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Dorstenia spp. are characterized by a herbaceous habit, the presence of rhizomes and inflorescences being coenanthia. They occur in different forest formations, and it is believed that there are two distribution centres for Dorstenia, one Palaeotropical (Africa) and one Neotropical (Central and South America). The present study aimed to undertake a comparative analysis of the karyotype diversity of Dorstenia, based on chromosome data and DNA C values of 13 Neotropical species, and an analysis of low-coverage genomic sequencing of three African and one Neotropical species. The most frequent chromosome number is 2n = 32, with variations for 2n = 28 and 30, suggesting an important role of polyploidy in the karyotype differentiation of Dorstenia. Karyotypes varied in the number of metacentric, submetacentric and acrocentric chromosomes, with evidence of rearrangements. Genome sizes (C values) varied almost two-fold, from 2C = 2.50 to 5.47 pg. There was no clear relationship between DNA C value variations and karyotype features, such as chromosome numbers and distribution of CMA/DAPI bands or rDNA sites. Proximal heterochromatin was most frequent, but CMA+ and DAPI+ bands predominated in relation to DAPI+ bands. 5S and 35S ribosomal DNA (rDNA) sites were also observed in the proximal regions in almost all species. Based on a chromosome modelling approach, our data suggest that dysploidy events may have been responsible for the chromosome number variations, whereas rearrangements involving repetitive fractions may have been associated with changes in genome size. There was great variability in the amount of rDNA between species, including a remarkable 5S rDNA amplification in D. bahiensis, and a diversity of transposable elements in the four genomes with genomic sequence data. The combination of data from karyotypes, genomes and phylogeny suggests that dysploidy has played a predominant role in the evolution of Neotropical Dorstenia spp.

Cestrum species present large genomes (2 C = ~ 24 pg), a high occurrence of B chromosomes and great diversity in heterochromatin bands. Despite this diversity, karyotypes maintain the chromosome number 2n = 16 (except when they present B chromosomes), and a relative similarity in chromosome morphology and symmetry. To deepen our knowledge of the Cestrum genome composition, low-coverage sequencing data of C. strigilatum and C. elegans were compared, including cytogenomic analyses of seven species.Bioinformatics analyses showed retrotransposons comprising more than 70% of the repetitive fraction, followed by DNA transposons (~ 17%), but FISH assays using retrotransposon probes revealed inconspicuous and scattered signals. The four satellite DNA families here analyzed represented approximately 2.48% of the C. strigilatum dataset, and these sequences were used as probes in FISH assays. Hybridization signals were colocalized with all AT- and GC-rich sequences associated with heterochromatin, including AT-rich Cold-Sensitive Regions (CSRs). Although satellite probes hybridized in almost all tested species, a satDNA family named CsSat49 was highlighted because it predominates in centromeric regions.Data suggest that the satDNA fraction is conserved in the genus, although there is variation in the number of FISH signals between karyotypes. Except to the absence of FISH signals with probes CsSat1 and CsSat72 in two species, the other satellites occurred in species of different phylogenetic clades. Some satDNA sequences have been detected in the B chromosomes, indicating that they are rich in preexisting sequences in the chromosomes of the A complement. This comparative study provides an important advance in the knowledge on genome organization and heterochromatin composition in Cestrum, especially on the distribution of satellite fractions between species and their importance for the B chromosome composition.

Coffea spp. chromosomes are very small and accumulate a variety of repetitive DNA families around the centromeres. However, the proximal regions of Coffea chromosomes remain poorly understood, especially regarding the nature and organisation of the sequences. Taking advantage of the genome sequences of C. arabica (2n = 44), C. canephora, and C. eugenioides (C. arabica progenitors with 2n = 22) and good coverage genome sequencing of dozens of other wild Coffea spp., repetitive DNA sequences were identified, and the genomes were compared to decipher particularities of pericentromeric structures. The searches revealed a short tandem repeat (82 bp length) typical of Gypsy/TAT LTR retrotransposons, named Coffea_sat11. This repeat organises clusters with fragments of other transposable elements, comprising regions of non-coding RNA production. Cytogenomic analyses showed that Coffea_sat11 extends from the pericentromeres towards the middle of the chromosomal arms. This arrangement was observed in the allotetraploid C. arabica chromosomes, as well as in its progenitors. This study improves our understanding of the role of the Gypsy/TAT LTR retrotransposon lineage in the organisation of Coffea pericentromeres, as well as the conservation of Coffea_sat11 within the genus. The relationships between fragments of other transposable elements and the functional aspects of these sequences on the pericentromere chromatin were also evaluated. Highlights: A scattered short tandem repeat, typical of Gypsy/TAT LTR retrotransposons, associated with several fragments of other transposable elements, accumulates in the pericentromeres of Coffea chromosomes. This arrangement is preserved in all clades of the genus and appears to have a strong regulatory role in the organisation of chromatin around centromeres.

In the present work, we study the ovule, seed, and fruit development in six Bulbostylis species in order to characterize the genus in a comparative approach and to identify the characteristics that can be used in taxonomy and phylogeny. Flowers and fruits at different developmental stages were analyzed using LM and SEM after processing according to standard techniques. The species studied have the following: anatropous and bitegmic ovules, weak crassinucellar ovules, obturator of integumentary origin, monosporic embryo sac of the Polygonum type, nuclear endosperm, hypostase formation, seed coat formed by tanniferous endotegmen and exotesta, and Bulbostylis-type embryo. On the other hand, the pericarp development constitutes the main variation within Bulbostylis since the cells of the exocarp may or may not present starch grains, and their inner periclinal walls may be slightly or deeply concave depending on the degree of development of the mesocarp sclereids. In a taxonomic context, the results herein obtained are in conflict with studies which suggest infrageneric groupings based on fruit micromorphology, and also with the relationship among the Bulbostylis species based on molecular analysis. This work contributes to a better understanding of the reproductive anatomy and embryology in Bulbostylis, and reveals the first insights about the origin of multiple embryos in Cyperaceae. Given the frequent presence of polyembryony in Bulbostylis, and the poor mention of this condition in the family, this work highlights an aspect in the anatomy of Cyperaceae that must be re-explored.

For a long time, the Cyperid clade (Thurniceae-Juncaceae-Cyperaceae) was considered a group of species possessing holocentromeres exclusively. The basal phylogenetic position of Prionium serratum (Thunb.) Drège (Thurniceae) within Cyperids makes this species an important specimen to understand the centromere evolution within this clade. In contrast to the expectation, the chromosomal distribution of the centromere-specific histone H3 (CENH3), alpha-tubulin and different centromere-associated post-translational histone modifications (H3S10ph, H3S28ph and H2AT120ph) demonstrate a monocentromeric organisation of P. serratum chromosomes. Analysis of the high-copy repeat composition resulted in the identification of two centromere-localised satellite repeats. Hence, monocentricity was the ancestral condition for the Juncaceae-Cyperaceae-Thurniaceae Cyperid clade, and holocentricity in this clade has independently arisen at least twice after differentiation of the three families, once in Juncaceae and the other one in Cyperaceae. In this context, methods suitable for the identification of holocentromeres are discussed. Supplementary Information The online version contains supplementary material available at 10.1007/s00412-020-00745-6.

Cyperaceae representatives present peculiar microsporogenesis and microgametogenesis, which raises the question of how regular the sedge pollen is. In order to answer this question, preanthesis pollen grains of Cyperaceae individuals were analyzed under different tools such as light and transmission electron microscopy, which included cytochemistry and immunogold procedures. The results showed that maturing pollen in Cyperaceae presents some unusual endomembrane behaviors. Endoplasmic reticulum and dictyosomes are concerned in classic secretion pathways in vegetative cells, and possibly the late breakdown of degenerative microspores. However, cortical and concentric endoplasmic reticulum are also present and are possibly related to other functions aside the biosynthetic pathway. Unconventional secretion of large membrane-bound bodies containing cell wall precursors was also observed and confirmed by immunogold. However, since these bodies most likely receive material from dictyosomes, as observed in silver nitrate reaction, the "unconventional" status of this secretion is debatable. Reports of the literature show that these unusual endomembrane formations are not exclusive of the sedge pollen, but little attention have been given to them so far. This could represent an opportunity to re-examine our understanding on the endomembrane system in pollen cells in general.

Made available in DSpace on 2020-12-12T02:28:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 The 35S and 5S ribosomal DNA (rDNA) organized in thousands of copies in genomes, have been widely used in numerous comparative cytogenetic studies. Nevertheless, several questions related to the diversity and organization of regulatory motifs in 5S rDNA remain to be addressed. The 5S rDNA unit is composed of a conserved 120 bp length coding region and an intergenic spacer (IGS) containing potential regulatory motifs (Poly-T, AT-rich and GC-rich) differing in number, redundancy and position along the IGS. The Cestrum species (Solanaceae) have large genomes (about 10 pg/1C) and conserved 2n = 16 karyotypes. Strikingly, these genomes show high diversity of heterochromatin distribution, variability in 35S rDNA loci and the occurrence of B chromosomes. However, the 5S rDNA loci are highly conserved in the proximal region of chromosome 8. Comparison of seventy-one IGS sequences in plants revealed several conserved motifs with potential regulatory function. The AT- and GC-rich domains appeared highly conserved in Cestrum chromosomes. The 5S genic and the GC-rich IGS probe produced FISH signals in both A (pair 8) and B chromosomes. The GC-rich domain presented a strong potential for regulation because it may be associated with CpG islands organization, as well as to hairpin and loop organization. Another interesting aspect was the ability of AT- and GC-rich motifs to produce non-heterochromatic CMA/DAPI signals. While the length of the 5S rDNA IGS region varied in size between the Cestrum species, the individual sequence motifs seem to be conserved suggesting their regulatory function. The most striking feature was the conserved GC-rich domain in Cestrum, which is recognized as a signature trait of the proximal region of chromosome pair 8. Laboratório de Citogenética e Diversidade Vegetal Departamento de Biologia Geral Centro de Ciências Biológicas Universidade Estadual de Londrina Departamento de Morfologia Instituto de Biociências Universidade Estadual Paulista (UNESP) Departamento de Morfologia Instituto de Biociências Universidade Estadual Paulista (UNESP)

Cyperaceae is a family of Monocotyledons comprised of species with holocentric chromosomes that are associated with intense dysploidy and polyploidy events. Within this family the genus Rhynchospora has recently become the focus of several studies that characterize the organization of the holocentric karyotype and genome structures. To broaden our understanding of genome evolution in this genus, representatives of Rhynchospora were studied to contrast chromosome features, C-CMA/DAPI band distribution and genome sizes. Here, we carried out a comparative analysis for 35 taxa of Rhynchospora, and generated new genome size estimates for 20 taxa. The DNA 2C-values varied up to 22-fold, from 2C = 0.51 pg to 11.32 pg, and chromosome numbers ranged from 2n = 4 to 61. At least 37% of our sampling exhibited 2n different from the basic number x = 5, and chromosome rearrangements were also observed. A large variation in C-CMA/DAPI band accumulation and distribution was observed as well. We show that genome variation in Rhynchospora is much larger than previously reported. Phylogenetic analysis showed that most taxa were grouped in clades corresponding to previously described taxonomic sections. Basic chromosome numbers are the same within every section, however, changes appeared in all the clades. Ancestral chromosome number reconstruction revealed n = 5 as the most likely ancestral complements, but n = 10 appears as a new possibility. Chromosome evolution models point to polyploidy as the major driver of chromosome evolution in Rhynchospora, followed by dysploidy. A negative correlation between chromosome size and diploid number open the discussion for holokinetic drive-based genome evolution. This study explores relationships between karyotype differentiation and genome size variation in Rhynchospora, and contrasts it against the phylogeny of this holocentric group.

For a long time, the Cyperid clade (Thurniceae-Juncaceae-Cyperaceae) was considered as a group of species possessing holocentromeres exclusively. The basal phylogenetic position of Prionium serratum L. f. Drège (Thurniceae) within Cyperids makes this species an important specimen to understand the centromere evolution within this clade. Unlike expected, the chromosomal distribution of the centromere-specific histone H3 (CENH3), alpha-tubulin and different centromere associated post-translational histone modifications (H3S10ph, H3S28ph and H2AT120ph) demonstrate a monocentromeric organisation of P. serratum chromosomes. Analysis of the high-copy repeat composition resulted in the identification of a centromere-localised satellite repeat. Hence, monocentricity was the ancestral condition for the Juncaceae-Cyperaceae-Thurniaceae Cyperid clade and holocentricity in this clade has independently arisen at least twice after differentiation of the three families, once in Juncaceae and the other one in Cyperaceae. Methods suitable for the identification of holocentromeres are discussed.

Background and Aims Long terminal repeat-retrotransposons (LTR-RTs) comprise a large portion of plant genomes, with massive repeat blocks distributed across the chromosomes. Eleocharis species have holocentric chromosomes, and show a positive correlation between chromosome numbers and the amount of nuclear DNA. To evaluate the role of LTR-RTs in karyotype diversity in members of Eleocharis (subgenus Eleocharis), the occurrence and location of different members of the Copia and Gypsy superfamilies were compared, covering interspecific variations in ploidy levels (considering chromosome numbers), DNA C-values and chromosomal arrangements. Methods The DNA C-value was estimated by flow cytometry. Genomes of Eleocharis elegans and E. geniculata were partially sequenced using Illumina MiSeq assemblies, which were a source for searching for conserved proteins of LTR-RTs. POL domains were used for recognition, comparing families and for probe production, considering different families of Copia and Gypsy superfamilies. Probes were obtained by PCR and used in fluorescence in situ hybridization (FISH) against chromosomes of seven Eleocharis species. Key Results A positive correlation between ploidy levels and the amount of nuclear DNA was observed, but with significant variations between samples with the same ploidy levels, associated with repetitive DNA fractions. LTR-RTs were abundant in E. elegans and E. geniculata genomes, with a predominance of Copia Sirevirus and Gypsy Athila/Tat clades. FISH using LTR-RT probes exhibited scattered and clustered signals, but with differences in the chromosomal locations of Copia and Gypsy. The diversity in LTR-RT locations suggests that there is no typical chromosomal distribution pattern for retrotransposons in holocentric chromosomes, except the CRM family with signals distributed along chromatids. Conclusions These data indicate independent fates for each LTR-RT family, including accumulation between and within chromosomes and genomes. Differential activity and small changes in LTR-RTs suggest a secondary role in nuclear DNA variation, when compared with ploidy changes.

Species of Cestrum (Linnaeus, 1753) have shown large diversity in the accumulation and distribution of repetitive DNA families, and B chromosomes have been described in seven species. Some types of repetitive DNA were identified in A and B chromosomes in species of this plant group, such as AT-rich SSR, 35S and 5S rDNA, C-Giemsa and C-CMA/DAPI bands and retrotransposons. To increase our understanding of the relationships of A and B chromosomes, the B of C. strigilatum Ruiz & Pavón, 1799 was microdissected, amplified and hybridized in situ against chromosomes of this species, and in six other species of this genus. FISH signals were observed in whole the B of C. strigilatum, including stretches of A chromosomes, as well as in some A chromosomes of all tested species. A strong FISH signal was seen adjacent to the 5S rDNA in the proximal region of pair 8 of all species and, due to this, we have searched for 5S rDNA fragments in the microdissected B chromosome. PCR and sequencing data evidenced 5S rDNA deletion along evolutionary pathways of the B of C. strigilatum. Although A and B chromosomes displayed redundancy in the repetitive DNA families in different species, the B of C. strigilatum seemed to differ from those Bs of other Cestrum species by the loss of rDNA fractions. A possible origin of Bs in Cestrum was discussed.

Polyploidy and hybridization are key events in plant evolution. Due to these events, complexes of species can be formed. Dysploidy, a frequent chromosome change in holocentric organisms, may add some difficulties to species delimitation, especially in Cyperaceae. The Eleocharis montana complex is known for its members with overlapping morpho-anatomical features and unclear circumscription. To understand its biological history, several tools were used to investigate different American populations, including morpho-anatomical analysis, genome size estimates, cytogenomic analysis, molecular marker characterization, and genomic in situ hybridization (GISH). Despite overlapping morphological features, it was possible to separate two groups, the first containing E. parodii and E. subarticulata, presenting diploid and dysploid karyotypes, respectively, and small DNA C-values. The second contained E. elegans, E. contracta, and E. montana, with large genomes created by polyploidy. All analyses suggest that E. montana with 2n = 40 is a cytotype of E. contracta with 2n = 20, and both evolved from a natural hybridization involving E. parodii (2n = 10) and a second progenitor that is yet unknown. Furthermore, the GISH results indicated that E. parodii may be an ancestor of E. elegans. All species occur in the probable center of diversification in Austral South America, where the hybridization zone is identified. Fieldwork and information obtained from herbaria indicate that diploid and dysploid species (E. subarticulata and E. parodii) do not occur beyond the center of diversification. However, E. elegans and specially E. montana occur more widely, occupying different flooded environments and landscapes.

Background Plant genomes are rich in repetitive sequences, and transposable elements (TEs) are the most accumulated of them. This mobile fraction can be distinguished as Class I (retrotransposons) and Class II (transposons). Retrotransposons that are transposed using an intermediate RNA and that accumulate in a “copy-and-paste” manner were screened in three genomes of peppers (Solanaceae). The present study aimed to understand the genome relationships among Capsicum annuum, C. chinense, and C. baccatum, based on a comparative analysis of the function, diversity and chromosome distribution of TE lineages in the Capsicum karyotypes. Due to the great commercial importance of pepper in natura, as a spice or as an ornamental plant, these genomes have been widely sequenced, and all of the assemblies are available in the SolGenomics group. These sequences were used to compare all repetitive fractions from a cytogenomic point of view. Results The qualification and quantification of LTR-retrotransposons (LTR-RT) families were contrasted with molecular cytogenetic data, and the results showed a strong genome similarity between C. annuum and C. chinense as compared to C. baccatum. The Gypsy superfamily is more abundant than Copia, especially for Tekay/Del lineage members, including a high representation in C. annuum and C. chinense. On the other hand, C. baccatum accumulates more Athila/Tat sequences. The FISH results showed retrotransposons differentially scattered along chromosomes, except for CRM lineage sequences, which mainly have a proximal accumulation associated with heterochromatin bands. Conclusions The results confirm a close genomic relationship between C. annuum and C. chinense in comparison to C. baccatum. Centromeric GC-rich bands may be associated with the accumulation regions of CRM elements, whereas terminal and subterminal AT- and GC-rich bands do not correspond to the accumulation of the retrotransposons in the three Capsicum species tested.

Made available in DSpace on 2020-12-10T17:38:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-21 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Capsicum annuum is one of the most important horticultural crops worldwide. Anthracnose disease (Colletotrichum spp.) is a major constraint for chili production, causing substantial losses. Capsidiol is a sesquiterpene phytoalexin present in pepper fruits that can enhance plant resistance. The genetic mechanisms involved in capisidiol biosynthesis are still poorly understood. In this study, a 3 ' RNA sequencing approach was used to develop the transcriptional profile dataset of C. annuum genes in unripe (UF) and ripe fruits (RF) in response to C. scovillei infection. Results showed 4,845 upregulated and 4,720 downregulated genes in UF, and 2,560 upregulated and 1,762 downregulated genes in RF under fungus inoculation. Four capsidiol-related genes were selected for RT-qPCR analysis, two 5-epi-aristolochene synthase (CA12g05030, CA02g09520) and two 5-epi-aristolochene-1,3-dihydroxylase genes (CA12g05070, CA01g05990). CA12g05030 and CA01g05990 genes showed an early response to fungus infection in RF (24 h post-inoculation-HPI), being 68-fold and 53-fold more expressed at 96 HPI, respectively. In UF, all genes showed a late response, especially CA12g05030, which was 700-fold more expressed at 96 HPI compared to control plants. We are proving here the first high-throughput expression dataset of pepper fruits in response to anthracnose disease in order to contribute for future pepper breeding programs. Univ Estadual Londrina, Programa Posgrad Agron, Lab Ecofisiol & Biotecnol Agr, Londrina, Parana, Brazil Boyce Thompson Inst Plant Res, Ithaca, NY USA Univ Estadual Paulista, Inst Biociencias, Rio Claro, Brazil Empresa Brasileira Pesquisa Agr, Brasilia, DF, Brazil Univ Estadual Londrina, Lab Citogenet & Diversidade Vegetal, Londrina, Parana, Brazil Univ Estadual Norte Fluminense, Genet & Melhoramento Plantas, Campos Goytacazes, Goytacazes, Brazil Univ Estadual Paulista, Inst Biociencias, Rio Claro, Brazil FAPESP: 2017/01455-2

Highlight The functional cell of an asymmetric tetrad is selected according to its replication capability, while degenerative cells progress to multistep PCD, including mitotic disorder and DNA elimination., Members of the Cyperaceae family exhibit an asymmetric microsporogenesis that results in the degeneration of three out of four meiotic products. Efforts have been made previously to describe the resulting structure, named the pseudomonad, but mechanisms concerning the establishment of cell domains, nuclear development, and programmed cell death are largely unknown. Using the Rhynchospora genus as a model, evidence for cell asymmetry, cytoplasmic isolation, and programmed cell death was obtained by a combination of electron microscopic, cytochemical, immunocytochemical, in situ hybridization, and flow cytometric methods. Degenerative cells were identified at the abaxial region, with the cytoskeleton marking their delimitation from the functional domain after meiosis. After attempting to initiate cell division with an unreplicated genome and abnormal spindle assembly, these cells exhibited a gradual process of cytoplasmic contraction associated with hypermethylation of cytosines and differential loss of DNA. These results indicate that the asymmetric tetrad establishes a functional cell, where one nucleus is preferentially selected to survive. Degenerative haploid cells are then eliminated in a multistep process associated with mitotic disorder, non-random elimination of repetitive DNA, vacuolar cell death, and DNA fragmentation.

Microsporogenesis and microgametogenesis are unusual in sedges (Cyperaceae), the third largest monocotyledonous family, as three microspores are aborted in favor of a single functional microspore. However, studies using light microscopy show that megasporogenesis and megagametogenesis occur normally. Nevertheless, the lack of ultrastructural details limits our knowledge of female gametophyte development in this family. Given the importance of morphological studies of reproductive structures, ovules and megagametophytes of Rhynchospora pubera were analyzed under transmission electron microscopy for the first time. Overall, ovules presented features similar to those described for the family, but ultrastructural details revealed an absence of a clear boundary between the egg cell and the central cell cytoplasm. Most interestingly, antipodal and nucellar cells showed several signs of vacuolar cell death, which suggest that programmed autolysis in sporogenous and gametophytic tissue is common in gametophyte development in the Cyperaceae. This may be related to the reproductive success of this family.

The genera Nothoscordum and Ipheion (Allioideae, Amaryllidaceae) are cytologically characterized by a dysploid series with variable numbers of metacentric and acrocentric chromosomes typical of karyotypes rearranged by Robertsonian translocations (RT). Since they have large chromosomes, low diploid numbers, and possess two telomeric motifs [the vertebrate-type (TTAGGG) n and the Arabidopsis-type (TTTAGGG) n ] they are suitable for investigating the occurrence and possible role of interstitial telomeric sites (ITS) associated with RT. We analyzed the distributions of telomeric sites in 12 species of Nothoscordum and Ipheion and found that both telomeric probes colocalized in all chromosome termini. Cloning and sequencing PCR products obtained using both telomeric primers simultaneously revealed long stretches of (TTAGGG) n and (TTTAGGG) n sequences together with degenerated telomeric sequences. Most acrocentric chromosomes have a 45S rDNA site at the terminal region of the short arms adjacent to the most distal telomeric sites. Telomeric signals were found at all chromosome ends, but ITS were also detected in a few proximal and subterminal regions in some Nothoscordum species. Although RT are common in this group of plants, our findings suggest that proximal positioning of telomeric motifs are not necessarily related to that kind of rearrangement. Rather, transposition of telomeric sequences followed by amplification, could better explain the presence of (TTAGGG) n and (TTTAGGG) n repeats at those sites. Furthermore, a few small interstitial sites found in some Nothoscordum species indicate that dispersion of these sequences was not restricted to the proximal region.

The objective of this study was to optimize the extraction of protein by applying a multi-enzymatic pretreatment to okara, a byproduct from soymilk processing. The multi-enzyme complex Viscozyme, containing a variety of carbohydrases, was used to hydrolyze the okara cell walls and facilitate extraction of proteins. Enzyme-assisted extraction was carried out under different temperatures (37–53 °C), enzyme concentrations (1.5–4%) and pH values (5.5–6.5) according to a central composite rotatable design. After extraction, the protein was concentrated by isoelectric precipitation. The optimal conditions for maximum protein content and recovery in protein concentrate were 53 °C, pH 6.2 and 4% of enzyme concentration. Under these conditions, protein content of 56% (dry weight basis) and a recovery of 28% were obtained, representing an increase of 17 and 86%, respectively, compared to the sample with no enzymatic pretreatment. The multi-enzyme complex Viscozyme hydrolyzed the structural cell wall polysaccharides, improving extraction and obtaining protein concentrate from the okara. An electrophoretic profile of the protein concentrate showed two distinct bands, corresponding to the acidic and basic subunits of the protein glycinin. There were no limiting amino acids in the protein concentrate, which had a greater content of arginine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-018-3067-4) contains supplementary material, which is available to authorized users.

Made available in DSpace on 2015-10-21T13:12:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-01. Added 1 bitstream(s) on 2015-10-22T09:52:02Z : No. of bitstreams: 1 WOS000351607800007.pdf: 567544 bytes, checksum: daec061ae5075b709c566ce85b2190bf (MD5) Agropolis Fondation Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Special Visiting Scientist grant from the Ciência sem Fronteiras program A novel structure of nonautonomous long terminal repeat (LTR) retrotransposons called terminal repeat with GAG domain (TR-GAG) has been described in plants, both in monocotyledonous, dicotyledonous and basal angiosperm genomes. TR-GAGs are relatively short elements in length (

Species of Cestrum L. (Solanaceae) exhibit large variability in the accumulation of repetitive DNA, although their species possess a stable diploid number with 2n = 16. In this study, we used chromosome banding and fluorescence in situ hybridization (FISH) to characterize the karyotypes and populations of two species, Cestrum nocturnum L. and C. mariquitense Kunth. We also performed a karyotype comparison using 16 idiograms, of which 4 were developed in this study and 12 were obtained from the literature. Cestrum nocturnum displayed more bands than C. mariquitense, but the latter exhibited greater interpopulational variation in the band patterns. There was a tendency for large bands to be located at intercalary/terminal regions and for small bands to be located at intermediate/proximal regions. The idiogram comparison revealed a large variation in the amount, distribution, and size of heterochromatic bands. FISH with rDNA probes revealed stability in the number and location of 5S sites, while 45S was more...

Megasporogenesis begins when one cell arising from the hypodermis of the nucellus differentiates into a megaspore mother cell. This cell undergoes meiosis, giving rise to a tetrad of megaspores, whose functional megaspore originates the megagametophyte. Representatives of the family Cyperaceae draw attention because both micro- and megasporogenesis culminate in the selection of a functional cell and the loss of the other three by programmed cell death after meiosis. We investigated in a more detailed manner the megasporogenesis and megagametogenesis in Eleocharis sellowiana Kunth using structural and ultrastructural tools. Although the megagametophyte of E. sellowiana is of the Polygonum type, we found some new features related to the megaspore mother cell, such as (1) absence of nuclear staining when exposed to toluidine blue and (2) presence of plastids and nucleus located at the micropilar pole, instead of the central region to the chalazal pole, as found in the literature for the Polygonum type. This opposite polarity could indicate that the cytoplasmic orientation in the megasporocyte is not a decisive event in the selection of the functional meiotic product for Polygonum type development.

Karyomorphological analysis of four species of Schoenoplectus (Cyperaceae) from north-central Mexico were carried out. Chromosome numbers ranged from 2n = 38 to 2n = 84. New records of counting are given for Schoenoplectus acutus var. occidentalis (2n = 38 and 2n = 84) and S. americanus (2n = 66). Intra-individual variation in chromosome number is reported for the first time for S. acutus, with a rare polyploid mixoploidy with a prevalence of cells with 2n = 38 (36 small + 2 compound, larger chromosomes) and a few cells with 2n = 84 small, dot-shaped chromosomes, this being the first record of polyploid mixoploidy for Cyperaceae. Mean length of the diploid set ranged from 51.5 μm (S. tabernaemontani) to 79.5 μm (S. acutus). The lowest average chromosome length for the dot-shaped chromosomes was 0.69 μm (S. acutus) and the highest 1.62 μm (S. tabernaemontani); the pair of large chromosomes in S. acutus reached 3.17 μm. A low interchromosomal asymmetry index (A2), 0.11 to 0.14 was found, very similar among ...

Heteroptera is the most numerous and diverse suborder of Hemiptera, with about 38,000 species. This diversity also involves cytogenetic features, including chromosome number and a sex determining system. Information about heterochromatin occurrence and distribution is scarce in heteropterans, but still, there is some evidence of variability. We determined the chromosome number and CMA3/DAPI-banding pattern of 179 individuals of 25 heteropteran species from Brazil. Eight species of Pentatomidae exhibited a constant chromosome number (2n = 12 + XY), but in Coreidae (12 species), Largidae (1 species), Rhopalidae (1 species), and Pyrrhocoridae (3 species), the numbers ranged from 2n = 10 + 2m + X0 to 2n = 24 + 2m + X0. Although there were no large differences in the chromosome size between species, the CMA3/DAPI-banding patterns differed markedly. Among the genera, species of Edessa, Spartocera, Hypselonotus, Phtia,Holhymenia and Euryophthalmus showed a large accumulation of heterochromatin, while the other species exhibited few or no heterochromatic bands. In general, when heterochromatin was more accumulated, this occurred preferentially at terminal positions, except in Holhymenia histrio, which exhibited intercalary bands. This study made it possible to identify some chromosome rearrangements and to enhance our knowledge of the evolutionary mechanisms that determine karyotype differentiation in Heteroptera.

Fluorescent in situ hybridization (FISH) with rDNA probes has been used for comparative cytogenetics studies in different groups of organisms. Although heteropterans are a large suborder within Hemiptera, studies using rDNA are limited to the infraorder Cimicomorpha, in which rDNA sites are present in the autosomes or sex chromosomes. We isolated and sequenced a conserved 18S rDNA region of Antiteuchus tripterus (Pentatomidae) and used it as a probe against chromosomes of 25 species belonging to five different families of Pentatomomorpha. The clone pAt05, with a length of 736 bp, exhibited a conserved stretch of 590 bp. FISH analysis with the probe pAt05 always demonstrated hybridization signals in sub-terminal positions, except for Euschistus heros. Apparently, there is a tendency for 18S rDNA sites to locate in autosomes, except for Leptoglossus gonagra and Euryophthalmus rufipennis, which showed signals in the m- and sex chromosomes, respectively. Although FISH has produced evidence that rearrangements are involved in rDNA repositioning, whether in different autosomes or between sex and m-chromosomes, we have no conclusive evidence of what were the pathways of these rearrangements based on the evolutionary history of the species studied here. Nevertheless, the diversity in the number of species analyzed here showed a tendency of 18S rDNA to remain among the autosomes.

The late stages of microsporogenesis in the family Cyperaceae are marked by the formation of an asymmetrical tetrad, degeneration of three of the four nuclei resulting from meiosis and the formation of pseudomonads. In order to understand the cytological changes involved in the development of pseudomonads, a combination of 11 different techniques (conventional staining, cytochemistry procedures, immunofluorescence, FISH and transmission electron microscopy: TEM) were used to study the later stages of microsporogenesis in Rhynchospora pubera. The results demonstrated the occurrence of two cytoplasmic domains in the pseudomonads, one functional and the other degenerative, which are physically and asymmetrically separated by cell plate with an endomembrane system rich in polysaccharides. Other changes associated with endomembrane behaviour were observed, such as a large number of lipid droplets, vacuoles containing electron-dense material and concentric layers of endoplasmic reticulum. Concomitant with the isolation of degenerative nuclei, the tapetal cells also showed evidence of degeneration, indicating that both tissues under programmed cell death (PCD), as indicated by immunofluorescence and TEM procedures. The results are significant because they associate cellular polarisation and asymmetry with different cytoplasmic domains, and hence open new possibilities for studying cellular compartmentalisation and PCD.

Satellite DNA repeats (or satDNA) are fast-evolving sequences usually associated with condensed heterochromatin. To test whether the chromosomal organisation of centromeric and non-centromeric satDNA differs in species with holocentric chromosomes, we identified and characterised the major satDNA families in the holocentric Cyperaceae species Rhynchospora ciliata (2n = 10), R. globosa (2n = 50) and R. tenuis (2n = 2x = 4 and 2n = 4x = 8). While conserved centromeric repeats (present in R. ciliata and R. tenuis) revealed linear signals at both chromatids, non-centromeric, species-specific satDNAs formed distinct clusters along the chromosomes. Colocalisation of both repeat types resulted in a ladder-like hybridisation pattern at mitotic chromosomes. In interphase, the centromeric satDNA was dispersed while non-centromeric satDNA clustered and partly colocalised to chromocentres. Despite the banding-like hybridisation patterns of the clustered satDNA, the identification of chromosome pairs was impaired due to the irregular hybridisation patterns of the homologues in R. tenuis and R. ciliata. These differences are probably caused by restricted or impaired meiotic recombination as reported for R. tenuis, or alternatively by complex chromosome rearrangements or unequal condensation of homologous metaphase chromosomes. Thus, holocentricity influences the chromosomal organisation leading to differences in the distribution patterns and condensation dynamics of centromeric and non-centromeric satDNA.

The genus Rhynchospora Vahl. (Cyperaceae) has about 270 species, two subgenera and 27 sections. Cytogenetic studies showed that polyploidy is the main mechanism of numeric changes in the karyotypes of Rhynchospora. However, some species have exhibited some evidence of agmatoploidy and symploidy. The occurrence of holocentric chromosomes seems to support more the events of fusion (symploidy) and fission (agmatoploidy), leading to the appearance and maintenance of intra- and interspecific variations based in disploidy. In this study, chromosome counts and recounts were performed in 14 species obtained from different Brazilian localities. New chromosome numbers have been reported for R. tenuis, R. globosa, R. pubera, R. nervosa, R. junciformis and R. asperula. The analysis showed the absence of primary constrictions on the chromosomes and numeric variation from 2n = 4 to 2n = 58. The primary (x = 5) and secondary (x = 6 and 9) basic numbers were again confirmed. Our results expand the information related to ...

Heterochromatin comprises a fraction of the genome usually with highly repeated DNA sequences and lacks of functional genes. This region can be revealed by using Giemsa C-banding, fluorochrome staining and cytomolecular tools. Some plant species are of particular interest through having a special type of heterochromatin denominated the cold-sensitive region (CSR). Independent of other chromosomal regions, when biological materials are subjected to low temperatures (about 0 °C), CSRs appear slightly stained and decondensed. In this study, we used Cestrum strigilatum (Solanaceae) to understand some aspects of CSR condensation associated with cytosine methylation levels, and to compare the behavior of different heterochromatin types of this species, when subjected to low temperatures.

Micromorphology of the achene surface of 26 Brazilian species of Eleocharis was studied by scanning electron microscopy in order to evaluate its usefulness in the taxonomy of the genus. The results point out two patterns of cell organization according to silica structures of achenes. The first corresponds to a group of species (group A) that have small to medium cells arranged vertically. The second is found in those species (group B) with medium to large cells arranged horizontally. These data were useful in separating species of Eleocharis subgenus Scirpidium and E. subgenus Limnochloa (group B) from E. subgenus Eleocharis (group A). However, group A shows considerable variation in silica wall arrangement. Eleocharis squamigera, previously considered as part of E. subgenus Eleocharis, shows features rather similar to those of Scirpidium, confirming recent phylogenies. The subgenus Limnochloa was clearly distinguished from others by achenes with large cells (over 55 μm width), presence of crenate or repand anticlinal walls, and some orifices near the wall in some species. The silica wall ornamentation seems to be a useful morphological tool for studying relationships between subgenera and distinguishes Limnochloa from the other subgenera.

Studies of rDNA location in holocentric chromosomes of the Cyperaceae are scarce, but a few reports have indicated the occurrence of multiple 45S rDNA sites at terminal positions, and in the decondensed state of these regions in prometaphase/metaphase. To extend our knowledge of the number 45S and 5S rDNA sites and distribution in holocentric chromosomes of the Cyperaceae, 23 Brazilian species of Eleocharis were studied. FISH showed 45S rDNA signals always located in terminal regions, which varied from two (E. bonariensis with 2n = 20) to ten (E. flavescens with 2n = 10 and E. laeviglumis with 2n = 60). 5S rDNA showed less variation, with 16 species exhibiting two sites and 7 species four sites, preferentially at terminal positions, except for four species (E. subarticulata, E. flavescens, E. sellowiana and E. geniculata) that showed interstitial sites. The results are discussed in order to understand the predominance of terminal rDNA sites, the mechanisms involved in the interstitial positioning of 5S rDNA sites in some species, and the events of amplification and dispersion of 45S rDNA terminal sites.

Copaifera langsdorffii possesses 2n = 24 large meta- and submetacentric chromosomes (5.97-2.60 microm) in comparison with other Caesalpinioideae trees. Chromosome banding revealed an abundance of GC-rich blocks with a few differences in the size and location of bands between different populations. Polymerase chain reaction and digestion with restriction enzyme RsaI were carried out in order to isolate repetitive DNAs, yielding three fragments of different size: (1) cp-rDNA-like, 109 bp (pCl03 clone); (2) Ty1-copia-like retroelement, 185 bp (pCl23 clone); and (3) Ty3-gypsy-like retroelement, 269 bp (pCl08 clone). The first clone produced unmistakable hybridization signals at subterminal and intercalary positions, coinciding with or adjacent to most of the GC-rich bands. The second clone (pCl23 clone) showed dispersed signals distributed throughout several chromosomes, while the pCl08 clone exhibited hybridization signals scattered and organized in blocks in all chromosomes of the complement. Fluorescent in situ hybridization and chromosome banding results allowed the detection of translocation events and unequal crossing-over involving 45S rDNA regions (pairs 7, 8, and 11). However, the most intriguing result was the insertion and subsequent overamplification of a cp-rDNA-like fragment and its distribution over the chromosomes of C. langsdorffii. Additionally, the results suggest that this segment behaves like satellite DNA. These different chromosome markers produced by cytomolecular techniques show that samples from different locations, although isolated, retain more karyotypic similarities than differences.

Valeriana scandens presents perfect and pistillate flowers, the latter with sterile anthers. The species is composed of two varieties with different ploidy; V. scandens var. scandens (2n = 28) and V. scandens var. candolleana (2n = 56), both of which occur in RS, Brazil. Crosses between these varieties may give rise to hybrids with pollen sterility. In this study, we analyzed the microsporogenesis and microgametogenesis of sterile and fertile anthers, and also investigate whether pollen sterility is caused by an irregular meiotic process. Developmental analysis using light microscopy and scanning electron microscopy showed that sterile anthers develop similarly to fertile anthers until the end of meiosis. After this stage, sterile tetrads do not separate as a consequence of exine fusion between adjacent microspores, which is similar to sterile pollen of Brassica ms-cdl1 mutants. In addition, vacuolated immature pollen grains degenerate after separation. The cytogenetic analysis of the microspore mother cell (MMC) showed that the diploid population of V. scandens var. scandens (2n = 28) has pollen sterility that is not caused by a cytogenetic disturbance. The MMCs analyzed from prophase I to tetrad stage showed a regular meiotic process, indicating the phenotype of V. scandens sterile pollen is a postmeiotic process formed by fusion of exine between opposite microspores.

Solanum comprises about 1,400 species of shrubs, trees and vines. This group is cytogenetically interesting because it possesses karyotypes apparently conserved in chromosome number and shape, but with diversity in the repetitive DNA. The objective of this study is to characterize 16 species of Solanum and two species of closely related genera (Lycianthes australe and Vassobia breviflora) using cytogenetic parameters. All the species presented 2n = 24, confirming previous chromosome counting. Additionally, nonreticulated nuclei, proximal condensation in prophase-metaphase and little variation in the karyotype symmetry were observed. Solanum corymbiflorum exhibited chromosomes approximately three times bigger in relation to the other species. GC-rich heterochromatin was preferentially located at terminal regions and AT-rich blocks always appear in the centromeric regions. The 45S rDNA sites were coincident with C/CMA3 + regions (satellites) and found in just one pair, except in S. corymbiflorum which presented two pairs. FISH with 5S rDNA showed signals in the paracentromeric region of one chromosome pair, except in S. trachytrichium and S. gemellum which showed two hybridization signals. The results point out to different ways of karyotype differentiation in Solanum and closely related genera and bring important issues on the value of the cytogenetical information for taxonomic studies.