M. Ruffini Castiglione

Nuclear DNA contents, rDNAs, chromatin organization, and karyotype evolution in Vicia sect. Faba.

SummaryAutomated karyotype analyses, nuclear DNA contents, and sequences of rDNA internal transcribed spacers of the nine species inVicia sect.faba are reported. As karyomorphological parameters are used to evaluate the karyotype evolution, so the determination of the heterochromatin by Feulgen absorption at different thresholds of optical density provided further evidence on the chromatin organization withinVicia sect.faba. The comparison of sequences of rDNA spacers has enabled the definition of the phylogenetic relationships between the analyzed species.

Chromatin organisation and computer aided karyotyping of Triticum durum Desf. cv. Timilia

Abstract The tribe Triticeae includes three cereal genera Secale, Hordeum and Triticum and because of their economical and agronomical importance, the relationships among species on the tribe have been extensively investigated. The wild wheat relatives are an important source of genetic variation for cultivated species and wheat is an important crop of the mediterranean region. Banding pattern of metaphase chromosomes and nuclear DNA content in root meristematic cells of an old sicilian landrace “Timilia” were determined. Microdensitometric evaluation of nuclear absorption at different thresholds of optical density indicates the organization of chromatin in the interphase nuclei. Chromosome morphometric data, karyotype simmetry, the TF% values and Syi indices were determined. The results are compared with the data of other durum wheat varieties as Capeiti and Simeto.

DNA methylation patterns on plant chromosomes

Abstract DNA methylation patterns of chromosomes belonging to several plant species have been investigated. We have reported previous data obtained by means of different indirect immunocytochemical approaches with specific antibodies against 5-methyl cytosine. The processed results have been comparatively discussed with the aim to provide further insight into certain aspects of the structure and organization on the chromosome level of the plant genome.

Distribution of 5-methylcytosine-rich regions in the metaphase chromosomes of Vicia faba

The DNA methylation pattern ofVicia faba metaphase chromosomes was examined with a specific monoclonal antibody. 5-methylcytosine (5-mC) residues are present in different chromosomal sites, and are particularly abundant in telomeric and/or subtelomeric regions and in certain intercalary bands. Chromosomal localization of methylated regions enables a better knowledge of the lengthwise differentiation of this chromosome complement. Our results also indicate that there may be differences in monoclonal antibody binding between corresponding regions of homologous chromosomes inV. faba. This behaviour is detectable in specific regions with different frequencies. The data support results previously obtained forAllium cepa metaphase chromosomes using the same monoclonal antibody.

Chromosome Banding and DNA Methylation Patterns, Chromatin Organisation and Nuclear DNA Content in Zingeria biebersteiniana

Chromosome structure and chromatin organisation of a two-chromosome model cereal Zingeria biebersteiniana (Claus) P. Smirnov were studied: nuclear DNA content was determined by microdensitometric analysis after Feulgen staining; Feulgen absorption at different thresholds of absorbance in interphase nuclei also provided evidence on the organisation of chromatin, allowing quantitative estimation of condensed chromatin within interphasic nucleus. The DNA methylation pattern of Z. biebersteiniana metaphase chromosomes was examined with a specific monoclonal antibody. 5-methyl-cytosine residues are present in several chromosome sites and differences may be present between corresponding regions of homologues. Chromosome banding pattern reveals large bands in the centromeric regions of each chromosome, showing constitutive heterochromatin; by fluorochromes staining pericentromeric blocks are evidenced. After the cold and 9-aminoacridine pre-treatments and after aceto-carmine and aceto-orceine staining, respectively, the metaphase chromosomes were analysed by image analysis system revealing a segmentation of the chromosome body that resembles Giemsa/Reverse banding in animal chromosomes.

Cytological investigation of Haplopappus gracilis (Nutt.) Gray: 5-methylcytosine-rich regions, fluorochrome banding and chromatin sensitivity to DNase I digestion

Summary.Haplopappus gracilis (Nutt.) Gray, one of the five known higher plants with a chromosome number of 2n = 4, was studied from a cytological point of view. The chromosome complement of this species was characterized by means of automated karyotype analysis. Moreover, the DNA methylation pattern and fluorochrome banding were determined and compared with cytological data present in the literature. DNA methylation distribution along metaphase chromosomes involved all chromosome territories evidenced by C-banding. Other methylated bands correlated positively with aceto-orcein-positive heterochromatic portions and/or with late replicating bands and/or fluorochrome bands. Some methylated bands showed differences between homologous chromosomes. These bands belonged partly to certain heterochromatic domains and partly to intercalary sites not defined by other standard banding techniques. Differences between the homologues were also indicated by our DNA content data obtained after DNase I digestion.

Cytological and molecular characterization of Vicia esdraelonensis Warb. & Eig: a rare taxon

Summary.Vicia esdraelonensis, a rare taxon belonging to section Hypechusa of subgenus Vicia, was recovered and analyzed by cytological, karyological, and molecular methods, with the aim of both characterizing this species and furthering our knowledge of the phylogeny of subgenus Vicia. Automated karyotype analysis, nuclear DNA content, and chromatin organization were determined by the Feulgen reaction, as well as chromosome banding after double staining with 4′,6-diamidino-2-phenylindole (DAPI) and chromomycin A3. The chromosome number and the nuclear DNA content were in agreement with the values of the species of section Hypechusa. The GC- and AT-rich preferential sites were determined by chromomycin A3 and DAPI staining. Karyomorphological parameters indicated that V. esdraelonensis is in an intermediate position in the spatial representation of the species of section Hypechusa on the basis of symmetry indices, as well as in the dendrogram of linkage distance constructed on 37 chromosome parameters. Molecular data based on internal transcribed spacer sequences show that V. esdraelonensis can doubtlessly be included in section Hypechusa and document its closeness to V. noeana. A cladistic analysis combining the molecular data set with karyological characters is also reported. Karyological, cytological, and molecular data allow characterization of the V. esdraelonensis genome and provide information about the phylogenetic position of this species within the Hyrcanicae series of section Hypechusa.

Cytology of Vicia species. X. Karyotype evolution and phylogenetic implication in Vicia species of the sections Atossa, Microcarinae, Wiggersia and Vicia

Automated karyotype analyses and sequence of rDNA spacers have been analysed for the species belonging to sections Atossa, Microcarinae, Wiggersia and Vicia. Karyomorphological parameters, based on Rec, Syi and TF% indices, have been determined and evidenced that, in term of symmetry, the karyotype of Vicia lathyroides was the most asymmetric one. A multivariate analysis using 34 karyological parameters, in addition to the symmetry indices, has been carried out and the corresponding dendrogram of linkage distances showed six different groups. Molecular investigations on the inclusive group in study by employing ITS DNA sequences indicated a different pattern of relationships. The cladistic analysis combining the molecular data set with karyological parameters evidenced that the species of sections Vicia and Atossa join closely to each other in a paraphyletic group, which includes the monophyletic section Wiggersia. Therefore, our karyological and molecular data provide information about the phylogenetic position of the analysed species inside the subgenus Vicia and are discussed in relation to previous results obtained by morphology, isozymes and ribosomal genes analyses.

Cytology of Vicia species. VI. Nuclear chromatin organization, karyomorphological analysis and DNA amount in Vicia serratifolia Jacq.

SUMMARYKaryomorphological features of Vicia serratifolia Jacq. were studied: the nuclear DNA content was determined in meristematic root cells, karyotype and morphometric data were generated by an automated image analysis system and the distribution and staining properties of heterochromatin were assessed in metaphase chromosomes. Moreover, Feulgen absorption at different thresholds of optical density provided evidence on the organization of the chromatin in interphase nucleus. The collected data were compared with those previously gathered in V. faba used as internal standard and V. narbonensis considered the reference species of the Narbonensis complex.

DNA methylation and chromosomal rearrangements in reconstructed karyotypes of Hordeum vulgare L.

Summary.One standard and two reconstructed barley karyotypes were used to study the influence of chromosomal rearrangements on the distribution pattern of DNA methylation detectable at the chromosome level. Data obtained were also compared with Giemsa N-bands and high gene density regions that had been previously described. The effect of chromosomal reconstruction in barley seems to be decidedly prominent in the repositioning of genomic DNA methylation along metaphase chromosomes. In comparison to the standard karyotype, the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complements not subjected to structural alterations. Moreover, differences may occur between corresponding regions of homologues. Some specific chromosomal bands, including the nucleolus-organizing regions, showed a relative constancy in the methylation pattern, but this was not the case when the two satellites were combined by translocation in chromosome 6H5H of line T-30. Our results suggest that epigenetic changes like DNA methylation may play an important role in the overall genome reorganization following chromosome reconstruction.