Luciano Vilela Paiva

Androgenetic haploids and SSR markers as tools for the development of tropical maize hybrids

The traditional process of obtaining maize hybrids involves the generation of inbred lines through successive generations of selfing and subsequent testcrosses in order to identify the best combining ability by allelic complementation. A fast alternative to obtain inbred lines is to induce the formation of haploids followed by chromosome doubling. However, even with the aid of haploid-inducing genetic sources, this strategy has not been widely used in maize breeding programs, partly due to difficulties inherent to haploid generation and identification. In order to evaluate the possibility of using dihaploids to generate homozygous maize tropical lines, we used the androgenetic haploid inducer line W23 as a female parent in crosses with the tropical single-cross hybrid BRS1010. Within the progeny of these crosses, 462 seeds were phenotypically selected as putative haploids by the purple-colored endosperm and colorless embryo conditioned by the R1-nj gene. Among these, only four individuals were confirmed as being haploids using SSR markers, chromosome counting and flow cytometry, showing that the phenotypic marker was not efficient in detecting haploids in the tropical maize genotype used. All four haploids as well as some diploid plants presented reduced size, corroborating the difficulties for haploid identification by phenotypic evaluation. Genetic diversity analysis revealed by SSR markers divided the haploids in two groups represented by flint and dent maize inbred lines, which could be helpful in identifying complementary dihaploid lines. The present article demonstrates that a combination of haploid production and SSR fingerprinting is a feasible strategy for maize hybrid development in tropical germplasm.

Molecular characterization and genetic diversity of potato cultivars using SSR and RAPD markers.

Este trabalho teve como objetivo avaliar a divergencia genetica e identificar cultivares de batata por meio de marcadores RAPD e SSR. O DNA genomico de 16 cultivares de batata foi amplificado com primers RAPD e com pares de primers SSR. Os 25 primers RAPD geraram 92 bandas polimorficas e os 20 pares de primers SSR produziram 136 bandas polimorficas. Os dendrogramas gerados pela analise de agrupamento permitiram uma distincao genetica das cultivares, entretanto nao houve uma correlacao entre os dendrogramas ao comparar os dois marcadores utilizados. Os valores de PIC demonstraram o alto conteudo informativo dos primers utilizados e, com a utilizacao de 6 primers RAPD e de 3 pares de primers SSR, identificou-se as 16 cultivares de batatas. Dessa forma, por meio de marcadores moleculares RAPD e SSR foi possivel avaliar a divergencia genetica e identificar as 16 cultivares comerciais de batata analisadas neste estudo.

In Silico and Quantitative Analyses of MADS-Box Genes in Coffea arabica

MADS-box genes comprise a family of transcription factors that act as key regulators in many cellular development processes of several organisms. Members of this family have highly conserved regions and play important roles as transcription factors, activating target genes. The present work aimed at analyzing the MADS-box gene family present in a database generated by the Brazilian Coffee Genome Project (CAFEST) as well as of observing their respective sites of expression within these data. Through the use of bioinformatics tools, it was possible to identify and classify 26 expressed sequence tag contigs, 13 of them were expressed exclusively in vegetative tissues, 11 in reproductive tissues, and two in both. Later, quantitative analysis by quantitative reverse transcriptase PCR was carried out for three of them belonging to the groups of genes APETALA3 (B genes), AGAMOUS (C genes), and SEPALLATAS (E genes), which could be compared with the expression profile of in silico analysis and of its putative orthologous genes. Therefore, this study aims at a better understanding of the development processes performed by this family of genes in Coffea arabica, mainly in reproductive organs, as well as to compare functions of MADS-box orthologous studied in other species.

PR gene families of citrus: their organ specific-biotic and abiotic inducible expression profiles based on ESTs approach

In silico expression profiles, of the discovered 3,103 citrus ESTs putatively encoding for PR protein families (PR-1 to PR-17), were evaluated using the Brazil citrus genome EST CitEST/database. Hierarchical clustering was displayed to identify similarities in expression patterns among citrus PR-like gene families (PRlgf) in 33 selected cDNA libraries. In this way, PRlgf preferentially expressed by organ and citrus species, and library conditions were highlighted. Changes in expression profiles of clusters for each of the 17 PRlgf expressed in organs infected by pathogens or drought-stressed citrus species were displayed for relative suppression or induction gene expression in relation to the counterpart control. Overall, few PRlgf showed expression 2-fold higher in pathogen-infected than in uninfected organs, even though the differential expression profiles displayed have been quite diverse among studied species and organs. Furthermore, an insight into some contigs from four PRlgf pointed out putative members of multigene families. They appear to be evolutionarily conserved within citrus species and/or organ- or stress-specifically expressed. Our results represent a starting point regarding the extent of expression pattern differences underlying PRlgf expression and reveal genes that may prove to be useful in studies regarding biotechnological approaches or citrus resistance markers.

A Phosphate Transporter Promoter from Arabidopsis thaliana AtPHT1;4 Gene Drives Preferential Gene Expression in Transgenic Maize Roots Under Phosphorus Starvation

Phosphorus (P) stress responsive genes have been identified and characterized, including the high-affinity phosphate transporter AtPHT1;4 from Arabidopsis thaliana. This gene encodes a membrane protein that is primarily expressed in roots under phosphorus deficiency. A 2.3-kb promoter region from AtPHT1;4 has been fused with the β-glucuronidase (GUS) encoding gene and introduced into maize via biolistic bombardment to evaluate its spatiotemporal activity in a heterologous system. AtPHT1;4::GUS expression is detected preferentially in transgenic maize roots under P deficiency. Further analysis of transgenic plants has also revealed that GUS activity is higher in roots than in leaves by about sixfold. These results demonstrate the ability of AtPHT1;4 promoter to direct expression of the reporter gene in a monocot root system under P stress. This property of AtPHT1;4 promoter makes it useful to engineer maize plants to modify the soil’s rhizosphere and increase efficiency of P acquisition under P stress conditions.

Transcriptome analysis of resistant soybean roots infected by Meloidogyne javanica

Soybean is an important crop for Brazilian agribusiness. However, many factors can limit its production, especially root-knot nematode infection. Studies on the mechanisms employed by the resistant soybean genotypes to prevent infection by these nematodes are of great interest for breeders. For these reasons, the aim of this work is to characterize the transcriptome of soybean line PI 595099-Meloidogyne javanica interaction through expression analysis. Two cDNA libraries were obtained using a pool of RNA from PI 595099 uninfected and M. javanica (J2) infected roots, collected at 6, 12, 24, 48, 96, 144 and 192 h after inoculation. Around 800 ESTs (Expressed Sequence Tags) were sequenced and clustered into 195 clusters. In silico subtraction analysis identified eleven differentially expressed genes encoding putative proteins sharing amino acid sequence similarities by using BlastX: metallothionein, SLAH4 (SLAC1 Homologue 4), SLAH1 (SLAC1 Homologue 1), zinc-finger proteins, AN1-type proteins, auxin-repressed proteins, thioredoxin and nuclear transport factor 2 (NTF-2). Other genes were also found exclusively in nematode stressed soybean roots, such as NAC domain-containing proteins, MADS-box proteins, SOC1 (suppressor of overexpression of constans 1) proteins, thioredoxin-like protein 4-Coumarate-CoA ligase and the transcription factor (TF) MYBZ2. Among the genes identified in non-stressed roots only were Ser/Thr protein kinases, wound-induced basic protein, ethylene-responsive family protein, metallothionein-like protein cysteine proteinase inhibitor (cystatin) and Putative Kunitz trypsin protease inhibitor. An understanding of the roles of these differentially expressed genes will provide insights into the resistance mechanisms and candidate genes involved in soybean-M. javanica interaction and contribute to more effective control of this pathogen.

Estabelecimento in vitro de estrelícia (Strelitzia reginae Banks.)

A estrelicia (Strelitzia reginae Banks.) e uma planta ornamental tropical de grande valor comercial. O seu desenvolvimento, no entanto, e bastante lento e, consequentemente, a producao de novas mudas tambem. Assim, a cultura de tecidos e uma alternativa para a formacao de novas mudas. Objetivou-se, assim, avaliar o comportamento in vitro, de estrelicia e a viabilidade de propagacao dessa especie por meio desse processo. Para o estabelecimento in vitro utilizaram-se como explantes gemas axilares, segmentos foliares e embrioes imaturos, nao se obtendo sucesso nos dois primeiros. No cultivo dos embrioes, avaliaram-se o periodo para coleta das sementes e o desenvolvimento dos embrioes em diferentes concentracoes de sacarose e do meio de cultura e ainda o uso de diferentes concentracoes de BAP. Determinou-se como melhor periodo para coleta das sementes e extracao dos embrioes 20 semanas apos a polinizacao. As diferentes concentracoes do meio MS nao alteraram o desenvolvimento dos embrioes, sendo esse favorecido pela adicao de 20,64 g/L de sacarose ao meio de cultura. A adicao de BAP proporcionou a formacao de plantas de menor tamanho.

Estudos sobre superação de dormência em sementes de Smilax japecanga Grisebach

A propagacao sexuada de Smilax japecanga Grisebach e limitada em razao da dormencia de suas sementes, as quais levam de 6 a 8 meses para germinar, dificultando, assim, a obtencao de mudas. Com o objetivo de promover a germinacao das sementes, experimentos foram conduzidos visando a superacao de dormencia, observando-se a germinacao nos 35 dias subsequentes aos tratamentos. Utilizaram-se escarificacao mecânica, com esmeril e escarificacao quimica, com acido sulfurico (98%), por 30 segundos, 1, 5 e 10 minutos; embebicao em acido giberelico (GA3), por 48 horas, as concentracoes de 0,55; 1,1; 1,65 e 2,2 mM; e uma combinacao da escarificacao quimica, por 6 minutos, com a embebicao em GA3 1,92 mM, por 48 horas. O uso de escarificacao quimica seguida de embebicao em GA3 promoveu a maior germinacao (86%). A embebicao em GA3 1,65 mM promoveu a germinacao de 56% das sementes. Germinacao inferior foi obtida por meio de escarificacao mecânica (8%) e escarificacao quimica por 1 minuto (7%).

Expression of genes involved in lipid metabolism in the muscle of beef cattle fed soybean or rumen-protected fat, with or without monensin supplementation.

Degree of unsaturation of fatty acids, which is influenced by lipid source and level of metabolism in the rumen, is a major determinant in how dietary lipids affect genes that regulate beef marbling. A total of 28 Red Norte bulls with an initial live weight of 361±32 kg (P>0.05) were used in a completely randomized experimental design to analyze the expression of genes that are involved in lipid metabolism in the longissimus dorsi (LD) when diets contained soybean grain or rumen-protected fat, with or without monensin. Treatments were arranged as a 2×2 factorial, with 4 treatments and 7 replicates per treatment. Half of the animals that received soybean or rumen-protected fat were supplemented with 230 mg head(-1) d(-1) of monensin. Gene expression was analyzed by reverse-transcription quantitative PCR (RT-qPCR). Expression of sterol regulatory element-binding protein-1c (SREBP-1c) in the LD muscle was not affected by lipid source or monensin (P>0.05). There was an interaction effect (P<0.05) between lipid source and monensin for peroxisome proliferator-activated receptor α (PPAR-α) and stearoyl-CoA desaturase (SCD) expression, where greater gene expression was found in animals fed soybean plus monensin and the lower gene expression was found in animals fed rumen-protected fat plus monensin. Expression of lipoprotein lipase (LPL) and fatty acid-binding protein 4 (FABP4) were greater (P<0.05) in the LD muscle of animals fed soybean. Monensin had no effect on LPL and FABP4 expression when soybean without monensin was fed, but when rumen-protected fat was fed, monensin increased LPL expression and decreased FABP4 expression (P<0.05). Linoleic and arachidonic acids had negative correlations (P<0.05) with the expression of PPAR-α, SCD, FABP4, and LPL genes. PPAR-α gene expression was not correlated with SREBP-1c but was positively correlated with SCD, FABP4, LPL, and glutathione peroxidase (GPX1) gene expression (P<0.001). Lipid sources and monensin interact and alter the expression of PPAR-α, SCD, acetyl CoA carboxylase α (ACACA), LPL, FABP4, and GPX1. These changes in gene expression were most associated with arachidonic and α-linolenic acids and the ability of lipid sources and monensin to increase these fatty acids in tissues.

Morphological characteristics and cell viability of coffee plants calli

O objetivo deste trabalho foi caracterizar e comparar dois tipos de calos de explantes foliares de Coffea arabica (cultivar Catigua). Celulas de diferentes tipos de calos foram caracterizadas quanto a viabilidade e caracteristicas morfologicas externas e internas. Foram obtidos dois tipos de calos morfologicamente distintos: (a) amarelo friavel e (b) transparente aquoso. Os calos amarelos friaveis apresentaram maior viabilidade celular e caracteristicas embriogenicas. Microscopia eletronica de varredura e transmissao mostraram caracteristicas embriogenicas em calos amarelos friaveis evidenciadas pela presenca de celulas pequenas e isodiametricas. Os calos transparentes aquosos apresentaram celulas alongadas e vacuolizadas.