Comparative cytogenetics of three economically important Piper L. species from the Brazilian Amazon.

The species Piper hispidinervum, Piper aduncum, and Piper affinis hispidinervum have essential oils with high levels of safrole, dillapiole, and sarisan, respectively. Safrole is important for pharmaceutical and chemical industries, while dillapiole and sarisan are promising compounds to control insects and fungi. These species are very similar morphologically and their taxonomy is controversial. Divergent hypotheses consider P. aduncum and P. hispidinervum either as a single species or as distinct taxa, while P. affinis hispidinervum is inferred to be a natural hybrid or a chemotype of P. hispidinervum. Delimiting the taxonomic boundaries would be helpful for germplasm conservation and breeding programs. This study aimed to undertake a detailed analysis of P. aduncum, P. hispidinervum, and P. affinis hispidinervum karyotype and rDNA sites. Genomic in situ hybridization (GISH) was used to establish genomic homology among species and to test the natural hybridization hypothesis for origin of P. affinis hispidinervum. Karyotype traits were similar for all three species: 2n = 26 small chromosomes, predominantly metacentric. All three species exhibited CMA+ bands on the secondary constriction of chromosome pair 4. A size-heteromorphic 35S rDNA site was co-localized with the CMA+ band. A 5S rDNA site was located in the proximal region of chromosome pair 7. The patterns of genomic hybridization revealed that the repetitive DNA fraction of the species is highly similar in terms of proportion of genome, sequence type, and distribution. Our findings did not allow us to differentiate the three species and point to the importance of deeper genomic studies to elucidate the taxonomic controversy.

Genomic constitution, allopolyploidy, and evolutionary proposal for Cynodon Rich. based on GISH.

Polyploidy is the main mechanism for chromosome number variation in Cynodon. Taxonomic boundaries are difficult to define and, although phylogenetic studies indicate that some species are closely related, the degree of genomic similarity remains unknown. Furthermore, the Cynodon species classification as auto or allopolyploids is still controversial. Thus, this study aimed to investigate the genomic constitution in diploid and polyploid species using different approaches of genomic in situ hybridization (GISH). To better understand the hybridization events, we also investigated the occurrence of unreduced gametes in C. dactylon diploid pollen grains. We suggest a genomic nomenclature of diploid species as DD, D1D1, and D2D2 for C. dactylon, C. incompletus, and C. nlemfuensis, and DDD2D2 and DD2D1D1 for the segmental allotetraploids of Cynodon dactylon and C. transvaalensis, respectively. Furthermore, an evolutionary proposal was built based on our results and previous data from other studies, showing possible crosses that may have occurred between Cynodon species.

Direct evidence for crossover and chromatid interference in meiosis of two plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei).

Crossing over, in addition to its strictly genetic role, also performs a critical mechanical function, by bonding homologues in meiosis. Hence, it is responsible for an orderly reduction of the chromosome number. As such, it is strictly controlled in frequency and distribution. The well-known crossover control is positive crossover interference which reduces the probability of a crossover in the vicinity of an already formed crossover. A poorly studied aspect of the control is chromatid interference. Such analyses are possible in very few organisms as they require observation of all four products of a single meiosis. Here, we provide direct evidence of chromatid interference. Using in situ probing in two interspecific plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei) during anaphase I, we demonstrate that the involvement of four chromatids in double crossovers is significantly more frequent than expected (64% versus 25%). We also provide a physical measure of the crossover interference distance, covering ~30-40% of the relative chromosome arm length, and show that the centromere acts as a barrier for crossover interference. The two arms of a chromosome appear to act as independent units in the process of crossing over. Chromatid interference has to be seriously addressed in genetic mapping approaches and further studies.

Identification of ribosomal sites and karyotype analysis in Festuca ulochaeta Steud. and Festuca fimbriata Ness., grasses native to Brazil.

Festuca L. has more than 600 perennial species described, which makes it the largest genus within the family Poaceae. In Brazil, only two native species of Festuca have been described, for which cytogenetic studies need to be strengthened: Festuca ulochaeta and Festuca fimbriata. The aim of this study was to characterize the karyotypes of F. ulochaeta and F. fimbriata based on the mapping of rDNA sites. The FISH was performed with 35S and 5S rDNA probes. Both species have 42 chromosomes, of which 36 were metacentric and six were submetacentric. Festuca fimbriata has two pairs of 35S rDNA sites, one located on the metacentric pair 4, in an interstitial position, and one at the submetacentric pair 14 in the proximal position. Festuca ulochaeta has one pair of 35S rDNA in interstitial-proximal position in the metacentric pair 3. Both species showed 5S rDNA sites only on chromosome pair 21 in the terminal position of the short arm. The analysis of the chromosomal characteristics indicates that these species have a symmetrical karyotype and allopolyploid origin.

Comparative meiosis and cytogenomic analysis in euploid and aneuploid hybrids of Urochloa P. Beauv.

The genus Urochloa includes most of the important grasses and hybrids currently used as pastures in the tropical regions. Cytogenetic analyzes have identified some aneuploid hybrids that provide new perspectives for genetic breeding. The objective was to analyze the meiotic behavior in euploid (2n = 4x = 36) and aneuploid (2n = 4x = 36 + 2) hybrids of U. ruziziensis x U. decumbens and U. ruziziensis x U. brizantha. Later, the chromosomes and respective genomes involved in pairing configurations and abnormalities were identified through GISH, with an emphasis on tracking the behavior of the additional chromosomes in the aneuploid hybrid U. ruziziensis x U. decumbens (B1B2B2B2 genomes). The aneuploid U. ruziziensis x U. decumbens shows a higher frequency of univalents, reduction of bivalents, and higher index of irregularities compared with the euploid hybrid. For the aneuploid U. ruziziensis x U. brizantha, there was a reduction in the frequency of univalents, an increase in bivalent and trivalent rates and a lower frequency of abnormalities when compared with the euploid hybrid. The rates of meiotic abnormalities and pairing configurations are parental genotype-dependent and influenced by trisomy. The chromosomes of the B1 and B2 genomes of the aneuploid hybrid (U. ruziziensis x U. decumbens) are involved in the formation of univalents, bivalents, and multivalents in inter-, intra- and inter-intragenomic pairings. In general, the segregation times of chromosomes of the genomes are different, since the chromosomes of the B1 genome segregate more slowly.

Comparative karyotype analysis among cytotypes of Cynodon dactylon (L.) Pers. (Poaceae).

Cynodon dactylon is characterized by taxonomic and systematic complexity, and polyploidy is one of the factors responsible for its genetic and morphological diversity. The aim of the present study was to compare karyotypes of C. dactylon cytotypes based on fluorescent banding and nuclear DNA content. The nine C. dactylon accessions evaluated were obtained from the Active Germplasm Bank (BAG) of the United States Department of Agriculture (USDA). Roots were pretreated with cycloheximide, fixed in Carnoy's solution and subjected to enzymatic digestion. Slides were prepared by the dissociation and air-drying technique. The fluorescent banding pattern was obtained using chromomycin A3 (CMA)/4,6-dimidino-2-phenylindole (DAPI) staining and DNA content was estimated by flow cytometry. The chromosome number of the accessions ranged from 2n = 2x = 18 to 2n = 5x = 45. Chromosomal polymorphism was observed based on the distribution and number of heterochromatic bands, with CMA+ bands located in the pericentromeric position and DAPI+ bands mainly in the terminal position. PI477004-26 (2n = 3x = 27) and PI291966-27 (2n = 4x = 36) had the highest and lowest number of DAPI+ bands, respectively. The number of CMA+ bands was stable, as only PI477004-26, PI291966-27 and PI289750-10 (2n = 5x = 45) showed variation. There was no direct correlation between an increase in the ploidy level and an increase in the percentage of heterochromatic regions, mainly in relation to A-T-rich blocks. The chromosomal banding variation found reinforces the notion of allopolyploidy occurrence in C. dactylon and demonstrates the genomic complexity of this species regard to repetitive DNA content.

Characterization of aneuploidy in interspecific hybrid between Urochloa ruziziensis (R. Germ. & Evrard) Crins and Urochloa decumbens (Stapf) R. D. Webster.

The aim of the study was to characterize the type of aneuploidy present in the hybrid Urochloa ruziziensis × Urochloa decumbens and to confirm the origin of the additional chromosomes through comparative analysis of the hybrid and parental karyotypes. C and CMA banding techniques were used for chromosome differentiation. The parental genotypes showed 36 chromosomes. The hybrid presented plants with 36 + 2 chromosomes and plants with 36 + 1 chromosomes. Urochloa ruziziensis (4x) presented four chromosomes with CMA and C bands co-located in the terminal position. In U. decumbens, four chromosomes presented terminal CMA bands, eight chromosomes were distinguished by C banding with pericentromeric and terminal bands, one chromosome with terminal band at both ends and one chromosome presented one C terminal band. For the hybrid, CMA bands were found on five chromosomes and C bands on seven chromosomes, all in terminal position. Aneuploidy was identified in pairs 3' and 4' in the hybrid plants with 36 + 2 chromosomes, characterizing it as double trisomy. The karyotype of hybrid plants with 36 + 1 chromosomes indicated elimination of the additional chromosome identified in pair 4' and maintenance of trisomy on pair 3'. The comparative analysis of karyotypes indicates that the additional chromosomes that characterize the trisomy were inherited from U. ruziziensis (artificial tetraploid).

45S rDNA sites in meiosis of Lolium multiflorum Lam.: variability, non-homologous associations and lack of fragility.

In this study, we evaluated the behavior of 45S ribosomal DNA (rDNA) sites during the meiosis of Lolium multiflorum. The reason to study it in this species is that 45S rDNA sites are usually visualized as gaps in mitotic metaphase chromosomes and were initially denominated fragile sites (FSs). In different species, FSs were related to rearrangements that alter the karyotype and affect the chromosome pairing in meiosis. However, our findings show that the chromosome pairing in L. multiflorum is regular and, as in mitosis, the number of sites is variable. In diakinesis with five sites, one of the bivalents was in hemizygous state while, in diakinesis with seven sites, one of the bivalents had three conspicuous signals, two in syntheny in one of the homologous. Only four cells had gaps in the region of the 45S rDNA. Owing to the lower number of signals observed at the initial stages of meiosis, it is assumed that they are involved both in homologous and non-homologous associations and that they might assist the chromosome pairing. Regarding segregation, only meiocytes with five and six 45S rDNA signals were observed, and they were characterized by the segregation of 2/3 signals in the poles of anaphases I up to metaphases II; 2/2 and 3/3 in anaphases II and telophases II; and also 2/2 and 4/4 in the nuclei of tetrads, unlike the number of 45S signals expected. The numerical non-equivalence of sites among nuclei at later stages of meiosis is explained by the presence of chromosomes with hemizygous sites.

Forage potential of Urochloa genotypes by using leaf anatomy / Avaliação do potencial forrageiro de genótipos de Urochloa por meio da anatomia foliar

ABSTRACT: The digestibility potential of leaves from forages depends on the amount of nutrition in their tissues, with low lignin deposition in the cell wall, mainly parenchyma and phloem. This research evaluated the leaf structure of different Urochloa genotypes and discussed its potential for evaluating digestibility. The cultivars U. brizantha, U. decumbens, U. ruziziensis and three clones of U. ruziziensis (1, 95 and 97), which are under development in breeding programs, were evaluated. Plants were grown under the recommended culture conditions for the Urochloa species. Plants were cut 60 days after sowing, and leaves were collected at 15 days of regrowth. Leaves were fixed in FAA 70 and further stored in 70% ethanol until being submitted to the usual microtechniques for the preparation of microscopy slides. The area of the tissues from the interveinal and midrib regions was measured using ImageJ software, and their proportions were calculated. In the interveinal region, the proportion of chlorophyll parenchyma was greater for U. decumbens and the Clone 1 genotypes. Urochloa brizantha and clones 95 and 97 showed a higher proportion of the vascular bundle compared to U. ruziziensis, U. decumbens and Clone 1. The proportion of the ground parenchyma in the midrib was greater in U. brizantha, Clone 95 and Clone 97. Thus, it can be concluded that the Clone 1 genotypes (from U. ruziziensis) showed leaf tissues (parenchyma and phloem) with higher digestibility potential; in addition, U. brizantha and U. decumbens showed a high percentage of xylem and sclerenchyma, which reduces their quality as forage.

RESUMO: O potencial de digestibilidade das folhas de forrageiras depende da quantidade de seus tecidos com baixa deposição de lignina nas paredes celulares, principalmente o parênquima e floema. O presente trabalho objetivou avaliar a estrutura foliar de diferentes genótipos de Urochloa e seu potencial de digestibilidade foliar. Foram avaliadas as cultivares U. brizantha, U. decumbens, U. ruziziensis e três clones de U. ruziziensis (1, 95 e 97). As plantas foram cultivadas em campo com condições de manejo recomendadas para a espécie, sendo cortadas aos 60 dias e as folhas coletadas aos 15 dias de rebrota. As folhas foram fixadas em FAA 70, armazenadas em etanol 70% e posteriormente submetidas à microtécnica usual para preparação de lâminas semipermanentes. Foram analisadas as regiões internervural e da nervura mediana com o auxílio do software Image J sendo mensurada a área de cada tecido da folha e depois calculada a sua proporção em relação à área total das secções. Na região internervural, a proporção de parênquima foi maior em U. decumbens e no Clone 1. Ainda nesta região, Urochloa brizantha e os Clones 95 e 97 apresentaram maiores médias para a proporção de feixes vasculares. Na nervura mediana, a proporção do parênquima foi maior em U. brizantha, Clone 95 e Clone 97. De maneira geral, o Clone 1 (proveniente de U. ruziziensis) apresentou parênquima e floema em maiores proporções, enquanto genótipos de U. brizantha e U. decumbens demonstram altas quantidades de xilema e esclerênquima que reduzem o seu potencial de digestibilidade.

Relationship between epigenetic marks and the behavior of 45S rDNA sites in chromosomes and interphase nuclei of Lolium-Festuca complex.

The grasses of the Lolium-Festuca complex show a prominent role in world agricultural scenario. Several studies have demonstrated that the plasticity of 45S rDNA sites has been recently associated with the possible fragility of the loci. Often, these fragile sites were observed as extended sites and gaps in metaphases. This organization can be evaluated in relation to their transcriptional activity/accessibility through epigenetic changes. Thus, this study aimed to investigate the relationship of the 5-methylcytosine and histone H3 lysine-9 dimethylation in different conformations of 45S rDNA sites in interphase nuclei and in metaphase chromosomes of L. perenne, L. multiflorum and F. arundinacea. The FISH technique using 45S rDNA probes was performed sequentially after the immunolocalization. The sites showed predominantly the following characteristics in the interphase nuclei: intra- and perinucleolar position, decondensed or partially condensed and hypomethylated and hyper/hypomethylated status. Extranucleolar sites were mainly hypermethylated for both epigenetic marks. The 45S rDNA sites with gaps identified in metaphases were always hypomethylated, which justifies it decondensed and transcriptional state. The frequency of sites with hypermethylated gaps was very low. The structural differences observed in these sites are directly related to the assessed epigenetic marks, justifying the different conformations throughout the cell cycle.

Location of low copy genes in chromosomes of Brachiaria spp.

Repetitive DNA sequences have been widely used in cytogenetic analyses. The use of gene sequences with a low-copy-number, however, is little explored especially in plants. To date, the karyotype details in Brachiaria spp. are limited to the location of rDNA sites. The challenge lies in developing new probes based on incomplete sequencing data for the genus or complete sequencing of related species, since there are no model species with a sequenced genome in Brachiaria spp. The present study aimed at the physical location of conserved genes in chromosomes of Brachiaria ruziziensis, Brachiaria brizantha, and Brachiaria decumbens using RNAseq data, as well as sequences of Setaria italica and Sorghum bicolor through the fluorescent in situ hybridization technique. Five out of approximately 90 selected sequences generated clusters in the chromosomes of the species of Brachiaria studied. We identified genes in synteny with 5S and 45S rDNA sites, which contributed to the identification of chromosome pairs carrying these genes. In some cases, the species of Brachiaria evaluated had syntenic segments conserved across the chromosomes. The use of genomic sequencing data is essential for the enhancement of cytogenetic analyses.

Regrowth age modifies the leaf anatomy of Brachiaria genotypes / A idade de regeneração modifica a anatomia foliar dos genótipos de Brachiaria

Changes in leaf anatomy were evaluated in genotypes of Brachiaria brizantha, Brachiaria decumbens, Brachiaria ruziziensis and three Brachiaria ruziziensis clones through tissue proportion in internerval and midrib regions at three regrowth ages. Plants were grown and cutting was performed after 60 days. Further, leaves were sampled at 8, 15 and 29 regrowth days and processed with usual plant microtechnique. The internerval region showed higher parenchyma percentage at 15 days for Clone 95 and similar values at 15 and 29 days for Clone 1. The proportion of vascular bundles was lower after 15 days in Clones 1 and 95 and 29 days in B. brizantha. In the midrib, the parenchyma proportion was higher at 29 days in B. brizantha and lower at 15 days in B. ruziziensis. The proportion of vascular bundles was higher at 8 days in B. decumbens, B. brizantha and Clone 1, and lower at 29 days for Clones 97 and 95 and at 8 days in B. ruziziensis. Therefore, the regrowth age modifies the percentage of leaf tissues in Brachiaria genotypes, in which the fibers and vascular bundles increase at 29 days and 8-day-old leaves are not fully developed.

A anatomia foliar foi avaliada em genótipos de Brachiaria brizantha, Brachiaria decumbens, Brachiaria ruziziensis e três clones de Brachiaria ruziziensis; quanto à proporção de tecidos foliares, em três idades de rebrota. As plantas foram cultivadas e cortadas após 60 dias. As folhas foram amostradas aos 8, 15, 29 dias de rebrota e analisadas pela metodologia de microtécnica vegetal. Na região internervural, a proporção de parênquima foi maior aos 15 dias para o Clone 95, e aos 15 e 29 dias para o Clone 1. Proporção de feixes vasculares foi menor aos 15 dias para os Clones 1 e 95 e aos 29 dias para genótipo B. brizantha. Na nervura central, a proporção de parênquima foi maior aos 29 dias para B. brizantha e menor, aos 15 dias, para B. ruziziensis. Proporção dos feixes vasculares foi maior aos 8 dias para genótipo B. decumbens, B. brizantha e para o Clone 1, e menor aos 29 dias para os Clones 97 e 95; aos 8 dias, para genótipo B. ruziziensis. Portanto, a idade de rebrota modifica a porcentagem de tecidos foliares em genótipos de Brachiaria; contudo, fibras e feixes vasculares aumentam aos 29 dias; aos 8 dias, as folhas não estão totalmente desenvolvidas.

Cytotoxic and genotoxic effects of Solanum lycocarpum St. -Hil (Solanaceae) on the cell cycle of Lactuca sativa and Allium cepa / Efeito citotóxicos e genotóxicos de extratos Solanum lycocarpum St. -Hil (Solanaceae) no ciclo celular de Lactuca sativa e Allium cepa

Solanum lycocarpum St.- Hil popularly known as 'fruta-de-lobo' or 'lobeira' is native to the Brazilian Cerrado, and used in folk medicine due to its phytotherapic properties. The action of S. lycocarpum on the cell cycle and chromosomes in order to demonstrate whether there are aneugenic and/or clastogenic effects is unknown. Thus, this study aimed at investigating the cytotoxic and genotoxic potential of methanol and hexane extracts of S. lycocarpum on growth and cell cycle of Lactuca sativa and Allium cepa. Roots from both species were exposed for 72 hours to methanol and hexane extracts with 50, 100, and 200 µg mL-1 of S. lycocarpum. Slides were prepared by the squash technique and then analyzed to determine the mitotic index and the total of chromosomal and nuclear abnormalities. The frequencies of chromosomal and nuclear abnormalities were high and significant with a dose-dependent effect, indicating that S. lycocarpum has a cytotoxic and genotoxic action depending on the dose used on meristem cells of A. cepa and L. sativa.

Solanum lycocarpum St.-Hil (Solanaceae) conhecida popularmente como fruta-de-lobo ou lobeira é uma planta nativa do Cerrado brasileiro, utilizada na medicina popular nos tratamentos de diabetes, obesidade e redução do colesterol. Ainda não é conhecida a ação de S. lycocarpum sobre o ciclo celular e os cromossomos, demonstrando se possuem efeitos aneugênicos e/ou clastogênicos. O objetivo desse estudo foi investigar o potencial citotóxico e genotóxico dos extratos metanólico e hexânico de S. lycocarpum sob o crescimento e ciclo celular de Lactuca sativa e Allium cepa. As raízes de ambas as espécies foram expostas por 72h aos extratos metanólico e hexânico com 50, 100 e 200 µg mL-1 de S. lycocarpum. As lâminas foram montadas pela técnica de esmagamento e em seguida foram analisadas, afim de determinar o índice mitótico e anormalidades cromossômicas e nucleares. As frequências de anormalidades cromossômicas e nucleares foram altas e significativas, com efeito dose-dependente e confirmando que S. lycocarpum tem ação citotóxica e genotóxica de acordo com a dose utilizada sobre as células meristemáticas de A. cepa e L. sativa.

Decondensation of chromosomal 45S rDNA sites in Lolium and Festuca genotypes does not result in karyotype instability.

Fragile sites (FSs) in plants have been described for species like Lolium and other grasses. Whereas in humans FSs were shown to be involved in genome instabilities; the consequences of FSs expression in plants are not known yet. To evaluate whether FSs cause karyotype instabilities, we assessed the frequency of micronuclei and lagging chromosomes in meristematic cells, the stability of the DNA content, and the occurrence of neocentromeres in the presumed chromosomal fragments of Lolium perenne, Lolium multiflorum, Festuca arrundinacea, and two Festulolium hybrids. The cell cycle analysis along with flow cytometric genome size measurements showed high stability in all genomes evaluated. Neocentromeric activity was neither observed in the presumed fragments nor in any other chromosomal region, then this is not the mechanism responsible by the stability. However, Fluorescence in situ hybridization (FISH) with a 45S ribosomal DNA (rDNA) probe in combination with YOYO staining of metaphasic chromosomes showed that many extended nucleolus organizing region (NOR) form very thin YOYO-positive chromatin fibers connecting the acentric 'fragment' with the centromere-containing chromosome region. The obtained data indicate that the expression of FSs does not result in genome instabilities or neocentromere formation. The FS-containing 45S rDNA carrying chromatin fibers undergo a cell cycle and gene activity-dependent dynamic decondensation process.

Stability in chromosome number and DNA content in synthetic tetraploids of Lolium multiflorum after two generations of selection / Estabilidade no número cromossômico e no conteúdo de DNA em tetraploides sintéticos de Lolium multiflorum após duas gerações de seleção

ABSTRACT: Chromosome doubling of Italian ryegrass genotypes ( Lolium multiflorum Lam.) adapted to the brazilian edaphoclimatic conditions is an important strategy used by breeders and aims to obtain more vigorous genotypes with better forage quality and disease resistance. The effectiveness of chromosome doubling can be measured by genetic stability and fertility rates of plants over generations. However, a common problem in the polyploidization process is the regeneration of mixoploid plants that have impaired fertility and genetic stability. The objective of this study was to verify if progenies of recently tetraploidized plants remain stable regarding DNA content and chromosome number, over two generations. Progenies of L. multiflorum plants artificially tetraploidized with colchicine treatment were evaluated. Chromosome counting and estimates of the DNA content were used to evaluate the genetic stability. The percentage of tetraploid plants (4X) increased over generations (18%, 34% and 91% in cycle 0, 1 and 2, respectively). All progenies identified as tetraploid by flow citometry showed variation in chromosome number (mixoploidy), but produced viable seeds. Results showed that stabilization in chromosome number and DNA content in tetraploidized plant progenies requires time and that the success of this procedure depends on a continuous and accurate screening and selection.

RESUMO: A duplicação cromossômica de genótipos de azevém anual ( Lolium multiflorum Lam.) adaptados às condições edafoclimáticas brasileira é uma estratégia importante usada pelos melhoristas e visa a obtenção de genótipos mais vigorosos com melhor qualidade de forragem e resistência a doenças. A eficiência da duplicação cromossômica pode ser medida pela estabilidade genética e taxas de fertilidade das plantas ao longo das gerações. No entanto, um dos problemas comumente encontrados no processo de poliploidização é a regeneração de plantas mixoploides que apresentam comprometimento na fertilidade e instabilidade genética. O objetivo deste estudo foi verificar se progênies oriundas de plantas tetraploidizadas recentemente se mantêm estáveis quanto ao conteúdo de DNA e ao número cromossômico, ao longo de duas gerações. Foram avaliadas progênies de plantas de L. multiflorum tetraploidizadas artificialmente com tratamento de colchicina. A estabilidade genética foi avaliada por meio de contagens cromossômicas e estimativa da quantidade de DNA. A porcentagem de plantas tetraploides (4X) aumentou ao longo das gerações (18%, 34% e 91% no ciclo de 0, 1 e 2, respectivamente). Todas as progênies identificadas como tetraploides por meio de citometria de fluxo apresentaram variação no número cromossômico (mixoploidia), entretanto, produziram sementes viáveis. Os resultados demonstraram que a estabilização no número cromossômico e no conteúdo de DNA em progênies de plantas tetraploidizadas requer tempo e que o sucesso desse procedimento depende de um contínuo e rigoroso monitoramento e seleção.

Fragile sites of 45S rDNA of Lolium multiflorum are not hotspots for chromosomal breakages induced by X-ray.

Sites of 45S rDNA of Lolium are regions denominated fragile sites (FSs), constituting regions slightly stained with DAPI due to increased DNA unpacking in metaphasic chromosomes. Considered to be fragile regions in the genome, the FSs might be more responsive to induced breaks and result in chromosomal fragments and rearrangements, unless repairing mechanisms such as recombination or de novo telomere formation play a role at the break site of the DNA. Thus, this study aimed at investigating if SFs from Lolium are hotspots for the occurrence of breakages induced by X-ray and if they are regions favorable to synthesize new telomeres, using Hordeum vulgare as a comparative model. Lolium multiflorum and H. vulgare seedlings were irradiated with 20 and 50 Gy X-ray and evaluated one day following the irradiation and at 7-days intervals for a 28-days period, using FISH technique with 45S rDNA and Arabidopsis-type telomere probes in order to investigate the presence of chromosomal breakages and new telomere formation. H. vulgare did not survive after a few days of irradiation due to the increased rate of abnormalities. L. multiflorum also exhibited chromosomal abnormalities following the exposure, yet over the 28-days trial it had a decrease in the chromosomal damage rate and formation of de novo telomere has not been detected along this time. Despite being considered to be fragile regions in the genome, the 45S rDNA sites of Lolium are not hotspots to chromosomal breakages after the induction of breakages.

Chromosomal distribution of H3K4me2, H3K9me2 and 5-methylcytosine: variations associated with polyploidy and hybridization in Brachiaria (Poaceae).

KEY MESSAGE: Assessment of chromosomal distribution of modified histones and 5-methylcytosine shown that there are diversification of chromosomal types among species of Brachiaria and its interspecific hybrids. Histone post-translational modifications and DNA methylation are epigenetic processes that are involved in structural and functional organization of the genome. This study compared the chromosomal distribution of modified histones and 5-methylcytosine (5-mCyt) in species and interspecific hybrids of Brachiaria with different ploidy levels and reproduction modes. The relation between H3K9me2 and 5-mCyt was observed in the nucleolus organizer region, centromeric central domain and pericentromeric region. H3K4me2 was detected in euchromatic domains, mainly in the terminal chromosomal regions. Comparison of chromosomal distribution among species and hybrids showed greater variation of chromosomal types for the H3K9me2 in B. decumbens (tetraploid and apomictic species) and the 963 hybrid, while, for the H3K4me2, the variation was higher in B. brizantha and B. decumbens (tetraploid and apomictic species) and 963 hybrid. The chromosome distribution of 5-mCyt was similar between B. brizantha and B. decumbens, which differ from the distribution observed in B. ruziziensis (diploid and sexual species). Significant alterations in DNA methylation were observed in the artificially tetraploidized B. ruziziensis and in the interspecific hybrids, possibly as result of hybridization and polyploidization processes. The monitoring of histone modifications and DNA methylation allowed categorizing nuclear and chromosomal distribution of these epigenetic marks, thus contributing to the knowledge of composition and structure of the genome/epigenome of Brachiaria species and hybrids. These data can be useful for speciation and genome evolution studies in genus Brachiaria, and represent important markers to explore relationships between genomes.

Effect of SPL (Spent Pot Liner) and its main components on root growth, mitotic activity and phosphorylation of Histone H3 in Lactuca sativa L.

Spent Pot Liner (SPL) is a solid waste from the aluminum industry frequently disposed of in industrial landfills; it can be leached and contaminate the soil, sources of drinking water and plantations, and thus may pose a risk to human health and to ecosystems. Its composition is high variable, including cyanide, fluoride and aluminum salts, which are highly toxic and environmental pollutants. This study evaluated the effect of SPL and its main components on root growth and the mitosis of Lactuca sativa, by investigating the mechanisms of cellular and chromosomal alterations with the aid of immunolocalization. To this end, newly emerged roots of L. sativa were exposed to SPL and its main components (solutions of cyanide, fluoride and aluminum) and to calcium chloride (control) for 48h. After this, root length was measured and cell cycle was examined by means of conventional cytogenetics and immunolocalization. Root growth was inhibited in the treatments with SPL and aluminum; chromosomal and nuclear alterations were observed in all treatments. The immunolocalization evidenced normal dividing cells with regular temporal and spatial distribution of histone H3 phosphorylation at serine 10 (H3S10ph). However, SPL and its main components inhibited the phosphorylation of histone H3 at serine 10, inactivated pericentromeric regions and affected the cohesion of sister chromatids, thus affecting the arrangement of chromosomes in the metaphase plate and separation of chromatids in anaphase. In addition, these substances induced breaks in pericentromeric regions, characterized as fragile sites.

Chromosomal distribution and evolution of abundant retrotransposons in plants: gypsy elements in diploid and polyploid Brachiaria forage grasses.

Like other eukaryotes, the nuclear genome of plants consists of DNA with a small proportion of low-copy DNA (genes and regulatory sequences) and very abundant DNA sequence motifs that are repeated thousands up to millions of times in the genomes including transposable elements (TEs) and satellite DNA. Retrotransposons, one class of TEs, are sequences that amplify via an RNA intermediate and reinsert into the genome, are often the major fraction of a genome. Here, we put research on retrotransposons into the larger context of plant repetitive DNA and genome behaviour, showing features of genome evolution in a grass genus, Brachiaria, in relation to other plant species. We show the contrasting amplification of different retroelement fractions across the genome with characteristics for various families and domains. The genus Brachiaria includes both diploid and polyploid species, with similar chromosome types and chromosome basic numbers x = 6, 7, 8 and 9. The polyploids reproduce asexually and are apomictic, but there are also sexual species. Cytogenetic studies and flow cytometry indicate a large variation in DNA content (C-value), chromosome sizes and genome organization. In order to evaluate the role of transposable elements in the genome and karyotype organization of species of Brachiaria, we searched for sequences similar to conserved regions of TEs in RNAseq reads library produced in Brachiaria decumbens. Of the 9649 TE-like contigs, 4454 corresponded to LTR-retrotransposons, and of these, 79.5 % were similar to members of the gypsy superfamily. Sequences of conserved protein domains of gypsy were used to design primers for producing the probes. The probes were used in FISH against chromosomes of accesses of B. decumbens, Brachiaria brizantha, Brachiaria ruziziensis and Brachiaria humidicola. Probes showed hybridization signals predominantly in proximal regions, especially those for retrotransposons of the clades CRM and Athila, while elements of Del and Tat exhibited dispersed signals, in addition to those proximal signals. These results show that the proximal region of Brachiaria chromosomes is a hotspot for retrotransposon insertion, particularly for the gypsy family. The combination of high-throughput sequencing and a chromosome-centric cytogenetic approach allows the abundance, organization and nature of transposable elements to be characterized in unprecedented detail. By their amplification and dispersal, retrotransposons can affect gene expression; they can lead to rapid diversification of chromosomes between species and, hence, are useful for studies of genome evolution and speciation in the Brachiaria genus. Centromeric regions can be identified and mapped, and retrotransposon markers can also assisting breeders in the developing and exploiting interspecific hybrids.

Functional repetitive sequences and fragile sites in chromosomes of Lolium perenne L.

Lolium perenne is considered a high-quality forage widely used in temperate regions to meet the shortage of forage during the winter. In this species, some peculiarities related to cytogenetic aspects have already been described, as the variability in number and position of 45S ribosomal DNA (rDNA) sites and the expression of fragile sites, which require further studies to support the understanding of their causes and consequences. In this way, this study aimed to evaluate the relationship between the expression of fragile sites and functional repetitive sequences (rDNA and telomeric) in chromosomes of diploid and polyploid cultivars of L. perenne. The techniques of FISH, Ag-NOR and fluorescence banding were used to assess the distribution of sites of 45S rDNA, 5S, telomeric sequences, and the transcriptional activity of the 45S ribosomal genes and the distribution of AT- and/or GC-rich sequences in L. perenne, respectively. There was variability in the number and location of 45S rDNA sites, which was not observed for 5S rDNA sites. One of the genotypes showed two 45S rDNA sites on the same chromosome, located in different chromosome arms. Breaks and gaps were found in 45S rDNA sites in most metaphases evaluated for both cultivars. Telomeric sequences were not detected at the end of the chromosomal fragments corresponding to the location of breaks at 45S sites. Apparently, the transcriptional activity was modified in fragile sites. Variation in the number and size of nucleoli, nucleolar fusions and dissociations were observed. All CMA(+) bands were colocalized with the 45S sites.