Artigos
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11845
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Item Nuclear DNA content and karyotype of Rosewood (Aniba rosaeodora)(Genetics and Molecular Biology, 2005-10) Contim, Luis Antônio Serrão; Carvalho, Carlos Roberto de; Martins, Franciele Alline; Freitas, Danival Vieira deRosewood (Aniba rosaeodora Ducke, Lauraceae) is ecologically and economically important to the Amazon region. As a consequence of its economic importance, rosewood populations have been decimated in the Amazon forest. Species of nine genera of the Lauraceae family have characterized karyotypes with n = x = 12 chromosomes in the gametophytic phase but the genus Aniba is one of the least studied Lauraceae genera with a previously undescribed genome. We used cytogenetic techniques to determine that the A. rosaeodora karyotype contained 12 pairs (2n = 24) of relatively small submetacentric chromosomes with lengths ranging from 1.34 to 2.25 mm and a nucleolar organizer region (NOR) in the short arm of chromosome 7. Flow cytometry gave 2C = 2.32 pg of DNA, equivalent to approximately 2.24 x 109 base pairs.Item Genome size diversity in stingless bees (Hymenoptera: Apidae, Meliponini)(Apidologie, 2012-11) Tavares, Mara Garcia; Carvalho, Carlos Roberto; Soares, Fernanda Aparecida Ferrari; Campos, Lucio Antonio de OliveiraThe first studies on the genome size of stingless bee species showed a range from 0.27 pg (Melipona subnitida and Melipona quadrifasciata) to 1.38 pg (Melipona capixaba). Considering this variation, we quantified the DNA content of 26 species of Meliponini, in order to provide input for future comparative studies in this tribe. Haploid genome size (1C) estimates, using flow cytometry analyses (FCM), ranged from 0.26 ± 0.003 pg (Paratrigona subnuda) to 0.98 ± 0.023 pg (Melipona flavolineata), with an average of 0.54 ± 0.17 pg. FCM analyses also demonstrated a small difference in the haploid genome size between males and females of the same species, with the males generally having a smaller genome than females. Our data also evidenciated that variations in the genome size of stingless bees do not correlate with changes in chromosome number and that in some genera the DNA content is more variable than in others.Item Ploidy instability in long-term in vitro cultures of Coffea arabica L. monitored by flow cytometry(Plant Growth Regulation, 2012-12) Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo; Mendonça, Maria Andréia CorrêaSomatic embryogenesis of Coffea arabica L. has been mainly carried out in liquid medium for clonal and mass propagation of elite lines. This in vitro system involves suspension cultures of embryogenic aggregates, with high multiplication rate and unorganized growth. These characteristics are linked to the occurrence of somaclonal variation (SV), especially considering that cell aggregates are usually maintained for long periods in media supplemented with the synthetic auxin 2,4-dichlorophenoxyacetic acid. Because SV detection has been considered essential in in vitro tissue cultures, flow cytometry (FCM) was applied to verify ploidy instability in embryogenic cell aggregates of C. arabica, throughout successive subcultures. FCM allowed us to detect the occurrence of non-true-to-type aggregates in all samples collected after approximately 4 months in liquid medium. These aggregates showed octaploid and/or aneuploid cells, with DNA ploidy level being corroborated by chromosome counting. Considering this result, we recommend a limit of <4 months for true-to-type mass propagation of C. arabica cell aggregate suspensions. Besides, FCM was an important tool to detect SV at an early stage of tissue culture in this species, proving to be very useful for quality control in clonal propagation and in the introduction of somaclones to breeding programs.Item A practical and reliable procedure for in vitro induction of tetraploid tomato(Scientia Horticulturae, 2009-10-01) Praça, Milene Miranda; Carvalho, Carlos Roberto de; Clarindo, Wellington RonildoA relatively rapid, practical and reliable procedure for in vitro production of tetraploid tomato using colchicine is described. Using this procedure 11.11% generation of tetraploids was obtained after exposure of seedling shoot meristem explants with 8 mM colchicine for 96 h. Confirmation of tetraploid production (2n = 48), was determined by flow cytometry and verified by cytogenetic analysis of root tip preparations. The results indicate that the procedure is adequate for induction and screening of tetraploid tomato plantlets.Item An integrated cytogenetic, flow and image cytometry procedure used to measure the DNA content of Zea mays A and B chromosomes(Plant Science, 2008-10-31) Rosado, Tatiana Barbosa; Clarindo, Wellington Ronildo; Carvalho, Carlos RobertoThe DNA content of some Zea mays chromosomes has been measured by flow karyotyping because similar chromosomes could not been discriminated. This technique requires suspensions with a high amount of intact chromosomes and protocols that provide enough resolution to quantify the DNA content of individual chromosomes. In order to measure the DNA content of A and B chromosomes of Z. mays ‘Black Mexican Sweet Corn’, we associated cytogenetic, flow and image cytometry tools. The cytogenetic methodology provided slides with A and B chromosomes adequate for karyogram assembly. The flow cytometry histograms exhibited distinct G0/G1 and G2 peaks for ‘Black Mexican Sweet Corn’ with (sample) and without (standard) B chromosomes. As a result, the nuclear DNA content of five plants showing B chromosomes was quantified. The nuclear genome size value was proportionally distributed according to the integrated optical density value, which was measured for individual chromosomes by image cytometry. Then, the DNA content of each A and B chromosome was calculated in picograms and base pairs. Based on qualitative (cytogenetic) and quantitative (flow and image cytometry) analyses, it was possible to discriminate and characterize the A and B chromosomes of ‘Black Mexican Sweet Corn’.Item From chromosome doubling to DNA sequence changes: outcomes of an improved in vitro procedure developed for allotriploid “Híbrido de Timor” (Coffea arabica L. × Coffea canephora Pierre ex A. Froehner)(Plant Cell, Tissue and Organ Culture (PCTOC), 2017-11) Sattler, Mariana Cansian; Carvalho, Carlos Roberto; Nunes, Andrei Caíque Pires; Sanglard, Natália Arruda; Silva, Paulo Marcos Amaral; Oliveira, Stéfanie Cristina de; Soares, Taís Cristina Bastos; Clarindo, Wellington RonildoSince 1966, chromosome doubling has been performed mainly in vitro, associating anti-tubulin treatment and different plant tissues showing proliferative cells. Despite the achieved improvements, some bottlenecks have been pointed out, such as the low rate of polyploids and high rate of mixoploid plantlets. To overcome these hurdles, some approaches have indicated that indirect somatic embryogenesis (ISE) constitutes an alternative trigger for chromosome doubling, especially for homoploid and anorthoploid germplasms. In this way, a guideline has been developed for hexaploidization of the Coffea line “Híbrido de Timor” (HT) ‘CIFC 4106’ (anorthoploid, 3x = 33 chromosomes, 1C = 2.10 pg, Coffea canephora × Coffea arabica) from friable embryogenic calli (FEC) treated with colchicine. From this, a relatively high percentage (49.3%) of HT hexaploids (6x = 66 chromosomes, 2C = 4.20 pg) was obtained, without recovery of mixoploids. Besides confirmation of endomitosis induction through the obtained hexaploids, SSR markers revealed that the FEC/colchicine strategy also resulted in loss of allelic diversity in 39 regenerated HT plantlets, demonstrating its genotoxic effect. Considering these results, the present procedure resolved the main bottlenecks for chromosome doubling, which have been reported since the discovery and isolation of the anti-tubulin colchicine in 1930. Hexaploid HT plantlets have enriched Coffea germplasm banks as a new genetic resource since the resolution of their karyotype and DNA sequence. Just as the true allotetraploid C. arabica and the allotriploid HT ‘CIFC 4106’, the hexaploid HT is relevant to investigate the genomic and phenotypic changes arising from polyploidization events.Item Cytogenetic and flow cytometry data expand knowledge of genome evolution in three Coffea species(Plant Systematics and Evolution, 2012-04) Clarindo, Wellington Ronildo; Carvalho, Carlos Roberto; Mendonça, Maria Andréia CorrêaKaryotype and nuclear 2C-value data are considered important in taxonomic and evolutionary approaches in Coffea. Still, new methods are needed to further support such studies, especially to determine the progenitors of Coffea arabica. In this work, new cytogenetic and flow cytometry data were used to compare Coffea arabica, Coffea canephora and Coffea congensis. These data corroborate the hypothesis that C. canephora and C. congensis originated from a single ancestor, whose basic chromosome number was x = 11. In agreement with the observations of other authors, the karyotype and mean 2C-values confirm that C. arabica is a true allotetraploid originating from two diploid Coffea species with similar genomes. Although C. canephora and C. congensis have been considered potential progenitors of C. arabica, karyotype comparison revealed that only one of these species may be parental to C. arabica. These accurate cytogenetic and flow cytometry data contribute to expand our knowledge of the Coffea genome, as well as of possible progenitors of C. arabica.Item Revisiting the DNA C-values of the genome size-standards used in plant flow cytometry to choose the “best primary standards”(Plant Cell Reports, 2011-07) Praça-Fontes, Milene Miranda; Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo; Cruz, Cosme DamiãoFlow cytometry (FCM) techniques have enabled characterization of the genome size for various plant species. In order to measure the nuclear genome size of a species, reference standards with well-established DNA content are necessary. However, different 2C-values have been described for the same species used as reference standard. This fact has brought about inaccurate genome measurements, making relevant the establishment of optimal DNA reference standards for plant cytometric analyses. Our work revisited the genome size of Arabidopsis thaliana and other seven plant standards, which were denominated “Doležel’s standard set” and have been widely used in plant DNA measurements. These eight plant standards were reassessed for a comparative measurement of their DNA content values, using each plant species as primary standard in a cascade-like manner, from A. thaliana to Allium cepa. The genome size values obtained here were compared to those reported in the literature by statistical analyses. As a result, Raphanus sativus and Drosophila melanogaster were considered the most inadequate primary standards, whereas A. thaliana, Solanum lycopersicum and Pisum sativum were found to be the most suitable.Item Flow cytometric analysis using SYBR Green I for genome size estimation in coffee(Acta Histochemica, 2009-10-19) Clarindo, Wellington Ronildo; Carvalho, Carlos RobertoPlant genome size has been measured by flow cytometry using propidium iodide as a dye for nuclear DNA staining. However, some authors have reported the occurrence of genome size estimation errors, especially in plants rich in secondary metabolites, such as the coffee tree. In this context, we tested an alternative cytometric protocol using the SYBR Green I as a fluorochrome for stoichiometrically staining nuclear double-stranded DNA in Coffea canephora (2x) and Coffea arabica (4x). The results showed that the respective mean genome size measured from nuclei stained with SYBR Green I and propidium iodide was statistically identical. However, the G0/G1 peaks of nuclei stained with SYBR Green I exhibited lower coefficient variations (1.57–2.85%) compared to those stained with propidium iodide (2.75–4.80%). Coefficient variation statistical data suggest that SYBR Green I is adequate for stoichiometric nuclei staining using this methodology. Our results provide evidence that SYBR Green I can be used in flow cytometry measurements of plants, with the advantages of minimizing errors in nuclear DNA content quantification, staining relatively quicker, with high affinity, and being less mutagenic than propidium iodide.Item Comparison of the Coffea canephora and C. arabica karyotype based on chromosomal DNA content(Plant Cell Reports, 2008-10-08) Clarindo, Wellington Ronildo; Carvalho, Carlos RobertoNuclear genome size has been measured in various plants, seeing that knowledge of the DNA content is useful for taxonomic and evolutive studies, plant breeding programs and genome sequencing projects. Besides the nuclear DNA content, tools and protocols to quantify the chromosomal DNA content have been also applied, expanding the data about genomic structure. This study was conducted in order to calculate the Coffea canephora and Coffea arabica chromosomal DNA content, associating cytogenetic methodologies with flow cytometry (FCM) and image cytometry (ICM) tools. FCM analysis showed that the mean nuclear DNA content of C. canephora and C. arabica is 2C = 1.41 and 2.62 pg, respectively. The cytogenetic methodology provided prometaphase and metaphase cells exhibiting adequate chromosomes for the ICM measurements and karyogram assembly. Based on cytogenetic, FCM and ICM results; it was possible to calculate the chromosomal DNA content of the two species. The 1C chromosomal DNA content of C. canephora ranged from 0.09 (chromosome 1) to 0.05 pg (chromosome 11) and C. arabica from 0.09 (chromosome 1) to 0.03 pg (chromosome 22). The methodology presented in this study was suitable for DNA content measuring of each chromosome of C. canephora and C. arabica. The cytogenetic characterization and chromosomal DNA content analyses evidenced that C. arabica is a true allotetraploid originated from a cross between Coffea diploid species. Besides, the same analyses also reinforce that C. canephora is a possible progenitor of C. arabica.