Resumo: A germinação da semente do café (Coffea arabica L.) é resultado de eventos que ocorrem simultaneamente no embrião e endosperma. Para entender os mecanismos moleculares responsáveis por estes eventos, nós realizamos uma análise transcricional do embrião e dos endospermas micropilar e lateral de sementes embebidas por 10 dias. O sequenciamento obteve contigs que codificam 16.813 proteínas. Destas, 14.005 (~ 83%) foram altamente similares a pelo menos uma sequência de proteína na base de dados nr. 162 genes foram significativamente expressos em embrião, 36 em endosperma micropilar e 72 em endosperma lateral. A análise de tecido-especificidade dos genes significativamente expressos mostrou que a maior proporção de genes específicos está no embrião (113/162, ~70%), enquanto 11 genes foram expressos nos endospermas lateral e micropilar. No embrião, os genes foram associados principalmente com estresse abiótico, crescimento celular e comunicação intercelular. Nos endospermas micropilar e lateral, eles foram associados com estresse abiótico e degradação da parede celular. A acurácia da análise de RNA-seq foi confirmada por RT-qPCR. Este trabalho acrescenta novas informações sobre os mecanismos moleculares envolvidos na germinação de sementes de café.

Abstract: Germination of the coffee (Coffea arabica L.) seed is the result of events that occur simultaneously in the embryo and endosperm. To understand the molecular mechanisms responsible for these events, we undertook a transcriptome analysis of embryo, micropylar and lateral endosperms from 10-day-imbibed seeds. The sequencing yielded contigs coding for 16,813 proteins. From those, 14,005 (~ 83%) were highly similar to at least one protein sequence in the nr database. 162 genes were significantly expressed in the embryo, 36 in the micropylar endosperm and 72 in the lateral endosperm. The tissue specificity analysis of the significantly expressed genes showed that the embryo had the highest proportion of specific genes (113/162, ~70%), while 11 were expressed in the micropylar and lateral endosperms. In the embryo, genes were mainly associated with abiotic stress, cell growth, and intercellular communication. In the micropylar and lateral endosperms, they were associated with abiotic stress and cell wall degradation. The accuracy of RNA-seq data was confirmed by RT-qPCR. This work adds new information about the molecular mechanism involved in coffee seed germination.

O objetivo geral deste trabalho foi avaliar o uso da técnica de PCR (Polimerase Chain Reaction) na caracterização de cultivares de soja. O estudo foi realizado no Departamento de Produção Vegetal da Faculdade de Ciências Agronômicas / UNESP e do Instituto de Biociências de Botucatu-SP. Foram utilizadas quatorze cultivares de soja comerciais, das quais seis foram selecionadas como reação positiva à peroxidase (BRS 320, BRS 284, BRS 232, BRS 7860RR, BRSMG 760SRR, BRS295RR), quatro como reação negativa (BRS 326, BRS 8160RR, BRSMG 800A (NutriSoy), BRS Valiosa RR) e quatro como reação positiva e negativa (BRS 8060, BRS 270RR, FTS Campo Mourão e BRS 239). Assim, as 14 cultivares foram submetidas ao ensaio bioquímico colorimétrico tradicional e os resultados obtidos foram comparados com o ensaio de PCR convencional. Para a análise de PCR, o DNA foi extraído de sementes inteiras, sendo que os primers foram testados por PCR e visualizados por eletroforese em gel de agarose. A combinação dos primers prx9 + prx10 confirmou a utilização da reação de PCR para caracterizar as cultivares de soja considerada duvidosa por teste convencional colorimétrico.

The aim of this work was to evaluate the use of the molecular biology technique of PCR (Polimerase Chain Reaction) in the characterization of soybean cultivars. The study was performed at the Department of Plant Production, Faculty of Agricultural Sciences/ UNESP and Institute of Bioscience, Botucatu-SP. Fourteen commercial soybean cultivars were used, of which six were selected as positive reaction to peroxidase (BRS 320, BRS 284, BRS 232, BRS 7860RR, BRSMG 760SRR, BRS295RR), four as negative reaction (BRS 326, BRS 8160RR, BRSMG 800A (NutriSoy), BRS Valiosa RR) and four as double reaction (BRSGO 8060, BRS 270RR, FTS Campo Mourão and BRS 239). Thus, the results attained by the traditional biochemical colorimetric test for the 14 cultivars were compared with the conventional PCR assay. For PCR analysis, DNA was extracted from whole seeds and the primers were tested, and subsequently PCR and agarose gel electrophoresis were performed. The combination of primers prx9 + prx10 confirmed the use of the PCR reaction to characterize soybean cultivars considered doubtful by conventional colorimetric text.

A broca da cana, Diatraea saccharalis pertence à família dos lepidópteros. A presença da larva pode ser extremamente destrutiva, chegando a inviabilizar a atividade canavieira, causando prejuízos consideráveis à agroindústria sucro-alcooleira. Atualmente a broca da cana vem sendo extinta da plantação por métodos de controle biológico, entretanto a evolução desses programas depende de maiores conhecimentos básicos da biologia molecular deste inseto. O estudo do segmento do genoma mitocondrial denominado região controle é amplamente utilizado em análises genéticas e filogenéticas em insetos. O objetivo desse trabalho foi sequenciar e analisar a região controle do genoma mitocondrial de Diatraea saccharalis. Esse segmento apresentou 338 nucleotídeos, menor que o observado em Bombyx mori, com conteúdo de 93,5% de A/T. As analises realizadas mostraram que Diatraea saccharalis apresenta 76% de similaridade com Cydia pomonella.

This study aimed at the sequence and analysis of the mtDNA control region (CR) of the Diatraea saccharalis. The genome PCR amplification was performed using the complementary primers to the flanking regions of Bombyx mori CR mitochondrial segment. The sequencing revealed that the amplified product was 568 bp long, which was smaller than that observed for B. mori (725 bp). Within the amplified segment, a sequence with 338 nucleotides was identified as the control region, which displayed a high AT content (93.5%). The D. saccharalis mtDNA CR multiple sequence alignment analysis showed that this region had high similarity with the Lepidoptera Cydia pomonella.

The Arachis section is the most important of the nine sections of the genus Arachis because it includes the cultivated peanut, Arachis hypogaea. The genetic improvement of A. hypogaea using wild relatives is at an early stage of development in spite of their potential as sources of genes, including those for disease and pests resistance, that are not found in the A. hypogaea primary gene pool. Section Arachis species germplasm has been collected and maintained in gene banks and its use and effective conservation depends on our knowledge of the genetic variability contained in this material. Microsatellites are routinely used for the analysis of genetic variability because they are highly polymorphic and codominant. The objective of this study was to evaluate the transferability of microsatellite primers and the assay of genetic variability between and within the germplasm of some species of the Arachis section. Fourteen microsatellite loci developed for three different species of Arachis were analyzed and 11 (78%) were found to be polymorphic. All loci had transferability to all the species analyzed. The polymorphic loci were very informative, with expected heterozygosity per locus ranging from 0.70 to 0.94. In general, the germplasm analyzed showed wide genetic variation.

The genus Arachis currently comprises 69 described species, some of which have potential and real value as human and animal foods. These Arachis species have been collected and maintained in germplasm banks to provide material that can be used as sources of genes in breeding programs and for the selection of new cultivars. One of the principal objectives of germplasm conservation is the evaluation of genetic variability, which is best conducted using molecular markers. We investigated the use of heterologous primers to amplify microsatellite loci that could be used to evaluate genetic variability in Arachis germplasm. Fifteen microsatellite primer pairs were tested in 76 accessions of 34 species from the nine Arachis sections. The data indicated that heterologous primers were very useful in Arachis since they had high transferability among the species (91%) and allowed the amplification of very polymorphic putative loci, which allowed both the characterization of most accessions and to make inferences about the mating systems of some species analyzed. Our data also revealed that the germplasm analyzed showed high variability, even when represented by few accessions.