Abstract 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.

Resumo 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.

Abstract Soybean is a key crop in many countries, being used from human food to the animal industry due to its nutritional properties. Financially, the grain chain moves large sums of money into the economy of producing countries. However, like other agricultural commodities around the world, it can have its final yield seriously compromised by abiotic environmental stressors, like drought. As flowers imply in pods and in grains inside it to minimize damages caused by water restriction, researchers have focused on understanding flowering-process related genes and their interactions. Here a review dedicated to the soybean flowering process and gene network involved in it is presented, describing gene interactions and how genes act in this complex mechanism, also ruled by environmental triggers such as day-light and circadian cycle. The objective was to gather information and insights on the soybean flowering process, aiming to provide knowledge useful to assist in the development of drought-tolerant soybean lines, minimizing losses due to delays or anticipation of flowering and, consequently, restraining financial and productivity losses.

Resumo 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.

Abstract 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.

Abstract Water deficit is an important climatic problem that can impair agriculture yield and economy. Genetically modified soybean plants containing the AtNCED3 gene were obtained aiming drought-tolerance improvement. The NCED3 gene encodes a 9-cis-epoxycarotenoid dioxygenase (NCED, EC 1.13.11.51), an important enzyme in abscisic acid biosynthesis. ABA activates the expression of drought-responsive genes, in water-deficit conditions, targeting defense mechanisms and enabling plants to survive under low water availability. Results from greenhouse experiments showed that the transgene AtNCED3 and the endogenous genes GmAREB1, GmPP2C, GmSnRK2 and GmAAO3 presented higher expression under water deficit (WD) in the event 2Ha11 than in WT-plants. No significant correlation was observed between the plant materials and WD conditions for growth parameters; however, gas exchange measurements decreased in the GM event, which also showed 80% higher intrinsic water use when compared to WT plants. In crop season 2015/16, event 2Ha11 showed higher total number of pods, higher number of pods with seeds and yield than WT plants. ABA concentration was also higher in GM plants under WD. These results obtained in field screenings suggest that AtNCED3 soybean plants might outperform under drought, reducing economic and yield losses, thus being a good candidate line to be incorporated in the soybean-breeding program to develop drought-tolerant cultivars.

Abstract Water deficit is one of the major limitations to soybean production worldwide, yet the genetic basis of drought-responsive mechanisms in crops remains poorly understood. In order to study the gene expression patterns in leaves and roots of soybean, two contrasting genotypes, Embrapa 48 (drought-tolerant) and BR 16 (drought-sensitive), were evaluated under moderate and severe water deficit. Transcription factors from the AP2/EREBP and WRKY families were investigated. Embrapa 48 showed 770 more up-regulated genes than BR 16, in eight categories. In general, leaves presented more differentially expressed genes (DEGs) than roots. Embrapa 48 responded to water deficit faster than BR 16, presenting a greater number of DEGs since the first signs of drought. Embrapa 48 exhibited initial modulation of genes associated with stress, while maintaining the level of the ones related to basic functions. The genes expressed exclusively in the drought-tolerant cultivar, belonging to the category of dehydration responsive genes, and the ones with a contrasting expression pattern between the genotypes are examples of important candidates to confer tolerance to plants. Finally, this study identified genes of the AP2/EREBP and WRKY families related to drought tolerance.

The loss of soybean yield to Brazilian producers because of a water deficit in the 2011-2012 season was 12.9%. To reduce such losses, molecular biology techniques, including plant transformation, can be used to insert genes of interest into conventional soybean cultivars to produce lines that are more tolerant to drought. The abscisic acid (ABA)-independent Dehydration Responsive Element Binding (DREB) gene family has been used to obtain plants with increased tolerance to abiotic stresses. In the present study, the rd29A:AtDREB2A CA gene from Arabidopsis thaliana was inserted into soybean using biolistics. Seventy-eight genetically modified (GM) soybean lines containing 2-17 copies of the AtDREB2A CA gene were produced. Two GM soybean lines (P1397 and P2193) were analyzed to assess the differential expression of the AtDREB2A CA transgene in leaves and roots submitted to various dehydration treatments. Both GM lines exhibited high expression of the transgene, with the roots of P2193 showing the highest expression levels during water deficit. Physiological parameters examined during water deficit confirmed the induction of stress. This analysis of AtDREB2A CA expression in GM soybean indicated that line P2193 had the greatest stability and highest expression in roots during water deficit-induced stress.

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.

Metabolomics analysis of wild type Arabidopsis thaliana plants, under control and drought stress conditions revealed several metabolic pathways that are induced under water deficit. The metabolic response to drought stress is also associated with ABA dependent and independent pathways, allowing a better understanding of the molecular mechanisms in this model plant. Through combining an in silico approach and gene expression analysis by quantitative real-time PCR, the present work aims at identifying genes of soybean metabolic pathways potentially associated with water deficit. Digital expression patterns of Arabidopsis genes, which were selected based on the basis of literature reports, were evaluated under drought stress condition by Genevestigator. Genes that showed strong induction under drought stress were selected and used as bait to identify orthologs in the soybean genome. This allowed us to select 354 genes of putative soybean orthologs of 79 Arabidopsis genes belonging to 38 distinct metabolic pathways. The expression pattern of the selected genes was verified in the subtractive libraries available in the GENOSOJA project. Subsequently, 13 genes from different metabolic pathways were selected for validation by qPCR experiments. The expression of six genes was validated in plants undergoing drought stress in both pot-based and hydroponic cultivation systems. The results suggest that the metabolic response to drought stress is conserved in Arabidopsis and soybean plants.

The study of tolerance mechanisms for drought stress in soybean is fundamental to the understanding and development of tolerant varieties. Using in silico analysis, four marker genes involved in the classical ABA-dependent and ABA-independent pathways of drought response were identified in the Glycine max genome in the present work. The expression profiles of the marker genes ERD1-like, GmaxRD20A-like, GmaxRD22-like and GmaxRD29B-like were investigated by qPCR in root samples of drought sensitive and tolerant soybean cultivars (BR 16 and Embrapa 48, respectively), submitted to water deficit conditions in hydroponic and pot-based systems. Among the four putative soybean homologs to Arabidopsis genes investigated herein, only GmaxRD29B-like was not regulated by water deficit stress. Distinct expression profiles and different induction levels were observed among the genes, as well as between the two drought-inducing systems. Our results showed contrasting gene expression responses for the GmaxRD20A-like and GmaxRD22-like genes. GmaxRD20A-like was highly induced by continuous drought acclimating conditions, whereas GmaxRD22-like responses decreased after abrupt water deprivation. GmaxERD1-like showed a different expression profile for the cultivars in each system. Conversely, GmaxRD20A-like and GmaxRD22-like genes exhibited similar expression levels in tolerant plants in both systems.

O objetivo deste trabalho foi validar, pela técnica de PCR quantitativo em tempo real (RT-qPCR) genes para serem utilizados como referência em estudos de expressão gênica em soja, em ensaios de estresse hídrico. Foram avaliados quatro genes comumente utilizados em soja: Gmβ-actin, GmGAPDH, GmLectin e GmRNAr18S. O RNA total foi extraído de seis amostras: três amostras de raízes em sistema de hidroponia com diferentes intensidades de déficit hídrico (0, 25, 50, 75 e 100 minutos de estresse hídrico), e três amostras de folhas de plantas cultivadas em areia com diferentes umidades do solo (15, 5 e 2,5% de umidade gravimétrica). Os dados brutos do intervalo "cycle threshold" (Ct) foram analisados, e a eficiência de cada iniciador foi calculada para uma analise da Ct entre as diferentes amostras. A aplicação do programa GeNorm foi utilizada para a avaliação dos melhores genes de referência, de acordo com a estabilidade. O GmGAPDH foi o gene menos estável, com o maior valor médio de estabilidade de expressão (M), e os genes mais estáveis, com menor valor de M, foram o Gmβ-actin e GmRNAr18S, tanto nas amostras de raízes como nas de folhas. Estes genes podem ser usados em RT-qPCR como gens de referência para análises de expressão gênica.

The objective of this work was to validate, by quantitative PCR in real time (RT-qPCR), genes to be used as reference in studies of gene expression in soybean in drought-stressed trials. Four genes commonly used in soybean were evaluated: Gmβ-actin, GmGAPDH, GmLectin and GmRNAr18S. Total RNA was extracted from six samples: three from roots in a hydroponic system with different drought intensities (0, 25, 50, 75 and 100 minutes of water stress), and three from leaves of plants grown in sand with different soil moistures (15, 5 and 2.5% gravimetric humidity). The raw cycle threshold (Ct) data were analyzed, and the efficiency of each primer was calculated for an overall analysis of the Ct range among the different samples. The GeNorm application was used to evaluate the best reference gene, according to its stability. The GmGAPDH was the least stable gene, with the highest mean values of expression stability (M), and the most stable genes, with the lowest M values, were the Gmβ-actin and GmRNAr18S, when both root and leaves samples were tested. These genes can be used in RT-qPCR as reference gene for expression analysis.

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.

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.

Análises morfo-anatômicas e micromorfométricas de duas cultivares de soja, MG/BR46 (Conquista) e BR16—tolerante e sensível à seca, respectivamente, em experimento conduzido em casa de vegetação—foram feitas para estudar as diferentes estratégias de tolerância ao déficit hídrico. Tratamentos de seca foram aplicados no estádio reprodutivo R2 e R7, onde avaliações foram conduzidas em 30 dias e 45 dias após o inicio do estresse, respectivamente. O comprimento total das plantas da cultivar Conquista (parte aérea e raiz) foi maior do que das plantas da cultivar BR16. A massa seca da vagem foi adversamente afetada pelo déficit hídrico, diminuindo a produtividade das plantas da cultivar Conquista. Ambas as cultivares tiveram o desenvolvimento de pêlos radiciais normais e, uma diminuição da razão córtex:cilindro central foi observada em BR16 estressada por 30 dias, além de apresentar uma espessura do folíolo e distribuição dos estômatos normais. Diferenças na tolerância à seca observada entre as duas cultivares devem estar relacionadas também a outros fatores, alm das características morfológicas, já que esta espécie possui um ciclo de vida curto.

In a greenhouse experiment, morpho-anatomical and micromorphometrical analyses of two soybean cultivars, MG/BR46 (Conquista) and BR16-tolerant and sensitive to drought, respectively-were used to study their water-deficit-tolerance strategies. Drought treatments were applied at reproductive stages from R2 to R7, where evaluations were conducted at 30 days and 45 days after stress started, respectively. The total length of Conquista plants (shoot + root) was greater than of BR16 plants. Pod dry weight was adversely affected due to the lack of moisture, decreasing productivity even of Conquista plants. Both the cultivars had normal development of root hairs; however, it was observed a decrease in the cortex:central cylinder ratio in BR16 stressed for 30 days, and they also showed similar leaflet thickness and stomata distribution. Differences in drought tolerance observed between the two cultivars seemed to be related to factors other than morphological traits since this species has a short lifecycle.

As proteínas α-RyR e β-RyR apresentam papéis distintos no mecanismo de excitação-contração com diferenças em seus mecanismos de ativação e respostas a ligantes. O RNA total de filé de peito (Pectoralis major m) com PSE (L*24h>53,0; pH 5,8) e não-PSE (44<"L*24h>53) de duas linhagens distintas, de corte e de postura, foram utilizadas para estudar a expressão gênica dos genes α-ryr β-ryr por PCR-em-tempo-real. Os valores médios de expressão gênicas relativas (RQ) foram inferiores (p<0,05) para em frangos PSE das duas linhagens quando comparadas aos frangos não-PSE. Por outro lado, não houve diferenças (p>0,05) na expressão do , independentemente da linhagem estudada. Os resultados de RQ para β-ryr indicaram nas amostras PSE, uma alteração na proporção (1:1) de α-RyR/β-RyR comumente encontrada em músculos de aves. Estes resultados originam a primeira evidência da ocorrência de carnes PSE como resultado de uma disponibilidade acentuada de Ca2+ no citosol pela expressão diferenciada de proteínas receptoras de rianodina.

Total RNA isolated from Pectoralis major muscle from PSE (L*24h>53.0, pH<5.8) and non-PSE (44<"L*24h>"53) meats of two phenotypically distinct chicken lines, broiler and layer, was used to investigate the α-ryr and β-ryr gene expression by real-time RT-PCR approach. Mean relative quantification (RQ) values were lower (p<0.05) for β-ryr in PSE chickens from both lines when compared to non-PSE chickens, while there was no difference (p>0.05) in α-ryr gene expression regardless of line studied. The β-ryr RQ results suggested that in PSE samples an alteration might occur in the regular ratio (1:1) of α-RyR/β-RyR normally found in avian muscles. These results provided the first evidence of PSE meat occurrence as a result of the differential expression of ryanodine receptor genes which might lead to an increased in Ca2+ availability at the cell milieu.

Amostras do músculo Pectoralis major foram coletadas e uma RT-PCR foi conduzida para avaliar a sequência do mRNA do αRYR, região compreendida entre os resíduos de aminoácido 386-540, numerado de acordo com a sequência de perus. Os resultados revelaram duas classes de transcritos. O primeiro teve 97% e 74% de identidade com as sequências de αRyR e RyR1 de perus e humanos, respectivamente, e incluiu todos os transcritos com substituições de nucleotídeos. A segunda classe de transcritos foi caracterizada pela deleção de bases que levaram a um stop códon prematuro e a uma proteína truncada não-funcional. Esses resultados são até o momento, o primeiro relato de sequenciamento do αRYR, região hotspot1 de frangos e podem servir como guia para estudos futuros na tentativa de se encontrar uma solução para os problemas na cadeia de produção de frangos relacionados com as carnes PSE (pálida, flácida e exsudativa).

Samples of Pectoralis major m. were collected, and an RT-PCR analysis of the a-Ryanodine receptor (a RYR) from chicken mRNA hotspot region spanning aminoacid residues 386 to 540, numbered according to the turkey sequence, revealed two classes of transcripts. The sequences of the first class were similar to turkey and human with 97% and 74% of identity, respectively, and included all transcripts with substitutions in the nucleotide sequence. The second class was characterized by the deletion of nucleotides, leading to a premature stop codon and coding for a truncated and nonfunctional protein. These results are to date the first report related to the sequencing of the chicken αRYR hotspot region 1, which will possibility serve as a guide for further studies regarding a solution in the poultry production chain related to the problem of pale, soft and exudative (PSE) meat.

An understanding of the relationship of geographically different soybean gene pools, based on selectively neutral DNA markers would be useful for the selection of divergent parental cultivars for use in breeding. We assessed the relationships of 194 Chinese, 59 Japanese, and 19 Brazilian soybean cultivars (n = 272) using 12 simple sequence repeat (SSR) markers. Quantification Theory III and clustering analyses showed that the Chinese and Japanese cultivars were genetically quite distant to each other but not independent, while Brazilian cultivars were distantly related to the cultivars from the other two countries and formed a cluster that was distant from the other two gene pool clusters. Our results indicated that the Brazilian soybean gene pool is different from the Chinese and Japanese pool. Exchanges of these gene pools might be useful to increase the genetic variability in soybean breeding.