Abstract Microsatellites have been widely used to genotype individuals and to address a myriad of biological questions in many research fields for decades. However, when implementing a microsatellite marker analysis routine from scratch, various problems can arise throughout the process from DNA extraction to allele scoring, including inputting errors in the database, decreasing the reliability of results and having profound negative impacts on the derived decisions. Therefore, correctly assigning a genotype to a sample is crucial and dependent on acquiring knowledge of the technique steps, such as the chemistry of reactions, software, and data curation. This study tested two previously constructed simple sequence repeat (SSR) sets containing ten primer pairs each (ten-plex) in 1142 maize genotypes. Here, we describe the challenges faced when implementing this microsatellite-based genotyping protocol in our laboratory and possible ways to overcome them, hopefully aiding other novice research teams in this field. decades However scratch scoring database decisions Therefore steps reactions software curation SSR (SSR tenplex plex (ten-plex 114 genotypes Here microsatellitebased based them field 11 1

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. CRISPRCas Cas Glycine max. max . max) final cassete expressão. expressão expressão) Detectaramse Detectaram se sequenciamento funcionando

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. CRISPRCas Cas Glycine max Agrobacteriummediated Agrobacterium mediated expression cassette. cassette . cassette) sequencing working CRISPRbased based

Abstract Recent advances in genome editing have enormously enhanced the effort to develop biotechnology crops for more sustainable food production. CRISPR/Cas, the most versatile genome-editing tool, has shown the potential to create genome modifications that range from gene knockout and gene expression pattern modulations to allele-specific changes in order to design superior genotypes harboring multiple improved agronomic traits. However, a frequent bottleneck is the delivery of CRISPR/Cas to crops that are less amenable to transformation and regeneration. Several technologies have recently been proposed to overcome transformation recalcitrance, including HI-Edit/IMGE and ectopic/transient expression of genes encoding morphogenic regulators. These technologies allow the eroding of the barriers that make crops inaccessible for genome editing. In this review, we discuss the advances in genome editing in crops with a particular focus on the use of technologies to improve complex traits such as water use efficiency, drought stress, and yield in maize. production CRISPRCas CRISPR Cas genomeediting tool allelespecific allele specific However regeneration recalcitrance HIEdit/IMGE HIEditIMGE HI Edit/IMGE Edit IMGE ectopictransient ectopic transient regulators review efficiency stress maize HIEdit EditIMGE

ABSTRACT Heirloom tomatoes are open-pollinated varieties bearing a wide diversity of colors and shapes that may be used by breeders aiming to improve physical and biochemical fruit traits. Hence, in this work heirloom tomato accessions were characterized, gathering information to genetic breeding programs focusing on human food. For that, 67 heirloom tomato accessions were evaluated for fruit size, fruit mass, fruit volume, color, vitamin C, titratable acidity, soluble solids content, phenolic compounds content, total flavonoid content, and antioxidant activity. The experiment was conducted in a randomized complete block design with three repetitions. Linear mixed model, Pearson’s correlation and hierarchical clustering were applied to data. Five groups were formed by Ward’s clustering method. The accession UEL 300 constituted group A, which had the greatest mass and volume fruit. Eight accessions formed group B and showed mostly yellow fruits. Group C was comprised of 13 accessions, which had the highest levels of carotenoids, vitamin C, and antioxidant activity. Thirty-three accessions that constituted group D did not stand out for any of the attributes, while 12 accessions into group E had the highest content of phenolic compounds and flavonoids, along with the highest ratio of soluble solids and acidity. Five accessions in this collection (UEL 296, UEL 146, UEL 238, UEL 231, and UEL 217) stood out for their biochemical traits. The wide diversity for physical and biochemical fruit traits can be explored in tomato breeding programs, seeking to develop new cultivars, and strengthening family farming.

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.

RESUMO O grande desafio para garantir a segurança alimentar global está na necessidade de aumentar a produção agrícola. Neste contexto, a biotecnologia destaca-se como uma ferramenta importante para gerar plantas geneticamente melhoradas, com maior resistência/tolerância a pragas, doenças e condições climáticas adversas, que utilizem de forma eficiente os recursos naturais. Uma abordagem avançada para a geração de variabilidade genética de maneira precisa e direcionada tem chamado a atenção dos melhoristas, a técnica de edição genômica CRISPR/Cas (repetições palindrômicas curtas agrupadas e regularmente interespaçadas/proteínas associadas ao CRISPR). O sistema de edição genômica CRISPR/Cas é formado por um RNA guia que direciona uma nuclease para gerar cortes específicos no DNA alvo. Esta ruptura ativa os sistemas de reparo celular e, eventualmente, leva a deleções ou inserções de nucleotídeos. Assim, a tecnologia CRISPR/Cas pode ser aplicada tanto na pesquisa científica básica, quanto no desenvolvimento de culturas com características agronômicas melhoradas, com o potencial de trazer soluções inovadoras para a agricultura. Esta técnica tem sido aplicada em um grande número de culturas agrícolas, incluindo algumas hortaliças. Nesta revisão, apresentamos detalhes do sistema natural e artificial CRISPR/Cas, suas possibilidades como ferramenta biotecnológica, vantagens sobre outras abordagens de melhoramento genético, questões regulatórias e sua aplicabilidade nas hortaliças, bem como desafios futuros.

ABSTRACT The burden of the current global challenge involving food security lies in the need to improve crop production. In this regard, biotechnology stands out as an essential tool to generate plants able to cope with pests, diseases, and harsh climatic conditions, and more efficient in the use of natural resources. An advanced approach to create genetic variability in a precise and targeted way, the genome-editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins), has drawn the attention of breeders. The genome editing CRISPR/Cas system relies on a guiding RNA that directs a nuclease to generate a double-strand break (DSB) at a target DNA, activating the cell repair systems and eventually leading to deletions or insertions of nucleotides. Therefore, CRISPR/Cas is a toolbox to achieve many goals, from basic science investigations to the development of crops with improved agronomic traits, with potential to bring innovative solutions to food production. The CRISPR/Cas system has been applied in a large number of plants, including some horticultural species. In this review, we present details of the CRISPR/Cas natural and artificial systems, its possibilities as a biotechnological tool, advantages over other breeding techniques, regulatory issues, and its applicability in horticultural crops, as well as future challenges.