Phosphorylation of histone H3S10 in animal chromosomes: is there a uniform pattern?

Phosphorylation of serine 10 in histone H3 (H3S10ph) has been extensively analyzed and appears to be a conserved chromatin change associated with chromosome condensation in different eukaryotic organisms. In this work, we report the distribution of H3S10ph during meiosis in monocentric and holokinetic chromosomes of 6 insect species and in mitotic chromosomes of 7 mammalian species, aiming to investigate the labeling patterns in phylogenetically distant groups. The results indicated a very similar phosphorylation timing and distribution pattern among insects. The sex chromosomes of insects analyzed were always undercondensed and hypophosphorylated. Similarly, the micro chromosomes of the bug Pachylis aff pharaonis were also undercondensed and hypophosphorylated. Holokinetic chromosomes of bugs and monocentric chromosomes of grasshoppers and beetles displayed identical phosphorylation pattern in spite of the difference in the centromere type. Among mammals, a uniform chromosome phosphorylation was observed in marsupials, whereas bat chromosomes displayed a longitudinal banding pattern. These data indicate that, in general, the intensity of H3S10 phosphorylation in animal chromosomes is variable among the distinct chromosome types and associated with the degree of chromatin condensation at metaphase, but it may vary between different groups of animals.     

The relationships among lemons, limes and citron: a chromosomal comparison

Lemons, limes and citron constitute a group of closely related Citrus species, whose species delimitations and taxonomic relationships are unclear. In order to identify karyotypic similarities and species relationships within this group, the CMA+/DAPI- banding pattern and the distribution of the 5S and 45S rDNA sites of 10 accessions of lime, lemon, and citron were investigated. The four cultivars of C. limon analyzed showed the same pattern of CMA+ bands and rDNA sites, suggesting that they originated from a single germplasm, later differentiated by distinct somatic mutations. The lemons C. jambhiri, C. limonia and C. volkameriana displayed karyotypes very similar to each other, but they differed from C. limon by the absence of a single chromosome with one band in each telomere. The limes, C. aurantifolia and C. limettioides, seemed less related to each other and exhibited different heteromorphic chromosome pairs. In C. aurantifolia, the presence of a chromosome type unknown in all other Citrus species cytologically known so far supports the assumption that this accession may be derived from a hybrid with a species from the subgenus Papeda or from another genus. Citrus medica was the only homozygous accession of this group and all of its chromosome types were clearly represented in limes and lemons, some of them forming heteromorphic pairs. The analysis of the distribution of rDNA sites allowed a further refinement of the comparison among accessions. The lemons and limes were heterozygous for all rDNA sites, whereas C. medica was entirely homozygous. These data support the hypothesis that C. medica is a true species while the other nine accessions are hybrids.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Mitotic instability in wheat×Thinopyrum ponticum derivatives revealed by chromosome counting, nuclear DNA content and histone H3 phosphorylation pattern

To evaluate the mitotic stability of Triticum aestivum×Thinopyrum ponticum derivatives (BC₂F₇ and BC₂F₅ doubled haploids), chromosome counting by both conventional and immunostaining techniques, and measurement of DNA content were performed. The wheat progenitor line, PF 839197, the wheat recurrent parent CEP 19 and the control Chinese Spring were also investigated. In the hybrid derivatives, chromosome number ranged from 2n=36 to 60, with a predominance of chromosome numbers higher than 2n=42, that was confirmed by determination of nuclear DNA content. Chinese Spring and PF 839197 were stable, but CEP 19 showed chromosome number variation (20%). Analyses of non-pretreated cells revealed the presence of anaphase bridges, lagging chromatids, chromosome fragments and micronuclei. Immunostaining with an antibody recognizing histone H3 phosphorylated showed dicentric chromatids forming anaphase bridges and pericentromeric phosphorylation at centric chromosome fragments but not at lagging chromatids. The possible causes of the observed mitotic instability are discussed.     

Intra- and interspecific chromosome polymorphisms in cultivated Cichorium L. species (Asteraceae)

Endive (Cichorium endivia L.) and chicory (C. intybus L.) both have 2n = 18, but until now, there has been no detailed karyomorphological characterization. The present work evaluated five accessions of each species using FISH with rDNA probes and fluorochrome staining with CMA and DAPI. Both species presented distinct banding patterns after fluorochrome staining: while endive had proximal CMA⁺⁺/DAPI⁻ bands in the short arms of pairs 1, 2 and 3, chicory had proximal CMA-positive bands in chromosomes 1 and 3 and interstitial in the short arm of chromosome 8. Among endive accessions, FISH procedures revealed conserved position and number of 5S and 45S rDNA sites (two and three pairs, respectively), associated with the CMA-positive bands. Notwithstanding, polymorphisms were detected within chicory accessions regarding the number and the distribution of rDNA sites in relation to the most frequent karyotype (two pairs with 45S and one with 5S rDNA). The karyological markers developed allowed karyotypic differentiation between both species, uncovering peculiarities in the number and position of rDNA sites, which suggest chromosome rearrangements, such as translocations in chicory cultivars. The interspecific and intraspecific polymorphisms observed emphasize the potential of karyomorphological evaluations, helping our understanding of the relationships and evolution of the group.     

Chromosomal markers distinguish hybrids and non-hybrid accessions of mandarin

Mandarin is the common name of a heterogeneous group of Citrus species with a large range of variation in morphological and molecular characters as well as in number of species. Aiming to identify chromosome markers and to clarify the relationship within this group, the karyotype of 13 mandarin accessions were analyzed using CMA/DAPI staining and in situ hybridization with 5S and 45S rDNA probes. The CMA band pattern together with the position of rDNA sites revealed that mandarins can be separated karyologically into three groups: a) C. sunki and C. reshni; b) the Mediterranean mandarin, C. deliciosa, and the closely related C. tangerina cv. Dancy and C. reticulata cv. Cravo; c) the remaining cultivars, which are cytologically heterozygous and most probably interspecific hybrids. The former two groups are assumed to be pure species together with C. medica and C. grandis. A chromosome marker for mandarin species was identified and the relationship among the pure species and some hybrids is discussed.     

Mitotic Microtubule Development and Histone H3 Phosphorylation in the Holocentric Chromosomes of Rhynchospora Tenuis (Cyperaceae)

In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.     

BAC- and oligo-FISH mapping reveals chromosome evolution among Vigna angularis,V. unguiculata,and Phaseolus vulgaris

Chromosoma - Cytogenomic resources have accelerated synteny and chromosome evolution studies in plant species, including legumes. Here, we established the first cytogenetic map of V. angularis (Va,...     

Genotoxic potential of the latex from cotton-leaf physicnut (Jatropha gossypiifolia L.)

Jatropha gossypiifolia L. (Euphorbiaceae), popularly known as cotton-leaf physicnut, is a milky shrub notable for its medicinal properties. The present study aimed to evaluate the toxic, cytotoxic and genotoxic effects of the latex of J. gossypiifolia, using Allium cepa L. as test system. Seeds of A. cepa were exposed to five concentrations of the latex (1.25; 2.5; 5; 10 and 20 mL/L) in order to evaluate parameters of toxicity (evaluation of root growth), cytotoxicity (mitotic index frequency) and genotoxicity (frequency of chromosome alterations). The latex showed a significant decrease in root mean growth value as well as mitotic index for the tested concentrations, except for 1.25 mL/L, when compared to results from the negative control. The 1.25, 2.5 and 5 mL/L concentrations induced significant chromo-some adherences, C-metaphases and/or chromosome bridges, as genotoxic effects. The significant frequency of chromosome bridges also indicated mutagenic potential for chromosomes of J. gossypiifolia as discussed in the paper. Considering that the latex is used in popular therapies, and that the test system A. cepa presents good correlation with tests carried out in mammals, it can be pointed out that its use for medicinal purposes may be harmful to human health especially if ingested.     

Chromatin differentiation between Vigna radiata (L.) R. Wilczek and V. unguiculata (L.) Walp. (Fabaceae)

A comparative chromosomal evaluation was carried out between Vigna unguiculata (cowpea) and V. radiata (mung bean) with chromomycin A₃ (CMA₃)/4’,6-diamidino-2-phenylindole (DAPI) banding and fluorescent in situ hybridization (FISH) using 5S/45S ribosomal DNA (rDNA) probes. Both species had symmetric karyotypes (2n = 22), with prevalence of centromeres in chromosomes at median (m) and submedian (sm) regions and chromosomes ranging in size from 2.1 to 1.25 μm (V. unguiculata) and 2.18 to 0.93 μm (V. radiata). Three different banding patterns were identified for V. unguiculata: CMA₃⁺/DAPI⁰, CMA₃⁺⁺/DAPI^–, and CMA₃⁺/DAPI^–. The CMA₃⁺/DAPI⁰ bands were observed in the pericentromeric regions of all chromosomes, while the CMA₃⁺⁺/DAPI^– and CMA₃⁺/DAPI^– bands were co-localized with the 45S rDNA in the subtelomeric position (chromosomes B, G, and D, J, respectively) and in the proximal position in chromosome F. Two pairs of chromosomes (D and I) bearing interstitial 5S rDNA have been also identified. Vigna radiata displayed CMA₃⁰/DAPI⁺ bands distributed in the centromeric region of chromosomes B, C, and F, while CMA₃⁺⁺/DAPI^– bands were co-localized with the 45S rDNA sites in the subtelomeric position of the short arm in the F and K chromosome pairs. Three pairs of 5S rDNA sites were identified, the first in the proximal region of the long arm in chromosome E and the two others in the proximal and subterminal positions in the long arm of chromosome J. These data highlight some divergences regarding the amount and composition of the heterochromatin in both species, allowing the identification of individual chromosomes in V. unguiculata and V. radiata, and a comparison with other members of the Phaseoloid clade.