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.

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.

Recentemente, foi descoberta uma raça do nematóide-de-cisto-da-soja (NCS; Heterodera glycines) que apresentou a capacidade de quebrar a resistência da cultivar Hartwig, até então considerada resistente a todas as raças conhecidas do nematóide. Essa população foi coletada no Município de Sorriso, Estado do Mato Grosso, e foi caracterizada como raça 4. Para verificar a diversidade genética entre esta e outras populações pertencentes às raças 4 e 9, foi feita uma caracterização molecular pela técnica de marcadores moleculares RAPD. Foram utilizadas nove populações do NCS, das quais quatro apresentavam a capacidade de parasitar 'Hartwig'. Foi verificado que as populações capazes de parasitar 'Hartwig' foram bastante diferentes das demais. Por meio de análise de agrupamento, com base nas distâncias genéticas encontradas, foram obtidos três grupos: o primeiro, constituído por indivíduos classificados como raça 4, mas que não parasitam 'Hartwig'; o segundo, constituído por quatro populações capazes de parasitar 'Hartwig', e o terceiro, por apenas uma população, classificado como raça 9, e que também não parasita 'Hartwig'. Este estudo confirmou que a população de NCS, encontrada em Sorriso, é geneticamente distinta das demais populações da raça 4 encontradas e constitui uma nova raça, denominada 4+.

Recently, a new race of soybean cyst nematode (SCN) (Heterodera glycines) was discovered, which breaks the resistance of cultivar Hartwig, resistant to all known races of SCN. This population was obtained from soybean plants collected in the Sorriso county, state of Mato Grosso, Brazil, and is characterized as race 4. To verify if this isolate was different from others classified as races 4 and 9, their genetic diversity was analyzed by using the RAPD technique. Nine populations of SCN were analyzed, and only four populations were able to parasitize soybean plants cultivar Hartwig. Based on this study, it was verified that the populations that break Hartwig resistance were different from the other populations. Based on cluster analysis, the populations were separated into three groups. Race 4 populations were placed in the first group, while the race 9 population was placed in the second group and those capable of breaking cultivar Hartwig's resistance were placed in group three. This work confirms that the SCN population found in Sorriso is different from other isolates classified as race 4, and was called race 4+.