Análisis de la expresión del gen FRIGIDA4-like (FRL4) de café (Coffea arábica L.)

ABSTRACT Coffee is one of the most economically important commodities. In Brazil, this crop is responsible for generating more than eight million jobs. In the foreign market, Brazil is the largest producer and exporter of coffee. Due to its economic importance, several studies aiming the improvement of coffee are conducted, but there are still problems related to its productivity and quality of the beverage, such as sequential flowering, which causes production losses and a low quality drink. Thus, understanding the molecular mechanisms involved in the flowering process is essential to elucidate how flowering occurs in the coffee crop. The FRI gene is one of the main genes involved in flowering, as it positively regulates the FLC gene at expression levels that inhibit flowering. Thus, the objective of this work was to identify and analyze the expression of the FRIGIDA4-like gene (FRL4) through Bioinformatics and real-time PCR (RT-qPCR). The CaFRL4 gene was identified and showed high expression levels in leaf during flowering, which corroborates with the literature. The results obtained provide the basis for future studies involving genetic transformation in model plants and coffee, permitting the functional characterization of this gene.RESUMO O café é uma das commodities de maior importância econômica. Em âmbito nacional, a cafeicultura é responsável pela geração de mais de oito milhões de empregos. No mercado externo, o Brasil é o maior produtor e exportador do café. Diante da sua importância econômica, vários estudos visando o melhoramento do cafeeiro são desenvolvidos, mas, ainda existem problemas relacionados a sua produtividade e qualidade da bebida, como o florescimento sequencial, que ocasiona perdas de produção e uma bebida de baixa qualidade. Deste modo, a compreensão dos mecanismos moleculares envolvidos no processo de florescimento é essencial para elucidar como o florescimento ocorre na cultura do café. O gene FRI é um dos principais genes envolvidos no florescimento, pois regula positivamente o gene FLC a níveis de expressão que inibem o florescimento. Dessa forma, o objetivo deste trabalho foi identificar e analisar a expressão do gene FRIGIDA4-like (FRL4) por meio da Bioinformática e da PCR em tempo real (RT-qPCR). O gene CaFRL4 foi identificado e apresentou altos níveis de expressão em folha durante o florescimento, o que corrobora com a literatura. Os resultados obtidos dão base para estudos futuros envolvendo transformação genética em plantas-modelo e em café, possibilitando a caracterização funcional desse gene. Palavras-chave: RT-qPCR; Café arábica; Florescimento.ABSTRACT Coffee is one of the most economically important commodities. In Brazil, this crop is responsible for generating more than eight million jobs. In the foreign market, Brazil is the largest producer and exporter of coffee. Due to its economic importance, several studies aiming the improvement of coffee are conducted, but there are still problems related to its productivity and quality of the beverage, such as sequential flowering, which causes production losses and a low quality drink. Thus, understanding the molecular mechanisms involved in the flowering process is essential to elucidate how flowering occurs in the coffee crop. The FRI gene is one of the main genes involved in flowering, as it positively regulates the FLC gene at expression levels that inhibit flowering. Thus, the objective of this work was to identify and analyze the expression of the FRIGIDA4-like gene (FRL4) through Bioinformatics and real-time PCR (RT-qPCR). The CaFRL4 gene was identified and showed high expression levels in leaf during flowering, which corroborates with the literature. The results obtained provide the basis for future studies involving genetic transformation in model plants and coffee, permitting the functional characterization of this gene. Keywords: RT-qPCR, Arabica coffee, Flowering

Eficiência de protocolos de extração de RNA em diferentes tecidos do cafeeiro

In order to use sensitive techniques of molecular biology, such as the study of differentially expressed genes, a highqualityRNA in suitable quantities is necessary. Due to the presence of several varieties and often expressive quantities of secondarycompounds in plants, there is no standard method for the isolation of nucleic acids that can be used for all species. Polyphenols andpolysaccharides are the compounds that interfere the most in the extraction process, and when they are present, a low-quality RNAis produced. Four RNA extraction methods (CTAB method, Hot Borate, CONCERT and Tri Reagent), in four different coffee tissues(root, leaf, flower and fruit) were tested in this work, aiming at determining which method is more efficient. It was observed that theCTAB and Hot Borate methods, in which PVP and/or -mercaptoethanol were added and precipitation with LiCl was performed,presented more pure RNA, with no degradation observed in any of the tissues, being suitable for further gene expression analysis.High-quality RNA was not obtained from any tissue in the extraction with Tri Reagent, which includes the use of phenol, and thusexpression analysis was disturbed. The CTAB macroextraction method presented samples with the highest RNA quality and largestquantities in all tissues. Future works need to be carried out aiming the standardization of this macroextraction method.Para a utilização de técnicas sensíveis de biologia molecular, como o estudo de genes diferencialmente expressos, énecessário a obtenção de um RNA de boa qualidade e em quantidades adequadas. Devido à presença de grandes variedades, efrequentemente grande quantidade de compostos secundários em plantas, não existe um método padrão para o isolamento de ácidosnucléicos que possa ser utilizado para todas as espécies. Os polifenóis e os polissacarídeos são os compostos de maior interferênciano processo de extração, e quando presentes geram um RNA de baixa qualidade. Nesse trabalho foram testados quatro métodos deextração de RNA (Método CTAB, Borato quente, CONCERT e Tri Reagente), em quatro diferentes tecidos de café (raiz, folha, flor efruto), objetivando-se determinar qual método é mais eficiente. Foi observado que os métodos, CTAB e Borato quente, que possuíama adição PVP e/ou -mercaptoetanol, e precipitação com LiCl, foram os que apresentaram RNAs mais puros e sem degradação emtodos os tecidos, e puderam ser utilizados para a análise de expressão gênica. Com a extração utilizando o TriReagente, que tem comobase o fenol, não foi obtido RNA de boa qualidade em todos os tecidos e consequentemente não foi possível a análise de expressão. Ométodo de macroextração CTAB foi o que apresentou amostras com RNA de melhor qualidade e em grandes quantidades em todosos tecidos. Trabalhos posteriores precisam ser realizados a fim de padronizar esse método para microextração

Computer Vision-Aided Intelligent Monitoring of Coffee: Towards Sustainable Coffee Production

Coffee which is prepared from the grinded roasted seeds of harvested coffee cherries, is one of the most consumed beverage and traded commodity, globally. To manually monitor the coffee field regularly, and inform about plant and soil health, as well as estimate yield and harvesting time, is labor-intensive, time-consuming and error-prone. Some recent studies have developed sensors for estimating coffee yield at the time of harvest, however a more inclusive and applicable technology to remotely monitor multiple parameters of the field and estimate coffee yield and quality even at pre-harvest stage, was missing. Following precision agriculture approach, we employed machine learning algorithm YOLO, for image processing of coffee plant. In this study, the latest version of the state-of-the-art algorithm YOLOv7 was trained with 324 annotated images followed by its evaluation with 82 unannotated images as test data. Next, as an innovative approach for annotating the training data, we trained K-means models which led to machine-generated color classes of coffee fruit and could thus characterize the informed objects in the image. Finally, we attempted to develop an AI-based handy mobile application which would not only efficiently predict harvest time, estimate coffee yield and quality, but also inform about plant health. Resultantly, the developed model efficiently analyzed the test data with a mean average precision of 0.89. Strikingly, our innovative semi-supervised method with an mean average precision of 0.77 for multi-class mode surpassed the supervised method with mean average precision of only 0.60, leading to faster and more accurate annotation. The mobile application we designed based on the developed code, was named CoffeApp, which possesses multiple features of analyzing fruit from the image taken by phone camera with in field and can thus track fruit ripening in real time

Bone Radiographic Changes in Slaughter Buffalos with Low Body Condition Index

Background: The largest buffalo herd in Brazil is located on the Island of Marajó, in the State of Pará, northern Brazil. The pastures of the Island of Marajó consist of low quality graminaceous plants, which are generally poor in protein and mineral content. Unbalanced diets associated with low quality pastures are responsible for latent, sub-clinical diseases and metabolic disorders in bovines which affect bone health, especially in periods such as pregnancy and lactation. The purpose of this study was to point out and to describe the radiographic bone changes of buffalos with low body index bred in extensive system and intended for slaughter on the Island of Marajó, Brazil.Materials, Methods & Results: Radiographic examinations of anatomical pieces were obtained from 34 animals of buffalo species, with no distinction of gender, age, or breed. The animals were selected among those that were in the stockyard waiting for slaughtering for the obtainment of the anatomical pieces. For this selection, low physical condition was considered, which mainly included individuals with body condition indexes (ICC) of 1 and 2, on a scale of 1 to 5. From this selection, 98 anatomical pieces were obtained, which included: 28 sets of ribs, 20 femurs, 26 metacarpus, 7 mandibles, 3 radius and ulnas, 4 sets of vertebrae, 4 sets of metacarpus and phalanges, 1 tarsus and 1 set of tarsus and metatarsus. All the pieces were separated, identified, packed in plastic bag and forwarded to the radiographic study. At least one radiographic projection was obtained of each anatomical piece. These were identified, manually processed and stored for subsequent assessment. A single observer, in order to identify and to describe the bone radiographic changes, subjectively performed the radiographic assessment.Discussion: Bone changes were remarkable and in animals of this study, reinforcing the nutritional aspect as being of great importance for the perfect mineral homeostasis and for the osteoarticular system maintenance. Consistent radiographic findings with osteopenia are most often related to nutritional disorders that affect bone metabolism, mainly involving the homeostasis of calcium (Ca) and phosphorus (P). The nutritional hyperparathyroidism, more commonly reported in dogs, cats and exotic animals is a common example of these affections, in which the bone radiopacity reduction is the most evident radiographic aspect. Copper (Cu) deficiency has been correlated with osteochondrosis, epiphyseal fracture of the femoral head and degenerative arthropathy of the hip joint, and erosion of the articular cartilage in a deer (Cervu selaphus). Degenerative arthropathy through radiographs was also found in this study. From the bone radiographic analysis, it is concluded that the osteodystrophic diseases of buffalos raised in pasture system on the Island of Marajó, Pará, Brazil, present a variety of pathological conditions and the most commonly found were: osteoporosis characterized at the radiographic examination for the bone decreased radiopacity, change in bone trabeculae (medullary expansion) and cortical thinning, followed by pathological fractures with high incidence in the ribs. The low body condition, the underdevelopment and cachexia states of the animals in this study indicate the lack of an appropriate prophylactic conduct and a proper feed management. Therefore, the low reserves of P and Cu in the organism may have contributed to the osteoporotic process and, possibly, also to the protein-energy deficit, leading to secondary bone changes and causing a lack of productivity in the herd

A genome-wide analysis of the RNA-guided silencing pathway in coffee reveals insights into its regulatory mechanisms

microRNAs (miRNAs) are derived from self-complementary hairpin structures, while small-interfering RNAs (siRNAs) are derived from double-stranded RNA (dsRNA) or hairpin precursors. The core mechanism of sRNA production involves DICER-like (DCL) in processing the smallRNAs (sRNAs) and ARGONAUTE (AGO) as effectors of silencing, and siRNA biogenesis also involves action of RNA-Dependent RNA Polymerase (RDR), Pol IV and Pol V in biogenesis. Several other proteins interact with the core proteins to guide sRNA biogenesis, action, and turnover. We aimed to unravel the components and functions of the RNA-guided silencing pathway in a non-model plant species of worldwide economic relevance. The sRNA-guided silencing complex members have been identified in the Coffea canephora genome, and they have been characterized at the structural, functional, and evolutionary levels by computational analyses. Eleven AGO proteins, nine DCL proteins (which include a DCL1-like protein that was not previously annotated), and eight RDR proteins were identified. Another 48 proteins implicated in smallRNA (sRNA) pathways were also identified. Furthermore, we identified 235 miRNA precursors and 317 mature miRNAs from 113 MIR families, and we characterized ccp-MIR156, ccp-MIR172, and ccp-MIR390. Target prediction and gene ontology analyses of 2239 putative targets showed that significant pathways in coffee are targeted by miRNAs. We provide evidence of the expansion of the loci related to sRNA pathways, insights into the activities of these proteins by domain and catalytic site analyses, and gene expression analysis. The number of MIR loci and their targeted pathways highlight the importance of miRNAs in coffee. We identified several roles of sRNAs in C. canephora, which offers substantial insight into better understanding the transcriptional and post-transcriptional regulation of this major crop

Reference gene selection for quantitative PCR in liver, skeletal muscle, and jejunum of Bos indicus cattle

ABSTRACT The objective of the present study was to evaluate the stability of candidate reference genes and select the genes that can be used for normalizing real-time polymerase chain reaction (PCR) in the liver, skeletal muscle, and jejunum tissues of Nellore or Nellore × Angus steers fed different diets. Fourteen purebred and 14 crossbred steers were used, in which half of the animals of each genetic group received a diet containing whole shelled corn (WSC) and the other half whole shelled corn and sugarcane bagasse (WSCB). Stability was analyzed by the RefFinder program. To validate the selection of candidate reference genes, the expression of target genes was evaluated using the different groups of reference genes. The most stable genes were 18S, ACTB, and CASC3 for skeletal muscle; HMBS, ACTB, and 18S for the liver; and GAPDH, ACTB, and CASC3 for the jejunum, regardless of breed and diet provided. Possible errors caused in data analyses were clarified comparing the more and less stable genes as reference for normalization of the target genes FASN, ACOX, SCD1, MGAM, and SLC2A1. The use of the more stable and less stable sets of reference genes may lead to different conclusions in respect to the expression profile of the target studied gene. The selection of more suitable reference genes for each experiment is of utmost importance to ensure the reliability of gene expression studies so that they can be applied in practice