CRISPR-transient expression in soybean for simplified gRNA screening in planta

O objetivo deste trabalho foi desenvolver um método para criar e validar sistemas CRISPR-Cas e diferentes gRNAs em embriões de soja (Glycine max). Dois genes modelo foram usados para mutação simples com um gRNA ou deleção parcial do gene com dois guias. Os gRNAs foram inseridos nos vetores de transformação CRISPR por uma enzima de restrição do tipo IIS ou por subclonagem e inserção do promotor + gRNA2 no vetor de transformação final, com uso do método clássico de clonagem por enzimas de restrição. Os vetores foram construídos com sucesso para um e dois gRNAs. A transformação transiente de soja por Agrobacterium foi realizada para testar a qualidade dos gRNAs e do próprio sistema (cassete de expressão). Detectaram-se mutação simples e deleção gênica nos embriões transformados após o enriquecimento do DNA por digestão seguida de reação em cadeia da polimerase e sequenciamento, o que indica que o sistema CRISPR-Cas e os guias estavam funcionando. Este protocolo pode ser usado para acelerar as estratégias de edição de genoma baseadas em CRISPR, para transformação genética em soja.The objective of this work was to develop a method to create and validate CRISPR-Cas systems and different gRNAs in soybean (Glycine max) embryos. Two model genes were used for simple mutation with one gRNA or partial gene deletion with two guides. The gRNAs were inserted into the CRISPR transformation vectors by a type IIS restriction enzyme or by subcloning and inserting the promoter + gRNA2 in the final transformation vector using the classic restriction enzyme cloning method. The vectors were successfully constructed for one and two gRNAs. Agrobacterium-mediated transient transformation in soybean was carried out to test the quality of gRNAs and of the system itself (expression cassette). Simple mutation and gene deletion were detected in the embryos transformed after DNA enrichment by enzyme digestion followed by polymerase chain reaction and sequencing, which indicates that the CRISPR-Cas system and guides were working. This protocol can be used to accelerate CRISPR-based genome editing strategies for genetic transformation in soybean

Respostas heliotrópicas de cultivares de soja em três estádios fenológicos e dois regimes hídricos

The objectives of this work were to determine the heliotropic movements of the upper trifoliates for two soybean cultivars, BR 16 and Embrapa 48, during a daily cycle, in three phenological stages and two water regimes, and to estimate the impact of irrigation and daily leaflet movements on agronomic characteristics and grain yield. Heliotropic movements were studied in three phenological stages: V4–V6, V7–V10, and R5 in irrigated and non-irrigated plots. For each stage, the leaflet elevation and azimuth were measured hourly. Under a low (V4–V6 stage) and mid (V7–V10 stage) leaf area index (LAI) the diaheliotropism was slightly more frequent and intensive in non-irrigated than in irrigated plants, only at early morning and late afternoon hours. At R5 stage (high LAI) the paraheliotropism of superior trifoliates was predominant and more intensive in non-irrigated plants. The heliotropic movements are correlated to carbon gain, but not to environment (light intensity or temperature), for measurements at 11h. 'Embrapa 48' expresses greater paraheliotropism than 'BR 16' at high LAI, while 'BR 16' displays lower heliotropic plasticity under irrigation. In spite of significant heliotropic differences, genotype and water availability treatments did not influence the final grain yield.Os objetivos deste trabalho foram determinar o movimento heliotrópico dos trifólios superiores de duas cultivares de soja, BR 16 e Embrapa 48, durante o dia, em três estádios fenológicos e em dois regimes hídricos e determinar o impacto da irrigação e do movimento foliar diário nas características agronômicas e na produção de grãos. Movimentos heliotrópicos foram determinados a cada hora, em três estádios fenológicos: V4–V6, V7–V10 e R5, em parcelas irrigadas e não irrigadas. Para cada estágio, a elevação do folíolo e o azimute foram medidos por hora. Em índices de área foliar (IAF) baixo e médio (V4–V6 e V7–V10), o diaheliotropismo foi levemente mais intenso e frequente nas plantas sem irrigação do que nas irrigadas, e somente nas primeiras horas matinais e últimas horas da tarde. Em R5 (alto IAF), o paraheliotropismo dos trifólios superiores foi predominante e mais intenso em plantas não irrigadas. Os movimentos heliotrópicos se correlacionaram com o ganho de carbono mas não com o ambiente (intensidade de luz ou temperatura), para medidas às 11 horas. 'Embrapa 48' apresenta maior paraheliotropismo do que 'BR 16', em plantas com alto IAF enquanto 'BR 16' apresenta menor plasticidade nas respostas heliotrópicas nas parcelas irrigadas. Apesar de diferenças no heliotropismo, o genótipo e disponibilidade de água não influenciaram a produção de grãos

Fragmentação florestal na Bacia Hidrográfica do Rio São Francisco, Brasil

The dynamics of forest cover in the São Francisco River Basin (BHRSF) can be assessed by metrics of landscape ecology. The aim of this work was to evaluate the forest fragmentation of the São Francisco River Basin, Brazil, through metrics of landscape ecology, and the quantification of forest cover in federal conservation units (UCs) for the Caatinga, Atlantic Forest and Cerrado biomes in the years 1997, 2007 and 2017. The mapping of forest fragments was calculated by the area and the quantity of forest fragments that were divided into the size classes: (1) small (up to 10 ha); (2) medium (between 10 and 100 ha); and (3) large (greater than 100 ha). The metrics were calculated in the Fragstats® 4.2 software, and three groups of metrics were used: area, shape and border. The metric fragment numbers (NP), class area (CA), average fragment size (MPS) and edge density (ED) of forest fragments from the Caatinga and Cerrado biomes at BHRSF prove that the Basin is in the process of forest fragmentation mainly in the large fragments, which had their areas reduced, and in those of medium size, which had an increase in the amount of edges. In general, the protection of forest areas in the Caatinga and Cerrado UCs is not effective in maintaining the forest cover, given the deforestation that has existed in these protected areas in the last 20 years. Measures such as payments for environmental services or forest restoration must be used by the government and society in conjunction with the creation and maintenance of protected forest areas. The Atlantic Forest was the only biome to show an increase in the forest cover and a decrease in the forest fragmentation at BHRSF. Initiatives such as the Atlantic Forest Restoration Pact and the 2006 Atlantic Forest Law may have favored this increase in the forest cover.A dinâmica da cobertura florestal na Bacia Hidrográfica do Rio São Francisco (BHRSF) pode ser avaliada por métricas de ecologia da paisagem. O objetivo deste trabalho foi avaliar a fragmentação florestal na Bacia Hidrográfica do Rio São Francisco, Brasil, por meio de métricas de ecologia da paisagem e quantificar a cobertura florestal nas unidades de conservação federais (UCs) presentes, para os biomas Caatinga, Mata Atlântica e Cerrado nos anos de 1997, 2007 e 2017. O mapeamento dos fragmentos foi realizado considerando área e quantidade dos fragmentos florestais, divididos nas seguintes classes de tamanho: (1) pequeno (até 10 ha); (2) médio (entre 10 e 100 ha); e (3) grande (maiores que 100 ha). As métricas, área, forma e borda, foram calculadas no software Fragstats® 4.2. As métricas números de fragmentos (NP), área de classe (CA), tamanho médio dos fragmentos (MPS) e densidade de borda (ED) dos fragmentos florestais dos biomas Caatinga e Cerrado na BHRSF comprovam que a Bacia está em processo de fragmentação florestal, principalmente nos grandes fragmentos, que tiveram suas áreas reduzidas, e nos de tamanho médio, que tiveram aumento da quantidade de bordas. De forma geral, a proteção de áreas florestais nas UCs da Caatinga e Cerrado não foram eficazes em manter a cobertura florestal, haja vista o desmatamento nestas áreas protegidas em 20 anos. Medidas como pagamentos por serviços ambientais ou restauração florestal devem ser utilizadas pelo poder público e sociedade em conjunto com a criação e manutenção de áreas florestais protegidas. A Mata Atlântica foi o único bioma a apresentar aumento na cobertura florestal e redução da fragmentação florestal na BHRSF. Iniciativas como o pacto da restauração da Mata Atlântica e Lei da Mata Atlântica de 2006 podem ter favorecido esse aumento da cobertura florestal

Tolerância à seca em cultivares-elite de soja com a introgressão do transgene AtAREB1

The objective of this work was to verify if the introgression of the AtAREB1 gene in the 'LS93-0375' and 'BMX Desafio RR' elite soybean germplasms increases the tolerance of these plants to water deficit. The F4 progenies of these two elite cultivars and of the AtAREB1 transgenic line (BR16-AtAREB1) and its background ('BR16') were subjected to water deficit assays. The water deficit bioassays were performed in a greenhouse using the following six soybean lines: the genetically modified BR16-AtAREB1 and its background 'BR16'; 'LS93' and its F4 progeny, LS93-AtAREB1; and 'BMX Desafio RR' and its F4 progeny, Desafio-AtAREB1. A randomized complete block experimental design was carried out in a 6x2 factorial arrangement, with  the six soybean genotypes and two water conditions – control (C) and water deficit (WD) treatments – with nine replicates. Soybean genotypes containing the AtAREB1 gene showed better physiological performances under drought stress and altered expressions of drought-responsive genes. The intogression of AtAREB1 in soybean increases the plant drought tolerance, regardless of the genetic background in which the gene was introduced.O objetivo deste trabalho foi verificar se a introgressão do gene AtAREB1 em dois germoplasmas-elite de soja, 'LS93-0375' e 'BMX Desafio RR', aumenta a tolerância dessas plantas ao deficit hídrico. As progênies F4 das duas cultivares-elite e da linhagem transgênica AtAREB1 (BR16-AtAREB1) e de seu background ('BR16') foram submetidas a deficit hídrico. Os bioensaios de deficit hídrico foram realizados em casa de vegetação, tendo-se utilizado as seis seguintes linhagens de soja: a geneticamente modificada BR16-AtAREB1 e seu background 'BR16'; 'LS93' e sua progênie F4, LS93-AtAREB1; e 'BMX Desafio RR' e sua progênie F4, Desafio-AtAREB1. Utilizou-se delineamento experimental de blocos completos com tratamentos casualizados, em arranjo fatorial 6x2, com os seis genótipos de soja e duas condições hídricas – controle (C) e tratamentos de deficit hídrico (WD) –, com nove repetições. Os genótipos de soja que contêm o gene AtAREB1 exibiram melhor desempenho fisiológico sob estresse hídrico e expressão alterada de genes responsivos à seca. A introgressão de AtAREB1 na soja aumenta a tolerância à seca, independentemente do background genético em que o gene foi introduzido

Identification of quantitative trait loci for resistance against soybean sudden death syndrome caused by Fusarium tucumaniae

O objetivo deste trabalho foi identificar as regiões genômicas responsáveis pela resistência ao Fusarium tucumaniae sp. nov., agente causador da síndrome da morte súbita (SDS) da soja na América do Sul, utilizando-se uma população com background genético diferente daqueles relatados, anteriormente, para o Fusarium virguliforme sp. nov. (F. solani f. sp. glycines), também responsável por SDS em soja. Embora genes de efeito maior e locos de características quantitativas (QTL) para resistência a SDS tenham sido identificados, pouco se conhece sobre essa mesma doença causada por Fusarium tucumanie sp. nov., na América do Sul. Com a finalidade de identificar fatores genéticos relacionados com a resistência a F. tucumaniae e marcadores de DNA associados a esses fatores, foram conduzidas análises de QTL em linhagens puras recombinantes. A localização de quatro locos identificados diferem de QTL anteriormente descritos para resistência à SDS. Uma linha heterozigótica residual (RHL) foi analisada e mostrou-se heterozigótica próxima ao QTL mais efetivo, RSDS1, e homozigótica para outras regiões genômicas. O efeito genético de RSDS1 foi confirmado, usando-se linhas quase-isogênicas derivadas da RHL. Observou-se que a linha homozigótica para o genótipo Misuzudaizu mostrou resistência similar à da linha homozigótica para o genótipo Moshidou Gong 503.The objective of this work was to identify genomic regions that underlie resistance to Fusarium tucumaniae sp. nov., the causing agent of sudden death syndrome (SDS) in soybean in South America, using a population with a genetic background different from that previously reported for Fusarium virguliforme sp. nov. (F. solani f. sp. glycines), also responsible for SDS in soybean. Although major genes and quantitative trait loci (QTL) for SDS resistance have been identified, little is known about the same disease caused by Fusarium tucumaniae sp. nov., in South America. To identify genetic factors related to resistance to F. tucumaniae and DNA markers associated with them, a QTL analysis was performed using recombinant inbred lines. The map locations of the four loci, here identified, differed from those SDS resistance QTL previously described. It was screened a residual heterozygous line (RHL), which was heterozygous around the most effective QTL, RSDS1, and homozygous for the other genomic regions. The genetic effect of RSDS1 was confirmed using near-isogenic lines (NIL) derived from the RHL. The line which was homozygous for the Misuzudaizu genotype showed resistance levels comparable with that of the line homozygous for the Moshidou Gong 503 genotype

Size of AT(n) Insertions in Promoter Region Modulates Gmhsp17.6-L mRNA Transcript Levels

During earlier experiments, an SSR molecular marker (176 Soy HSP) showing high correlation (70%) with resistance/susceptibility to javanese root-knot nematode Meloidogyne javanica was identified in soybean. After being sequenced, results indicated that the SSR 176 Soy HSP marker was inserted in the promoter region of Gmhsp17.6-L gene. It was also detected in this region that resistant genotypes presented insertions between AT(31) and AT(33) in size and susceptible genotypes, AT(9). Gmhsp17.6-L gene coding region presented a perfect match in amino acid sequence in all soybean genotypes. A ribonuclease protection assay showed that Gmhsp17.6-L gene mRNA transcripts were present in all genotypes. A real-time relative quantification (qPCR) indicated in the resistant individuals higher mRNA transcripts levels, which presented in the sequencing more AT(n) insertions. These results suggest that the number of AT(n) insertions inside this promoter region could modulate up or down gene levels. Those findings can lead to the possibility of manipulating, between some limits, the mRNA transcripts levels using different sizes of AT(n) insertions

Análise de transcriptoma de experimentos de RNA- Seq com e sem repetições biológicas: revisão.

The discovery of nucleic acids opened new frontiers of knowledge, enablingresearchers to access an enormous amount of data, through large-scale sequencing methodologiesand bioinformatics tools. Amongst these new possibilities, RNA-Seq has been used to identify andquantify RNA molecules. To obtain more accurate biological responses from RNA-Seq data somequestions should be considered such as experimental design, type ofsynthesized library, size ofthefragments generated, number ofbiological replicates, depth, and coverage ofthe sequencing, speciesgenome availability and, the choice of software to properly perform the computational analyzes.Accurate bioinformatics analyzes allow the selection ofgenes with a lower error rate, increasing thevalidation assertiveness via RT-qPCR and thus, reducing costs. The objective of this review was topresent the analysis stages of RNA-Seq data, from experimental design to systems biology,considering relevant points, as well as to pointed out some software currently available to carry theseanalyzes out. Besides, with this review, we aimed to help the academic community to understand allsteps and biases involved in RNA-Seq data analysis, from experiments with or without biologicalreplicates.A descoberta de ácidos nucléicos abriu novas fronteiras de conhecimento, permitindoque os pesquisadores acessassem uma enorme quantidade de dados, através de metodologias desequenciamento em larga escala e ferramentas de bioinformática. Entre essas novas possibilidades,o RNA-Seq (sequenciamento de RNA) tem sido usado para identificar e quantificar moléculas deRNA. Para obter respostas biológicas mais precisas a partir dos dados de RNA-Seq, algumasquestões devem ser consideradas, como o desenho experimental, o tipo de biblioteca sintetizada, otamanho dos fragmentos gerados, o número de repetições biológicas, a profundidade e cobertura dosequenciamento, a disponibilidade do genoma da espécie e, a escolha dos softwares para executaradequadamente as análises computacionais. Análises bioinformáticas precisas permitem a seleçãode genes com menor taxa de erro, aumentando a assertividade da validação via RT-qPCR e, assim,reduzindo custos. O objetivo desta revisão foi apresentar as etapas de análise de dados de RNA-Seq,desde o projeto experimental até a biologia dos sistemas, considerando pontos relevantes, bemcomo apontar alguns softwares atualmente disponíveis para realizar essas análises. Além disso, comesta revisão, objetivamos ajudar a comunidade acadêmica a compreender todas as etapas e viesesenvolvidos na análise de dados de RNA-Seq, a partir de experimentos com ou sem réplicasbiológicas

Subtractive libraries for prospecting differentially expressed genes in the soybean under water deficit

Soybean has a wide range of applications in the industry and, due to its crop potential, its improvement is widely desirable. During drought conditions, soybean crops suffer significant losses in productivity. Therefore, understanding the responses of the soybean under this stress is an effective way of targeting crop improvement techniques. In this study, we employed the Suppressive Subtractive Hybridization (SSH) technique to investigate differentially expressed genes under water deficit conditions. Embrapa 48 and BR 16 soybean lines, known as drought-tolerant and -sensitive, respectively, were grown hydroponically and subjected to different short-term periods of stress by withholding the nutrient solution. Using this approach, we have identified genes expressed during the early response to water deficit in roots and leaves. These genes were compared among the lines to assess probable differences in the plant transcriptomes. In general, similar biochemical processes were predominant in both cultivars; however, there were more considerable differences between roots and leaves of Embrapa 48. Moreover, we present here a fast, clean and straightforward method to obtain drought-stressed root tissues and a large enriched collection of transcripts expressed by soybean plants under water deficit that can be useful for further studies towards the understanding of plant responses to stress.304314Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Implications of ethylene biosynthesis and signaling in soybean drought stress tolerance

Abstract\ud \ud Background\ud Ethylene is a phytohormone known for inducing a triple response in seedlings, leaf abscission and other responses to various stresses. Several studies in model plants have evaluated the importance of this hormone in crosstalk signaling with different metabolic pathways, in addition to responses to biotic stresses. However, the mechanism of action in plants of agricultural interest, such as soybean, and its participation in abiotic stresses remain unclear.\ud \ud \ud Results\ud The studies presented in this work allowed for the identification of 176 soybean genes described elsewhere for ethylene biosynthesis (108 genes) and signal transduction (68 genes). A model to predict these routes in soybean was proposed, and it had great representability compared to those described for Arabidopsis thaliana and Oryza sativa. Furthermore, analysis of putative gene promoters from soybean gene orthologs permitted the identification of 29 families of cis-acting elements. These elements are essential for ethylene-mediated regulation and its possible crosstalk with other signaling pathways mediated by other plant hormones.\ud From genes that are differentially expressed in the transcriptome database, we analyzed the relative expression of some selected genes in resistant and tolerant soybean plants subjected to water deficit. The differential expression of a set of five soybean ethylene-related genes (MAT, ACS, ACO, ETR and CTR) was validated with RT-qPCR experiments, which confirmed variations in the expression of these soybean target genes, as identified in the transcriptome database. In particular, two families of ethylene biosynthesis genes (ACS and ACO) were upregulated under these experimental conditions, whereas CTR (involved in ethylene signal transduction) was downregulated. In the same samples, high levels of ethylene production were detected and were directly correlated with the free fraction levels of ethylene’s precursor. Thus, the combination of these data indicated the involvement of ethylene biosynthesis and signaling in soybean responses to water stress.\ud \ud \ud Conclusions\ud The in silico analysis, combined with the quantification of ethylene production (and its precursor) and RT-qPCR experiments, allowed for a better understanding of the importance of ethylene at a molecular level in this crop as well as its role in the response to abiotic stresses. In summary, all of the data presented here suggested that soybean responses to water stress could be regulated by a crosstalk network among different signaling pathways, which might involve various phytohormones, such as auxins, ABA and jasmonic acid. The integration of in silico and physiological data could also contribute to the application of biotechnological strategies to the development of improved cultivars with regard to different stresses, such as the isolation of stress-specific plant promoters.This research was financially supported by grants from the Brazil Higher\ud Education Personnel Training Coordination (CAPES), the Brazil National\ud Council for Scientific and Technological Development (CNPq), the Brazilian\ud Foundation for Research Support (FAP-DF) and Embrapa Genetic Resources\ud and Biotechnology (Brazil)

Inquérito sobre o HIV entre HSH no Brasil em 2016 usando respondent-driven sampling (RDS) : descrição dos métodos e dos diagnósticos do RDS

Introdução: Este artigo detalha os métodos utilizados na segunda Pesquisa Nacional de Vigilância Biológica e Comportamental (BBSS) do HIV, sífilis e hepatite B e C entre os homens que fazem sexo com homens no Brasil. Métodos: O método Respondent-driven Sampling (RDS) foi utilizado em 12 cidades em 2016. A amostra foi iniciada com cinco a seis sementes em cada cidade. Testes rápidos para o HIV, sífilis e Hepatite B e C foram oferecidos aos participantes. O software RDS Analyst com o estimador de amostragem sucessiva (SS) de Gile foi utilizado para ajustar os resultados como recomendado, gerando um peso para cada indivíduo para análises. Osdados das 12cidades foram unidos em um único banco e analisados usando as ferramentas de dados complexos do Stata 14.0, com cada cidade sendo tratada como seu próprio estrato. Resultados: A duração da coleta de dados variou de 5,9 a 17,6 semanas e 4.176 homens foram recrutados nas 12 cidades. Dois sites não alcançaram o tamanho da amostra alvo devido a uma demora de seis meses na aprovação local do Comitê de Ética. Todas as cidades atingiram a convergência na principal variável estudada (HIV). Conclusão: O BBSS foi representativo e concluído conforme planejado e dentro do orçamento. A descrição dos métodos aqui é mais detalhada do que o habitual, devido às novas ferramentas e requisitos de diagnóstico das novas diretrizes do STROBE-RDS.Introduction: This paper details the methods used in the second national Biological and Behavioral Surveillance Survey (BBSS) of HIV, syphilis, and hepatitis B and C among men who have sex with men in Brazil. Methods: Respondent-driven sampling (RDS) was used in 12 cities in 2016. The targeted sample size was initiated with five to six seeds in each city. HIV, syphilis, and Hepatitis B and C rapid tests were offered to participants. RDS Analyst with Gile’s successive sampling (SS) estimator was used to adjust results as recommended and a weight for each individual was generated for further analysis. Data for the 12 cities were merged and analyzed using Stata 14.0 complex survey data tools with each city treated as its own stratum. Results: Duration of data collection varied from 5.9 to 17.6 weeks. 4,176 men were recruited in the 12 cities. Two sites failed to achieve targeted sample size due to a six-month delay in local IRB approval. No city failed to reach convergence in our major outcome variable (HIV). Conclusion: The comprehensive BBSS was completed as planned and on budget. The description of methods here is more detailed than usual, due to new diagnostic tools and requirements of the new STROBE-RDS guidelines