loci

Abstract Mandarin is the major type of citrus grown in Jeju, Korea and has a long history of cultivation there. Landrace Citrus species represent broad and natural genetic variability, which are important and valuable as genetic material. In this study, metaphase chromosomes of six Korean landrace mandarins were analyzed to understand the phylogenetic relationship among them and to compare these characteristics with those of other Citrus species at a cytogenetic level using CMA banding patterns and rDNA loci. Chromosomes were categorized into five types according to the distribution and number of heterochromatic CMA-positive bands; type A chromosomes have two telomeric and one proximal bands, type B have one telomeric and one proximal bands, type C have two telomeric bands, type D have one telomeric band, and type E have no band. Gamza mandarin (C. benikoji) displayed the 1A/45S + 2B/45S + 2C + 5D + 1D/45S + 1D/5S-45S + 6E pattern, without a solitary 5S rDNA locus that differentiated the gamza mandarin from other accessions. A solitary 5S rDNA locus was observed in the chromosomes of byungkyul mandarin (C. platymamma) (1A/45S + 2B/45S + 1C + 6D + 1D/45S + 1D/5S-45S + 5E + 1E/5S) and cheongkyul mandarin (C. nippokoreana) (1A/45S + 1B/45S + 1C + 7D + 1D/5S + 1D/45S + 1D/5S-45S + 5E). The chromosome composition of byungkyul mandarin based on CMA banding pattern and rDNA loci suggests that byungkyul mandarin may be related to pummelo, sweet orange, and members of the Citrus subgenus Papeda during its evolution. Cheongkyul mandarin possessed a distinct marker chromosome (D/45S) that can be used to distinguish cheongkyul madrarin from the other Korean landrace mandarins. Jinkyul mandarin (C. sunki) (1A/45S + 1B/45S + 1C + 10D + 2D/5S-45S + 3E) may be related to pummelos. The karyotype of pyunkyul mandarin (C. tangerina) (3B/45S + 2C + 7D + 1D/5S-45S + 5E) suggests that the pyunkyul mandarin is a hybrid between C. grandis and C. reticulata. Binkyul mandarin (C. leiocarpa) (1A/45S + 1C + 6D + 2D/45S + 2D/5S-45S + 6E) seemed to be related to mandarins and pummelos. Heterozygous karyotypes of six accessions separated and differentiated each of the six Korean landrace mandarins and potential marker chromosomes were identified.

Karyotype, RAPD and ISSR Analysis of Four Specimens in <i>Gynura nepalensis</i> DC.

Four specimens of Gynura nepalensis DC. were investigated for authentic characterization by differential cytogenetical and PCR based molecular analysis. Three specimens out of four were collected from different localities of Bangladesh viz. i) from Netrokona district (specimen A), ii) from Dhaka district (specimen B) and iii) from Bogra district (specimen D). On the other hand, specimen C was collected from Canada. The diploid chromosome number of four specimens were 2n=20 with different centromeric formulae such as 14m+6sm in specimens A, B and D while 20m in specimen C. Total length of 2n chromosome complement was 211.61 µm in specimen A, 217.58 µm in specimen B, 168.82 µm in specimen C and 197.80 µm in specimen D. The maximum range of chromosome length was found in specimen B (6.67–14.30 µm) and minimum in specimen C (6.44–10.12 µm). The four specimens have distinct CMA- and DAPI-banding pattern which confined to the terminal regions of respective chromosomes. Karyotype analysis revealed that the number, location and distributions of GC- and AT-rich repeats are different in these four specimens. Heteromorphicity in respect of occurrence of CMA- and DAPI-banding pattern in the homologue members were observed in specimen A, C and D. The heteromorphicity might be a result of deletion of banded region from the respective homologue members. One interstitial CMA band was found on the short arm in a member of pair V in specimen A and in pair IV of specimen C. Two terminal DAPI bands were found in two chromosomes of specimens A and B. These CMA- and DAPI-banded chromosomes were unique and thus could be used as marker chromosomes for the respective specimens. Only specimen C showed RAPD bands with three primers. However, the four specimens had distinguishable ISSR-banding patterns with seven primer combinations. Specimen C showed maximum ISSR bands. The combined RAPD and ISSR analysis placed specimen C in a separate cluster with the highest genetic distance. In spite of very distant location specimens A and D were placed in a sub-cluster with narrow genetic distance.

CMA band variability and physical mapping of 5S and 45S rDNA sites in Brazilian Cactaceae: Pereskioideae and Opuntioideae

Representatives of the Cactaceae subfamilies Pereskioideae and Opuntioideae from northeastern Brazil were studied using banding with the fluorochromes, CMA3 and DAPI, as well as with fluorescent in situ hybridization using 45S and 5S rDNA probes to identify the distributions of their heterochromatin and rDNA sites. Pereskia aculeata, P. bahiensis, P. grandifolia (Pereskioideae), Brasilopuntia brasiliensis, Tacinga funalis, and T. palmadora showed 2n = 22, while Opuntia dillenii showed 2n = 44, and O. ficus-indica 2n = 88. The karyotypes of all of the species were symmetric, with average chromosome lengths varying from 1.94 µm in O. dillenii to 3.17 µm in P. aculeata. One pair of terminal CMA+ bands corresponding to NORs occurred in all of the diploid cytotypes (except O. ficus-indica, which has two pairs of terminal CMA+ bands) as well as in O. dillenii (tetraploid). CMA+ bands were also observed in the interstitial region of the long arm of a chromosome pair in B. brasiliensis, while a number of variable proximal bands were observed on three chromosome pairs in O. dillenii and on most of the chromosomes of O. ficus-indica. The 45S rDNA sites corresponded to the terminal CMA+ bands, while the 5S rDNA sites were located in the interstitial regions of the long arms of the chromosome pairs of P. aculeata, P. bahiensis, P. grandifolia, and B. brasiliensis. Our data, and earlier publications, suggest that the subfamily Opuntioideae can be characterized as having proximal/interstitial CMA+ heterochromatin in at least one chromosome pair (except in Tacinga). The absence of proximal heterochromatic bands, however, appears to be a synapomorphy of the basal lineages of Cactaceae (subfamily Pereskioideae + Maihuenioideae), suggesting that karyotypes with heterochromatin restricted to the terminal region of a chromosome pair (45S rDNA) represent a plesiomorphic character of the family.

Karyological, morphological, and phylogenetic diversification in Leucocoryne Lindl (Allioideae, Amaryllidaceae)

The genus Leucocoryne (Amaryllidaceae) comprises approximately 15 species and is the only genus of the tribe Ipheieae endemic to the western region of Chile in sclerophyll and arid winter-rainfall biomes in the Andes. To better understand the karyotype diversification of this genus, nine species of Leucocoryne were analyzed in terms of their chromosome number and morphology, CMA and DAPI heterochromatic bands, distribution of rDNA sites, and their molecular phylogenetic relationships. Based on phylogenetic analyses Leucocoryne was divided in four clades: I (L. alliacea and L. conferta); II (L. dimorphopetala and L. narcisoides); III (L. coquimbensis, L. macropetala, L. appendiculata, L. talinensis, L. angustipetala, L. purpurea, and L. violacescens); and IV (L. vittata, L. ixioides, L. foetida, and L. pauciflora). The species were diploids with 2n = 10 (6M + 4A) or tetraploids with 2n = 18 (14M + 4A)/2n = 19 (13M + 6A). 45S rDNA sites were co-localized with CMA+/DAPI− bands on the short arms of acrocentric chromosomes in all species. 5S rDNA sites were observed on one, two, or four pairs of metacentric chromosomes per species. Estimations of the ancestral karyotype suggest that 2n = 10 (6M + 2A) with one 5S rDNA site per monoploid complement was the plesiomorphic condition of the genus Leucocoryne. Increases in number of 5S rDNA sites in the same species associated with biome shifts from sclerophyll to arid winter-rainfall region occurred independently in clade II and clade III. The two species of clade II retained the six fertile stamens (plesiomorphic condition) in contrast to the other Leucocoryne species that reduced to only three functional stamens. Based on these findings, we discuss the role of environmental change on the diversification of this group.

Functional repetitive sequences and fragile sites in chromosomes of Lolium perenne L.

Lolium perenne is considered a high-quality forage widely used in temperate regions to meet the shortage of forage during the winter. In this species, some peculiarities related to cytogenetic aspects have already been described, as the variability in number and position of 45S ribosomal DNA (rDNA) sites and the expression of fragile sites, which require further studies to support the understanding of their causes and consequences. In this way, this study aimed to evaluate the relationship between the expression of fragile sites and functional repetitive sequences (rDNA and telomeric) in chromosomes of diploid and polyploid cultivars of L. perenne. The techniques of FISH, Ag-NOR and fluorescence banding were used to assess the distribution of sites of 45S rDNA, 5S, telomeric sequences, and the transcriptional activity of the 45S ribosomal genes and the distribution of AT- and/or GC-rich sequences in L. perenne, respectively. There was variability in the number and location of 45S rDNA sites, which was not observed for 5S rDNA sites. One of the genotypes showed two 45S rDNA sites on the same chromosome, located in different chromosome arms. Breaks and gaps were found in 45S rDNA sites in most metaphases evaluated for both cultivars. Telomeric sequences were not detected at the end of the chromosomal fragments corresponding to the location of breaks at 45S sites. Apparently, the transcriptional activity was modified in fragile sites. Variation in the number and size of nucleoli, nucleolar fusions and dissociations were observed. All CMA(+) bands were colocalized with the 45S sites.

Cytogenetic studies in South American species of Serjania (Sapindaceae: Paullinieae)

Serjania Mill. (Paullinieae) is considered the most important neotropical genus of Sapindaceae due to species number and its widespread distribution. In this study, 14 species belonging to three sections were analyzed using conventional staining, C/CMA/DAPI banding, and fluorescence in situ hybridization (FISH) with a 18S-5.8S-26S rDNA probe. New chromosome counts are reported for Serjania crassifolia, Serjania platycarpa, and Serjania regnellii, all with 2n = 24, which is remarkably constant for Serjania. The karyotypes are moderately asymmetric, and variations observed in A1 and A2 indices show resemblances between S. platycarpa, Serjania hebecarpa, and S. crassifolia, and between Serjania communis, Serjania gracilis, and S. regnellii. The banding pattern was homogeneous in Serjania. C/DAPI bands (AT-rich sites) were not clearly evidenced, but changes in the number and position of GC-rich sites (CMA bands) were observed. These segments were associated with 18S-5.8S-26S rDNA sites. The significance of the results is discussed in relation to chromosomal data available for the genus and in regard to the infrageneric treatment of Serjania.

Comparative cytogenetics between the species Passiflora edulis and Passiflora cacaoensis

Passiflora edulis Sims is the most economically important species of the genus Passiflora. A new species was described recently, Passiflora cacaoensis Bernacci & Souza, which displayed morphologic characteristics very similar to P.edulis. Due to the need for delimitation of the two species, karyomorphological and banding analyses were carried out. Both species have 2n = 18, with the same karyotype formula 16 m + 2sm. There was variation between the species regarding the location of satellites and the width of chromosome pairs 2, 4 and 8. C banding revealed the presence of constitutive heterochromatin in the centromeric and telomeric regions of all chromosomes in both species. However, only in P.cacaoensis did chromosomes 3 and 9 have a large quantity of heterochromatin. Fluorochrome banding revealed CMA(+) bands only in the satellites, but no DAPI(+) bands. Fluorescence in situ hybridisation (FISH) showed that in P.cacaoensis the rDNA 5S probe is located in a single site in the subterminal position of the long arm of chromosome 5. However, for the rDNA 45S probe, two sites were detected in terminal positions of the long arms of chromosome 7, with a bigger and stronger signal, and of chromosome 9. According to the asymmetry index and the quantity of heterochromatin, P.cacaoensis is a more basal species than P.edulis. The cytogenetic data indicate that P.cacaoensis is closely related to P.edulis, but is a different species.

Cytogenetic and molecular evidence suggest multiple origins and geographical parthenogenesis in Nothoscordum gracile (Alliaceae).

Nothoscordum gracile is an apomitic tetraploid widely distributed throughout the Americas and naturalized in many temperate regions of other continents. It has been suggested to form a species complex with sexual and apomictic N. nudicaule and N. macrostemon. Tetraploids of these species also share a structurally heterozygous chromosome complement 2n = 19 (13M + 6A). In this work, the origin of N. gracile and its relationships with its related species was investigated based on cytological and molecular data. Cytogenetic analyses were based on meiotic behaviour, CMA bands, localization of 5S and 45S rDNA sites, and genomic in situ hybridization (GISH). Nuclear ITS and plastidial trnL-trnF sequences were also obtained for most individuals. Proximal CMA bands were observed in the long arms of all acrocentrics of 2x and 4x N. macrostemon but not in diploid and some tetraploid cytotypes of N. nudicaule. Samples of N. gracile showed a variable number of CMA bands in the long arms of acrocentrics. Analysis of ITS sequences, dot-blot, GISH, and 5S and 45S rDNA sites, revealed no differentiation among the three species. The trnL-trnF cpDNA fragment showed variation with a trend to geographical structuring irrespective of morphospecies and fully congruent with karyotype variation. The 2n = 19 karyotype was probably formed by a centric fusion event occurring in N. nudicaule and later transmitted to tetraploid cytotypes of N. macrostemon. Diploids of N. nudicaule and N. macrostemon appeared as consistent recently diverged species, whereas tetraploid apomicts seem to constitute an assemblage of polyploid hybrids originating from multiple independent hybridization events between them, part of which are morphologically recognizable as N. gracile.

Cytogenetical and Molecular Characterization of Five Commercial Varieties in <i>Trichosanthes anguina</i> L.

Five commercial varieties of Trichosanthes anguina (Snake Gourd), namely Turag, Dhaka Green, Super Long Green, Anika and Apurbo, were investigated cytogenetically and at the molecular level for authentic characterization. The variety Super Long Green was found to possess 2n=23 chromosomes and considered as a primary trisomic. 2n=22 chromosomes were found in the other 4 varieties. The total length of the 2n chromosome complement was largest in Anika and smallest in Apurbo. Only one extra large chromosome was found in Turag and Anika suggesting it had either been transferred from different genome or was a case of repeated duplication. No decrease in chromosomal length was found in Turag, a moderate decrease was found in Dhaka Green and Anika, and a sharp decrease in Super Long Green and Apurbo. A pair of satellites was found in Turag. Only a stain specific satellite was found in Dhaka Green. No CMA band was found in Turag and Super Long Green. The other 3 varieties showed distinct CMA banding pattern in respect of number, size, location and percentage of GC-rich area. The 5 varieties showed distinct RAPD bands with 6 different primer combinations. In addition, the Apurbo and Turag varieties showed unique DNA fragments with the primer combinations OPA-3 and OPA-4 respectively. A combination of cluster and karyotytpe analysis clearly indicated that Turag and Apurbo were distinctly related from the other 3 varieties. Therefore, with the help of cytogenetical and RAPD analysis, it was possible to characterize each variety.

Karyotype characterization of two populations of Vernonia geminata (Asteraceae, Vernonieae) using banding and FISH techniques

In order to extend our knowledge concerning karyotypes of the genus Vernonia, we applied various techniques of chromosome banding, including AgNOR and triple staining with the fluorochromes CMA/DA/DAPI (CDD), and of fluorescent in situ hybridization (FISH) for the 45S rDNA probe to specimens of two populations of Vernonia geminata collected from an open-pasture area, in southern Brazil. B chromosomes were observed in one of the populations. Both populations of V. geminata presented a pair of CMA(3)(+) terminal bands and one pair of chromosomes with terminal AgNOR banding. The FISH evidenced, in one population, two pairs of small sites of 45S rDNA; these being two small terminal sites and two centromeric sites. In the other population, there was only one pair of small terminal sites and two sites in two B chromosomes, one in each chromosome. There was coincidence of localization between CMA(+) and NOR bands with one of the small terminal sites of 45S rDNA of one chromosome of the normal complement, but not in B chromosomes.