Artigos
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11845
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Item Unraveling the complex genome of Saccharum spontaneum using Polyploid Gene Assembler(DNA Research, 2019-02) Nascimento, Leandro Costa; Yanagui, Karina; José, Juliana; Camargo, Eduardo L. O.; Grassi, Maria Carolina B.; Cunha, Camila P.; Bressiani, José Antônio; Carvalho, Guilherme M. A.; Prado, Paula F.; Mieczkowski, Piotr; Pereira, Gonçalo A. G.; Carazzolle, Marcelo F.; Carvalho, Carlos RobertoThe Polyploid Gene Assembler (PGA), developed and tested in this study, represents a new strategy to perform gene-space assembly from complex genomes using low coverage DNA sequencing. The pipeline integrates reference-assisted loci and de novo assembly strategies to construct high-quality sequences focused on gene content. Pipeline validation was conducted with wheat (Triticum aestivum), a hexaploid species, using barley (Hordeum vulgare) as reference, that resulted in the identification of more than 90% of genes and several new genes. Moreover, PGA was used to assemble gene content in Saccharum spontaneum species, a parental lineage for hybrid sugarcane cultivars. Saccharum spontaneum gene sequence obtained was used to reference-guided transcriptome analysis of six different tissues. A total of 39,234 genes were identified, 60.4% clustered into known grass gene families. Thirty-seven gene families were expanded when compared with other grasses, three of them highlighted by the number of gene copies potentially involved in initial development and stress response. In addition, 3,108 promoters (many showing tissue specificity) were identified in this work. In summary, PGA can reconstruct high-quality gene sequences from polyploid genomes, as shown for wheat and S. spontaneum species, and it is more efficient than conventional genome assemblers using low coverage DNA sequencing.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 Vertical heterogeneity of DNA ploidy level assessed by flow cytometry in calli of Passiflora Cincinnata(In Vitro Cellular & Developmental Biology - Plant, 2014-03) Silva, Thaís Cristina Ribeiro; Carvalho, Carlos RobertoDuring in vitro culture conditions, callus tissue is exposed to different intensities of environmental stress, which may induce somaclonal variation. Among the possible resulting abnormalities, callus cells can exhibit distinct DNA ploidy levels, a type of somaclonal variation associated with euploidy and/or aneuploidy. As somaclonal variation has been regarded as both a positive and negative phenomenon, the development of strategies to carefully assess the stability of DNA ploidy level within callus tissue is highly valuable. To this end, the present work aimed to evaluate the presence of intra- and inter-calli heterogeneity in relation to DNA ploidy level and nuclei density by flow cytometry. Calli were induced from cotyledonary leaves of Passiflora cincinnata, a wild passion fruit species. Embryogenic friable calli cultivated for 2, 6, and 9 mo were classified as young, intermediary, and old, respectively. These calli were horizontally sliced from the bottom-up at approximately the same thickness, and a total of 160 layers were evaluated by flow cytometry. Inter- and intra-calli heterogeneities were detected in relation to nuclei density and DNA ploidy level. Additional analysis was performed to identify the most proliferative layer. We conclude that care must be taken when using callus as source material for flow cytometry, since one portion cannot represent the whole cell mass. Moreover, in order to prevent the emergence of undesired ploidies during clonal propagation, callus culture time should not be prolonged.Item Indirect somatic embryogenesis in Coffea with different ploidy levels: a revisiting and updating study(Plant Cell, Tissue and Organ Culture (PCTOC), 2019-02) Sattler, Mariana Cansian; Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo; Sanglard, Natália Arruda; Amaral-Silva, Paulo Marcos; Oliveira, Stéfanie Cristina de; Cesário, Letícia Miranda; Ferreira, AdésioIndirect somatic embryogenesis (ISE) is required for plant propagation and a prerequisite for applications that may provide new germplasms. Genetic, epigenetic and physiological features of the explant donor are barriers for ISE establishment, hindering its wide use. Despite the identification and/or expression analysis of genes during ISE, no approach to establish the karyotype aspects has been performed so far. So, this study aims to establish the ISE and compare the in vitro responses between diploid (Coffea canephora and Coffea eugenioides), allotriploid (“Híbrido de Timor”—HT) and true allotetraploid (Coffea arabica) Coffea in a taxonomic and evolutive scenario. Under the same in vitro conditions, the four Coffea differed from each other during ISE. Leaf explants of the true allopolyploids yielded the highest mean number of friable calli (FC) in relative short time and visually exhibiting more pronounced length. FC of the allotetraploid C. arabica presented the highest mean number of mature cotyledonary somatic embryos (MCSE), which were also recovered faster in this species. However, MCSE mean number in HT was the same or lower than diploid Coffea. Besides, intraspecific variation related to the ISE responses was observed in each Coffea, mainly the mean number of FC obtained from ex vitro and in vitro C. arabica and C. eugenioides explants. So, epigenetic and physiologic features may also have influenced the ISE responses. The findings provide the basis for performing other approaches considering the ploidy level, epigenetic and physiological backgrounds. Besides, the data also contributed for understanding about the consequences of polyploidy.Item In vitro responses in Passiflora species with different chromosome numbers, ploidy levels and nuclear 2C values: revisiting and providing new insights(Plant Cell, Tissue and Organ Culture (PCTOC), 2019-03) Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo; Ferreira, Adésio; Praça-Fontes, Milene Miranda; Vieira, Ariane Tonetto; Ferreira, Darley Aparecido Tavares; Leite, Cristiana TorresTissue culture in Passiflora has emerged as a strategy to propagate species with agronomic relevance, which is the main focus of most in vitro studies. Different morphogenic responses have been obtained under the same environmental in vitro conditions, mainly for species of the subgenus Passiflora with distinct 2n chromosome numbers. The aims of this study were to verify and compare the in vitro responses in Passiflora species with distinct 2n chromosome numbers, ploidy levels and nuclear 2C values. Under the same in vitro conditions, only friable calli occurred from mature zygotic embryo explants of Passiflora coriacea (2n = 2x = 12 chromosomes, 2C = 1.00 pg), Passiflora lindeniana (2n = 4x = 24, 2C = 2.42 pg) and Passiflora contracta (2n = 8x = 48, 2C = 4.78 pg). In contrast, plantlets were regenerated from Passiflora foetida (2n = 20, 2C = 1.04 pg) and Passiflora miniata (2n = 18, 2C = 3.40 pg) via indirect organogenesis and indirect somatic embryogenesis, respectively. By now, from mature zygotic embryo explants, de novo shoot organogenesis and somatic embryos have been recovered for Passiflora species with 2n = 18 chromosomes and relative high nuclear 2C value (more than 2C = 2.93 pg—Passiflora cincinata), and only de novo shoot organogenesis for P. foetida with 2n = 20 chromosomes and relative low 2C value (2C = 1.04 pg). Therefore, in a taxonomic and evolutive context, this study showed that the in vitro morphogenic pathways pretty varied between the Passiflora species with distinct karyotype features.Item Flow cytometry and cytogenetic tools in eucalypts: genome size variation × karyotype stability(Tree Genetics & Genomes, 2017-10) Carvalho, Guilherme Mendes Almeida; Carvalho, Carlos Roberto; Soares, Fernanda Aparecida FerrariThe eucalypts comprise a group of woody plants used in commercial forest plantations owing to their high growth rates, adaptability to various ecological conditions and multiple applications. Despite the enormous amount of molecular data available for eucalypts, a basic understanding of the nature of its genome still requires information regarding the DNA amount in the genus. In this work, we estimated the genome size and base composition of 25 eucalypt species. With a comparative karyotype approach, we aimed to identify possible chromosomal alterations correlated with the genome size variation. Classical cytogenetic and genomic in situ hybridization experiments were conducted for this purpose. The studied species showed genome size ranging from 2C = 0.91 (Corymbia intermedia) to 2C = 1.37 pg (Eucalyptus paniculata) and AT/CG ratios varying from AT = 61.3 (Eucalyptus urophylla) to AT = 62.85% (C. intermedia). Comparative karyotype analysis revealed no remarkable differences in chromosome number (2n = 22) or morphology among eucalypt species despite considerable differences in nuclear DNA content. The genome in situ hybridization method did not distinguish non-homologous chromosomal regions of Eucalyptus baileyana and Corymbia citriodora, despite the difference of 0.45 pg between their genome sizes. The results found in the present work corroborate the consideration of small and dispersed DNA changes as the main cause of genome size variation in eucalypts.Item Karyotype revised of Pisum sativum using chromosomal DNA amount(Plant Systematics and Evolution, 2014-08) Carvalho, Carlos Roberto; Fontes, Milene Miranda Praça; Clarindo, Wellington RonildoPisum sativum was one of the first plants for which the mitotic karyotype was recognized and the karyogram assembled. These achievements were required owing to the physical mapping of P. sativum, providing data for evolutionary approaches and breeding programs. In spite of significant advances, precise morphometric characterization of chromosomes and karyogram assembly of P. sativum have become a topical problem. The present study proposes an unambiguous classification for the chromosomes of P. sativum, based on classical cytogenetic rules and chromosomal DNA amount. Cytogenetic procedure yielded mitotic cells showing morphologically preserved and stoichiometrically stained chromosomes. Twelve mitotic cells were selected, and the mean values for total, short- and long-arm lengths and DNA amount were measured for each chromosome. Chromosomal DNA amount fully correlated with total chromosome length, whose value proportionally decreases with the amount of DNA. Considering these data, all seven chromosomes could be unambiguously identified, yielding a new cytogenetic classification for P. sativum chromosomes. Moreover, the chromosome pairs were ordered according to the classical cytogenetic rule for assembly of karyograms. Since P. sativum is considered a model plant, it was possible to correlate the newly outlined karyotype with other cytogenetic data and linkage groups.Item Embryogenic potential of immature zygotic embryos of Passiflora: a new advance for in vitro propagation without plant growth regulators(Plant Cell, Tissue and Organ Culture (PCTOC), 2015-09) Carvalho, Carlos Roberto; Ferreira, Darley Aparecido Tavares; Sattler, Mariana Cansian; Clarindo, Wellington RonildoIn vitro strategies for Passiflora have been developed owing to its economic and ecological importance. However, plantlet regeneration through somatic embryogenesis has presented some problems, such as the reproducibility of the protocol and formation of abnormal embryos and plantlets. Thus, this study aimed to establish a protocol exploring the embryogenic potential of immature zygotic embryos (IZE) of the wild species Passiflora miniata Vanderpl. and Passiflora speciosa Gardn. Friable calli, which formed on the abaxial surface of the cotyledons, yielded globular, heart-shaped, torpedo and cotyledonary somatic embryos, characterising the embryogenic response as asynchronous. A high percentage of normal regenerants (90 %) was obtained from IZE in media lacking 2,4-dichlorophenoxyacetic acid (2,4-D) in comparison to the value of normal plantlets (60 %) regenerated from mature zygotic embryos inoculated in media with 2,4-D. This result demonstrates that IZE of P. miniata and P. speciosa possess sufficient levels of endogenous phytohormones to trigger a high rate of indirect somatic embryogenesis. All regenerated plantlets had the same genome size and chromosome number as the explant donor plants. Therefore, the indirect embryogenic pathway, employing IZE inoculated into media free of growth regulators, did not cause changes in the karyotype and morphology. Based on these results, IZE should be considered as explant for the establishment of somatic embryogenesis in other species. Besides, a new, reliable and relatively rapid protocol to recover plantlets of P. miniata and P. speciosa yielded several plants, which were acclimatised and used for ornamental purposes and breeding programs, and for reintroduction into biological reserves.Item The first karyogram of a Bromeliaceae species: an allopolyploid genome(Plant Systematics and Evolution, 2013-06) Carvalho, Carlos Roberto; Nunes, Andrei Caíque Pires; Nogueira, Ester Ujiie; Gontijo, Andreia Barcelos Passos Lima; Clarindo, Wellington RonildoThe Bromeliaceae family has been traditionally distributed in the subfamilies Bromelioideae, Tillandsioideae and Pitcairnioideae. However, phylogenetic studies have provided other classifications, highlighting the need for analyses in order to characterize the genome of different species from this family. In this sense, the present work aimed to determine nuclear 2C-value and base composition, characterize the chromosomes and establish the karyogram of Pitcairnia flammea. Flow cytometry yielded 2C = 1.44 pg, AT = 64.28 % and GC = 35.72 % for this species, indicating its relatively small genome size. Despite reduced length and morphological similarity of the chromosomes, P. flammea metaphases presented well-spread chromosomes, with well-defined primary constriction, without chromatin damage and cytoplasmic background. These aspects allowed morphometric chromosomal characterization and assembly of the first karyogram of a Bromeliaceae species. The karyogram displayed 2n = 50 chromosomes, of which all were submetacentric. Karyomorphological analysis revealed grouped pairs of cytogenetically identical chromosomes (2–3, 4–5, 6–9, 10–17, 18–19, 20–23 and 24–25), plus one isolated chromosome (1), not identical to any other. This result suggests an allopolyploid origin for the P. flammea genome. Thus, the present investigation contributed with karyotype data for taxonomic and evolutionary aspects of this group.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.
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