Jéferson Nunes Fregonezi

Bio: Jéferson Nunes Fregonezi is an academic researcher from Universidade Estadual de Londrina. The author has contributed to research in topics: Cestrum & Karyotype. The author has an hindex of 5, co-authored 5 publications receiving 121 citations.

Karyotype differentiation of four Cestrum species (Solanaceae) based on the physical mapping of repetitive DNA

Abstract: We studied the karyotypes of four Brazilian Cestrum species (C. amictum, C. intermedium, C. sendtnerianum and C. strigilatum) using conventional Feulgen staining, C-Giemsa and C-CMA3/DAPI banding, induction of cold-sensitive regions (CSRs) and fluorescent in situ hybridization (FISH) with rDNA probes. We found that the karyotypes of all four species was 2n = 2x = 16, with, except for the eighth acrocentric pair, a predominance of meta- and submetacentric chromosomes and various heterochromatin classes. Heterochromatic types previously unreported in Cestrum as neutral C-CMA30/DAPI0 bands, CMA3+ bands not associated with NORs, and C-Giemsa/CSR/DAPI- bands were found. The heterochromatic blocks varied in size, number, position and composition. The 45S rDNA probe preferentially located in the terminal and subterminal regions of some chromosomes, while 5S rDNA appeared close to the centromere of the long arm of pair 8. These results suggest that karyotype differentiation can occur mainly due to changes in repetitive DNA, with little modification in the general composition of the conventionally stained karyotype.

Chromosomal organization and phylogenetic relationships in Hypochaeris species (Asteraceae) from Brazil

Abstract: The association of cytogenetic and molecular techniques has contributed to the analysis of chromosome organization and phylogeny in plants. The fluorochrome GC-specific CMA 3 , fluorescent in situ hybridization (FISH) and RAPD (Random Amplified Polymorphic DNA) markers were used to investigate chromosome structure and genetic relationships in Hypochaeris (Asteraceae). Seven species native to South America, and two species introduced from Europe (H. glabra and Hypochaeris sp) were studied. FISH with rDNA probes identified one or two loci of 18S-5.8S-25S rDNA in the South American Hypochaeris species and one locus in the European species. Only one 5S rDNA locus was seen in all species studied. Blocks of GC-rich heterochromatin (CMA-positive bands) associated to 18S-5.8S-25SrDNA loci were detected in all species investigated. Co-location of 5S rDNA and CMA bands was also observed, except for three South American species and Hypochaeris sp. In two South American species, additional CMA bands not related to rDNA were observed on the long arm of chromosome 2, near to the centromere. Hypochaeris glabra exhibited additional CMA-positive signals distributed at pericentromeric regions, on the short arms of all chromosomes. A total of 122 RAPD markers were used to determine the genetic relationships among species. The level of polymorphism was very high, revealing two genetic groups comprising the South American and the European species, thus supporting a previous hypothesis of monophyly of the South American Hypochaeris species. The coefficients of genetic similarity between European and South American species were 0.35, on average. Polymorphism was also high within the two groups. The genetic associations observed with RAPD markers were consistent with chromosome characteristics. Species carrying similar distribution of 45S rDNA loci and CMA-positive signals were included in the same group revealed by RAPDs. Cytogenetic and molecular data support the view that not only chromosome rearrangements, but also changes in DNA sequence took place during the diversification of the South American Hypochaeris species.

The occurrence of different Bs in Cestrum intermedium and C. strigilatum (Solanaceae) evidenced by chromosome banding

Abstract: In this study, we examine the morphology, mitotic stability, meiotic behavior and the composition of heterochromatin of B chromosomes in Cestrum intermedium and C. strigilatum. The results showed that B chromosome number shows intraindividual variation in the root meristem, which seems to lead to a slight rate of B elimination in this somatic tissue. B chromosomes in both species were similar in size and shape, but differed with regard to the type, size and distribution of heterochromatin. Possible evolutionary pathways for B chromosome origin in Cestrum are discussed.

A karyotypic study of three southern Brazilian Asteraceae species using fluorescence in situ hybridization with a 45S rDNA probe and C-CMA3 banding

Abstract: The Asteraceae, one of the largest families of flowering plants, contains about 1,100 genera and 20,000 species, and is well known for its extensive karyotypic variation. In this study, conventional Feulgen staining, C-CMA3 banding, and fluorescence in situ hybridization with a 45S rDNA probe were used to determine the chromosome number and the number and physical position of GC-rich heterochromatin and 45S rDNA sites in three Asteraceae weed species (Crepis japonica, Galinsoga parviflora and Chaptalia nutans). The three species exhibited karyotype differences in the chromosome number and shape, as a commom feature of Asteraceae. However, the 45S rDNA sites always occurred on the short chromosomal arms, associated with GC-heterochromatin. Althought of these differences, it suggests that commom features of plant karyotype are maintained.

Distribution of a Ty3/gypsy-like retroelement on the A and B-chromosomes of Cestrum strigilatum Ruiz & Pav. and Cestrum intermedium Sendtn. (Solanaceae)

Abstract: Retroelements are a diversified fraction of eukaryotic genomes, with the Ty1/copia and Ty3/gypsy groups being very common in a large number of plant genomes. We isolated an internal segment of the Ty3/gypsy retroelement of Cestrum strigilatum (Solanaceae) using PCR amplification with degenerate primers for a conserved region of reverse transcriptase. The isolated segment (pCs12) was sequenced and showed similarity with Ty3/gypsy retroelements of monocotyledons and dicotyledons. This segment was used as probe in chromosomes of C. strigilatum and Cestrum intermedium. Diffuse hybridization signals were observed along the chromosomes and more accentuated terminal signals in some chromosome pairs, always associated with nucleolus organizer regions (NORs). The physical relationship between the hybridization sites of pCs12 and pTa71 ribosomal probes was assessed after sequential fluorescence in situ hybridization (FISH). Hybridization signals were also detected in the B chromosomes of these species, indicating an entail among the chromosomes of A complement and B-chromosomes.

The meaning of DAPI bands observed after C-banding and FISH procedures

Abstract: Under specific technical conditions chromosome staining with 4',6-diamidino-2-phenylindole (DAPI) permits characterization of heterochromatic regions as AT-rich (DAPI(+)) or AT-poor (DAPI(-)), especially when the chromosomes are counterstained with chromomycin A(3) (CMA), which preferentially binds to GC-rich DNA. DAPI(+) bands also often have been observed after C-banding or FISH. In these cases, however, it is not clear whether only AT-rich regions stain positively with DAPI or other heterochromatins with different base compositions also are stained. We evaluated the meaning of DAPI bands observed after C-banding and FISH using three plant species bearing different types of heterochromatin: DAPI(+)/CMA(-), DAP(-)/CMA(+) and DAPI(0)/CMA(0) (neutral bands). Additional tests were performed using propidium iodide, a fluorochrome without preferential affinity for AT or GC. Our results indicate that AT-rich heterochromatin stains as DAPI(+) bands after C-banding or FISH, but other kinds of heterochromatin also may be stained by DAPI.

Propidium iodide for making heterochromatin more evident in the C-banding technique.

Abstract: The detection of regions of heterochromatin has been the subject of intense investigation. We investigated an adaptation of the commonly used technique by replacing the nonfluorescent dye, Giemsa, by a fluorescent one, propidium iodide. This adaptation produces greater contrast of the heterochromatic bands in metaphase chromosomes and can be especially valuable when the organisms studied possess heterochromatin that is pale and difficult to visualize. We discuss the interactions of these two dyes with DNA and the excitation of the fluorescent dye when irradiated with ultraviolet light.

Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae)

Abstract: We investigated four orchids of the genus Maxillaria (M. discolor, M. acicularis, M. notylioglossa and M. desvauxiana) in regard to the position of heterochromatin blocks as revealed using chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI) fluorochrome staining and 5S and 45S rDNA sites using fluorescence in situ hybridization (FISH). The species showed differences in chromosome number and a diversified pattern of CMA+ and DAPI+ bands, including heteromorphism for CMA+ bands. The 5S and 45S rDNA sites also varied in number and most of them were co-localized with CMA+ bands. The relationship between 5S rDNA sites and CMA+ bands was more evident in M. notylioglossa, in which the brighter CMA+ bands were associated with large 5S rDNA sites. However, not all 5S and 45S rDNA sites were co-localized with CMA+ bands, probably due to technical constraints. We compare these results to banding data from other species and suggest that not all blocks of tandemly repetitive sequences, such as 5S rDNA sites, can be observed as heterochromatin blocks.