Andréa Mittelmann

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.

Potencial da população de milho ESA23B para o melhoramento dos teores de proteína e óleo

Among the traits that may add commercial value to maize (Zea mays L.), those related to nutritional quality, specially protein and oil content, are of great interest to the feed industry. The objective of this work was studying the variability of protein and oil content, as well as yield, in a group of maize testcrosses. One hundred and twenty S1 families of the ESA23B maize population were crossed with two testers, an open-pollinated population (BR108) and an exotic line (CML269). Testcrosses were evaluated at two locations under a completely randomized block design with three replications. Ear and grain yield, protein and oil content were evaluated. The three-way interaction location x tester x progeny was significant for all traits, except for oil content. Differences among progenies were detected for all traits. Testcross means varied from 8.40% to 11.82% for protein content and from 3.77% to 5.10% for oil content. Hybrids with similar or superior means to the best check were identified for protein content, ear yield, and grain yield. Estimates of the interpopulation additive variance ranged from 0.553 to 1.124 for protein content; 0.034 to 0.057 for oil content (percent data); 132.13 to 521.74 for ear yield and 116.33 to 381.73 for grain yield (data in grams per plant). The population ESA23B can potentially be improved for all the traits studied. Associations among traits were weak, thus concomitant selection of quality and yield can be feasible.

Comportamento agronômico de populações de azevém anual (Lolium multiflorum L.) para cultivo invernal na região sudeste

Na Regiao Sudeste o periodo do inverno e caracterizado pela escassez e perda de qualidade das pastagens, sendo o azevem anual uma das forrageiras invernais mais recomendadas para suplementacao da dieta dos rebanhos leiteiros nesta epoca do ano. Com este trabalho, buscou-se avaliar o comportamento de populacoes de azevem resultantes de coleta de germoplasma. Foram avaliadas 30 populacoes, sendo 22 resultantes de coletas recentes realizadas na Regiao Sul e oito pertencentes a colecao da Embrapa. Foi utilizado o delineamento experimental de blocos ao acaso com tres repeticoes, sendo realizados sete cortes. Foram avaliados: altura da planta, porcentagem e producao de materia seca, rebrota, numero de dias ate o florescimento e producao de sementes. Observou-se variacao entre as populacoes para todas as caracteristicas avaliadas. A estimativa da producao total de materia seca variou de 3654 kg/ha (populacao LE 284) a 8544 kg/ha (CNPGL 164). Os resultados demonstraram elevado potencial de producao de forragem entre as populacoes de azevem coletadas, sendo que algumas delas podem ser recomendadas para cultivo invernal na Regiao Sudeste.

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.

Morphology and pollen viability of Lolium multiflorum Lam.

O estudo e a caracterizacao do grao de polen sao fundamentais para diferentes areas, em especial, para a taxonomia, o melhoramento genetico, a filogenia e a paleobotânica. Para genotipos de Lolium multiflorum Lam., naturalizados no Brasil, cultivares introduzidas ou populacoes de melhoramento, diploides e poliploides, nao existem relatos sobre a caracterizacao morfopolinica. Nesse trabalho foram analisados dez gentotipos de azevem anual (L. multiflorum). A viabilidade dos graos de polen foi verificada por coloracao com os corantes carmim propionico e Alexander e a morfologia por meio da tecnica de acetolise, sendo obtidas medidas do eixo polar (P), do diâmetro equatorial (E), e da espessura da exina, e analise dos graos de polen por microscopia eletronica de varredura (MEV). Todos os genotipos avaliados apresentaram alta taxa de viabilidade polinica (>89%) para os dois corantes utilizados. Houve diferenca entre os genotipos para os caracteres quantitativos eixo polar, diâmetro equatorial na vista equatorial, exina, endoexina, ectoexina e razao P/E. Os graos de polen foram caracterizados como pequenos, monoaperturados, com abertura nao proeminente e com âmbito circular. As diferencas morfometricas entre os graos-de-polen podem auxiliar na distincao entre os mesmos e, posteriormente, serem utilizadas para comparacao entre os niveis de ploidia, auxiliando, assim, nos programas de melhoramento da especie.

Quantitative variation for grain quality in Brazilian maize populations

Development of maize (Zea mays L.) cultivars with high grain protein and oil concentrations and an appropriate amino acid composition, without losses in grain yield, represents a challenge in breeding programs. The objective of this work was to study the nutritional quality of ten Brazilian maize populations evaluated in three environments. A randomized block design with six replications was used. Ear yield and the concentrations of protein, oil, serine, glutamic acid, alanine, leucine, tyrosine and phenylalanine were evaluated. Individual and combined analyses of variance were performed. Populations diverged for most of the traits. Environmental effects influenced variation for most of the traits, unlike genotype-environment interaction, allowing the selection on the average of environments. Positive association exists among protein and most of the amino acids, when considered on a dry matter basis and there is no association between nutritional quality and yield; therefore, the development of cultivars that are superior for both traits is expected to be feasible.

Meiotic and mitotic behaviour of B chromosomes of ryegrass

Mitotic and meiotic analyses using conventional and fluorescent stains were employed in plants (accession ETBAZ 055 - origin: Capao do Leao, Rio Grande do Sul State, Brazil) from the Germplasm Active Bank of Ryegrass (Banco Ativo de Germoplasma de Azevem) of Embrapa, for observing the behaviour of B chromosomes. In several meiotic stages, there were observed up to two B chromosomes, which have presented an unstable behaviour regarding their precocious ascension to metaphase I or delays during anaphase I. At the end of the process, the Bs showed predominantly segregation for the nuclei under formation in order to guarantee their propagation. Concerning the cells that comprise the anther tapetum and the root meristems, the B chromosomes have presented a more stable behaviour.

Variabilidade entre plantas de azevém para caracteres relacionados à precocidade

The objective of this study was to verify intrapopulation variability for traits related to early forage production and heading date in an annual ryegrass population (Lolium multiflorum). A total of 500 plants were evaluated for heading date. Vigor, plant height and tiller number were evaluated 90 days after sowing. There was great variability for all the traits and correlations between early production traits and heading date were low. It is possible to obtain ryegrass cultivars with a larger period of utilization.

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.

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.