Genetic characterization of Paspalum notatum accessions by AFLP markers

Paspalum notatum Flügge is a warm-season forage grass with sexual diploid and apomictic tetraploid races. Genetic improvement was achieved in out-breeding diploids. The acquisition of artificial sexual tetraploids has raised the possibility of performing crosses and plant improvement at the tetraploid level. The objective of our study was to obtain a genetic and cytoembryological characterization of a germplasm collection of P. notatum, including 31 accessions from seven countries of America and 11 experimentally obtained genotypes. Morphology of mature gametophytes was observed to assess the mode of reproduction of the accessions. A total of 1342 AFLP fragments were generated across the 42 genotypes and from two reference taxa: P. urvillei and P. procurrens. AFLP data were converted into a binary matrix and similarity relationships were established. The genetic distance among all the accessions showed a maximum value of 0.36. In addition, eleven AFLP fragments were observed exclusively in apomictic plants, which could be linked to genomic regions implicated in the control of apospory.     

Cytogenetic studies on Brazilian Xyris species (Xyridaceae)

Cytological features are presented for the first time for 12 species and one natural hybrid from 16 Brazilian populations of the genus Xyris , including members of two sections ( Xyris and Nematopus ). The results are compared with a review of all available chromosome numbers previously reported for the genus. All analysed species show semi-reticulate interphase nuclei and prophase to prometaphase chromosomes with heterogeneous condensing behaviour. Chromosome numbers varied between 2 n = 26 and 2 n ≈ 108. In Brazil especially, the karyological differentiation of Xyris seems to be generally characterized by dysploid and polyploid increase of the chromosome number, possibly after interspecific hybridization. This is the first cytological study including species of the section Nematopus and the first karyological investigation including South American species of the genus Xyris . © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 138 , 245–252.     

Cytogenetic characterization reveals differences in nuclear organization of cystic cells among Brazilian species of Triatoma (Heteroptera,Reduviidae)

Cytogenetic studies in triatomines have described the occurrence of holokinetic chromosomes, heterochromatin distribution and the location of rDNA (ribosomal DNA) sites, but few aspects of nuclear organization in this group have been discussed. We have focused on ultrastructural and cytogenetic features and differences in cystic cells of seminiferous tubules between five species of Triatoma. Cystic cells showed evidence of polyploidy events and heterochromatic blocks appeared predominantly in the central region of the nuclei. Cytogenetic analyses showed that there was variation in chromocenter number between species, and that the central regions were AT‐rich [DAPI⁺ (4′,6‐diamidino‐2‐phenylindole⁺)], whereas the periphery was CG‐rich (CMA⁺). Another characteristic was the distribution of 45S rDNA, which differed according to the chromosomal location of this sequence. In all we have compared aspects of nuclear organization, polyploidy, heterochromatin, rDNA site distribution and methylation levels, as well as the relationships between five species of Triatoma from a cystic cell perspective.     

Cytogenetic analysis of some Brazilian marsupials (Didelphidae: Marsupialia)

Three species of marsupials from the Amazon region (Marmosa cinerea, Caluromys lanatus, and Didelphis marsupialis) and two from the region of S?o Paulo (Didelphis marsupialis and Didelphis albiventris) were studied. The G-banding pattern of the species with 2n = 14 (M. cinerea and C. lanatus) was very similar, as well as the pattern of G-bands in the species with 22 chromosomes (Didelphis). All of the autosomes of M. cinerea and D. albiventris have centromeric C-bands and the Y chromosome is totally C-band positive. The long arm of the M. cinerea X chromosome is completely C-band positive except for a negative band close to the centromeric region. In D. albiventris the long arm of the X chromosome is C-band positive except for a negative band close to the telomeric region. In M. cinerea the silver-stained nucleolar organizer regions (Ag-NORs) are found in the acrocentric chromosomes, being located in the telomeric region of one pair and in the centromeric region of the other pair. Caluromys lanatus has centromeric Ag-NORs in one acrocentric and in one submetacentric chromosome pairs. Didelphis marsupialis has three chromosome pairs with telomeric Ag-NORs. In D. albiventris the Ag-NORs are terminal and located in both arms of one pair and in the long arm of two pairs of chromosomes.     

Cytogenetic characterization of radiosensitive mouse mutants.

In order to develop mouse models for human mutagen-sensitive syndromes, we carried out cytogenetic characterization of several mouse mutants and MS/Ae mice showing enhanced radiosensitivities. The applied cytogenetic techniques include chromosomal analysis of in vitro cell cultures and lymphocyte cultures as well as in vivo UDS in hepatocytes, induction of micronuclei in polychromatic erythrocytes and translocation induction in spermatogonial stem cells. Among the mutations studied, namely the contrasted allele of steel (Slcon), viable dominant spotting (Wc), wasted (wst), varitint-waddler (Va) and dystonia musculorum (dt) as well as MS/Ae mice, various iso-, hyper- or hypo-sensitive conditions were recorded. Only Va and dt appear to be associated with some deficiency in DNA repair.     

Cytogenetic characterization of Chinese hamster-sheep somatic cell hybrids

The methods used to characterize cytogenetically Chinese hamster x sheep somatic cell hybrids have been reported. G and C banding patterns on hybrid metaphases allowed the discrimination between hamster and sheep chromosomes, and in addition to establish the unidirectional loss of sheep chromosomes in hybrid cells.     

Cytogenetic characterization of endemics: past and future

Abstract

Following an overview of the classification scheme for endemic species, the authors propose to complement it based on modern molecular cytogenetic techniques. Molecular cytogenetics provides new possibilities in the study of chromosomal evolution and genome organization, and contributes to a better characterization of the karyotype of endemic species. Through several examples, the authors demonstrate the advantages of molecular cytogenetic techniques in resolving relationships between endemic and related species, leading to a more precise categorisation, especially in the absence of polyploidy. The authors recommend some caution in the interpretation of cytogenetic data alone, and stress the importance of comparing cytogenetic results with those obtained from independent sources (i.e., molecular phylogenetics, phytogeography, ecology). Finally, the current classification of endemic species is revised in the light of molecular cytogenetics and other newly available evidence.     

Cytogenetic studies in Brazilian marine Sciaenidae and Sparidae fishes (Perciformes)

Fishes from the families Sciaenidae and Sparidae, the former comprising coastal species associated with shallow waters on the continental shelf and the latter composed of typically marine species, are of significant economic value. Karyotypic data are available for about 20% of the total number of species in these groups. In the present study, cytogenetic analyses were carried out in three Sciaenidae species, Menticirrhus americanus, Ophioscion punctatissimus and Pareques acuminatus, as well as in the sparid fish, Archosargus probatocephalus, using conventional staining (Giemsa) and Ag-nucleolar organizer regions (NORs) and C-banding techniques. The diploid values (2n) and number of chromosome arms were equal to 48 in all species analyzed. NORs were located at pericentromeric positions, equivalent to large heterochromatic blocks, in M. americanus (1st pair), O. punctatissimus (10th pair), P. acuminatus (2nd pair), and A. probatocephalus (3rd pair). Heterochromatin was detected at the centromeric position in most chromosome pairs, being more conspicuous among Scianidae members. The remarkable karyotypic conservativeness detected in these species is similar to that observed in other perciform groups previously studied, regarding both the number of acrocentric chromosomes and NOR location. However, unusual events of heterochromatinization seem to have taken place along the karyotypic evolution of members of the family Sciaenidae. For the family Sparidae, distinct cytotypes between samples of Northeast Brazil and those previously analyzed on the southeastern coast were identified, suggesting that putative biogeographic barriers could be present throughout both regions on South Atlantic coast.     

Cytogenetic damage in the buccal epithelium of Brazilian aviators occupationally exposed to agrochemicals

The frequency of micronuclei in both buccal cells and peripheral blood lymphocytes is extensively used as a biomarker of chromosomal damage and genome stability in human populations. We examined whether prolonged exposure to complex mixtures of pesticides leads to an increase in cytogenetic damage. The exposed group comprised 50 agricultural aviators, mainly from Central and Southeast regions of Brazil, who had inhaled agrochemicals for more than 10 years without personal protection equipment; the control group consisted of 17 men from the same regions, without indication of exposure to pesticides, There were three times higher frequencies of micronuclei (P < 0.05) and 2.5 times higher frequencies of binucleated cells in the aviators when compared to controls. However, cytotoxic alterations such as broken eggs and karyorrhexis did not present statistically significant differences between the exposed and control groups. Therefore, diverse agrochemicals used to combat pests in agriculture possess genotoxic effects in the oral mucosa of the agricultural pilots, as showed in this study.     

Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions1

In the last few years, genome‐based studies in diatoms have received a major boost following the genome sequencing of the centric species Thalassiosira pseudonana Hasle et Heimdal and the pleiomorphic raphid pennate diatom Phaeodactylum tricornutum Bohlin. In addition, molecular tools, such as genetic transformation, have been developed for both species. Despite these molecular advances, relatively little is known regarding the genetic diversity of the available strains of these diatoms. In this study, we have compiled a historical summary of the known P. tricornutum species resources and have provided a genetic and phenotypic overview of 10 different axenic strains. Examination of intraspecies genetic diversity based on internal transcribed spacer 2 (ITS2) sequence and amplified fragment length polymorphism (AFLP) analyses indicate four different genotypes. Seven strains are predominantly fusiform, whereas one strain is predominantly oval, and another is predominantly triradiate. Another is defined as a tropical strain because it appears better acclimated to growth at higher temperatures. Observations in the natural environment indicate that P. tricornutum is a coastal marine diatom that is able to adapt to unstable environments, such as estuaries and rock pools. Because it has rarely been noted in nature, we have developed specific primers to amplify ITS2 sequences and have successfully identified it in environmental samples. These resources should become useful tools for the diatom community when combined with the whole genome sequence and will open up a range of new possibilities for experimental investigations that can exploit the genotypic and phenotypic characteristics described.     

Antigenic characterization of Brazilian isolates of Anaplasma marginale

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.     

Cytogenetic characterization of three Hypericum species by in situ hybridization

The chromosomal positions of the 5S/25S rRNA genes of Hypericum perforatum (2n=32), H. maculatum (2n=16) and H. attenuatum (2n=32) were comparatively determined by FISH, and six, three and seven chromosome pairs of the respective karyotypes were subsequently distinguished. The rDNA loci between H. perforatum and H. maculatum seem to be identical (with respect to the ploidy difference), indicating that H. perforatum probably arose by autotetraploidization from an ancestor closely related to H. maculatum. The positional differences between the 5S rRNA gene loci of H. perforatum and H. maculatum on the one hand and H. attenuatum on the other argue against a previous hypothesis according to which H. perforatum originated from a remote interspecific hybridization between H. maculatum and H. attenuatum. Received: 7 October 1999 / Accepted: 23 November 1999     

Cytogenetic characterization of HB2 epithelial cells from the human breast

HB2 is a cell line originated by subcloning of MTSV1-7 mammary luminal epithelial cells isolated from human milk and immortalization via introduction of the gene encoding simian virus 40 (SV40) large T antigen. Despite its wide utilization as non-neoplastic counterpart in assays aimed to elucidating various biochemical and genetical aspects of normal and tumoral breast cells, to our knowledge no literature data have so far appeared concerning the chromosomal characterization of the HB2 cells. Here, we report the cytogenetic characterization of the karyotype of HB2 cells, which puts in evidence the occurrence of changes in chromosomal number and structure and the presence of unidentified chromosomal markers in variable amount. Our results do not detract from the utility of HB2 cells in illustrating fundamental aspects of breast cell biology, but rather interject a note of caution into generalizing results obtained with this cell line to other non-immortalized epithelial cell populations from the human breast. Therefore, this work represents a useful resource for all who want to perform appropriate and focused future studies on this cell line and proposes precise indications for a knowledgeable use of HB2 cells.     

Cytogenetic and molecular characterization of heterochromatin gene models in Drosophila melanogaster

In the past decade, genome-sequencing projects have yielded a great amount of information on DNA sequences in several organisms. The release of the Drosophila melanogaster heterochromatin sequence by the Drosophila Heterochromatin Genome Project (DHGP) has greatly facilitated studies of mapping, molecular organization, and function of genes located in pericentromeric heterochromatin. Surprisingly, genome annotation has predicted at least 450 heterochromatic gene models, a figure 10-fold above that defined by genetic analysis. To gain further insight into the locations and functions of D. melanogaster heterochromatic genes and genome organization, we have FISH mapped 41 gene models relative to the stained bands of mitotic chromosomes and the proximal divisions of polytene chromosomes. These genes are contained in eight large scaffolds, which together account for approximately 1.4 Mb of heterochromatic DNA sequence. Moreover, developmental Northern analysis showed that the expression of 15 heterochromatic gene models tested is similar to that of the vital heterochromatic gene Nipped-A, in that it is not limited to specific stages, but is present throughout all development, despite its location in a supposedly "silent" region of the genome. This result is consistent with the idea that genes resident in heterochromatin can encode essential functions.     

Cytogenetic markers for the characterization of Capsicum annuum L. cultivars

Karyotypic studies with conventional staining have been unsuccessful due to the uniformity of Capsicum chromosomes. In this study, we found diagnostic chromosome characters that permit to characterize cultivars; this is the first cytological characterization of both rDNAs (18S and 5S) in a species of Capsicum using a genus-specific probe and the most exhaustive in C. annuum to date. The heterochromatic banding patterns enabled us to identify cultivars, and fluorescent in situ hybridization (FISH) showed one 5S rDNA locus largely conserved within the cultivars, whereas high variation in the number of 18S rDNA loci was observed. One of the most obvious differences is the presence of an additional active nucleolar organizer region in pair #12 and the dispersal of inactive 18S rDNA signals. These results indicate that fluorochrome banding together with silver impregnation and FISH procedures are very useful for the identification of chromosomes and the interpretation of chromosomal variation between cultivars. The functional role of this variation is still uncertain, but our results show that copy number variation of repetitive DNA during the course of evolution might provide an excellent experimental system for studying genome rearrangements accompanying functional divergence in domesticated C. annuum.     

Cytogenetic characterization of the ionizing radiation-sensitive Chinese hamster mutant irs1

The X-ray-sensitive mutant V79 cell line irs1 was characterized with respect to chromosomal aberrations induced by 137Cs, mitomycin C (MMC), and decarbamoyl mitomycin C (DCMMC). To measure chromosome damage induced at different cell cycle stages, irs1 and the parental V79-4 cell lines were pulse-labeled with bromodeoxyuridine (BrdUrd) at the time of exposure and harvested at various intervals corresponding to exposure in G1, S, and G2 phases of the cell cycle. Metaphase spreads were stained with an anti-BrdUrd antibody, followed by a fluorescein-conjugated second antibody. With propidium iodide as a counter stain, cells were scored for aberrations. Compared to the parental V79 cells, irs1 cells had: (1) greatly increased sensitivity to all 3 agents; (2) a high frequency of chromatid exchanges after exposure in each phase of the cell cycle; and (3) more sensitivity to the agent causing crosslinks (MMC) than its monofunctional analog (DCMMC). The finding of chromatid-type damage in cells exposed to ionizing radiation during G1 is atypical of normal cells, but is similar to observations made in several mutant rodent cell lines and in ataxia telangiectasia cells. Our results suggest that the defect in irs1 cells can manifest itself as misrepair or misreplication during all phases of the cell cycle and leads to a high incidence of chromatid exchanges and deletions.     

2n+n Hybridization of Apomictic Paspalum dilatatum with Diploid Paspalum Species

Common dallisgrass (Paspalum dilatatum) is an apomictic pentaploid (2n=5x=50) of hybrid origin with irregular meiosis and with the genome formula IIJJX. The I and J genomes are homologous to those of diploid P. intermedium and P. jurgensii, respectively, but the source of the X genome is unknown. Members of the X genome may have genes of special biological significance, including those controlling apomixis. Common dallisgrass was crossed with several diploid Paspalum species in an attempt to identify the source of the X genome. Since common dallisgrass is apomictic, all hybrids produced will be formed by fertilization of an unreduced egg (2n+n). Any hybrid showing 30 chromosome bivalents at meiosis would indicate that the male diploid parent has a chromosome set that is homologous to the X genome of dallisgrass. Over 36,000 spikelets of dallisgrass were emasculated and dusted with pollen of 15 different diploid species (diploid species bearing I or J genomes were excluded). Only five (P. chaseanum, P. equitans, P. fasciculatum, P. notatum, and P. simplex) produced 2n+n hybrids with P. dilatatum. Meiotic chromosome behavior was similar in all hexaploid hybrids showing ca. 20 bivalents and 20 univalents. Results indicated a very low rate of 2n+n hybridization; none of the five diploid species possessed the X genome. Because several diploid species failed to hybridize with 5x dallisgrass, other methods should be attempted. Molecular markers specific for the X genome may help solve the question.     

Isolation and characterization of actinomycetes from Brazilian tropical soils

Actinomycetes have been isolated from three Brazilian tropical soils. The dispersion and differential centrifugation procedure revealed count values 1.5 to 5.0 times greater than those obtained by the conventional dilution plate technique for all soils and media tested. Eighteen strains, promising for biotechnological applications, were submitted to chemotaxonomic procedures and numerical taxonomy for identification. Two were identified as Amycolatopsis orientalis, one as Streptomyces misakiensis, and two tentatively included or associated to S. chromofuscus and S. griseoruber. The others, all belonging to the Streptomyces genus, could not be fitted into any known species, and were arranged by the UPGMA analysis for classification, as an isolated group. This suggests that the actinomycetes in tropical soils may represent a vast unexplored resource for biotechnology.