RNA-Seq analysis and de novo transcriptome assembly of Coffea arabica and Coffea eugenioides : P0068

Coffee is one of the most important agricultural commodites in the world. Coffea arabica, an allotetraploid from a recent hybridization of two diploid species (C.canephora and C.eugenioides) is responsible for 70% of world commercial production. Recent studies have been done to analyse transcriptome data of Coffea spp. in order to improve our knowledge in genetics and gene expression of those species. However, there is few data on coffee fruit transcriptome. In this work, RNA-Seq of two organs (whole fruit - cherry stage and mature leaves) from allotetraploid C. arabica cv. Mundo Novo and its diploid ancestor C.eugenioides, were done using Illumina HiSeq 2000 (100 bp single-end sequences). We report the generation of near 10 million reads for C.arabica (4,747,049 of fruits and 6,156,750 reads of leaves) and 7,5 million reads for C.eugenioides (3,688,364 of fruits and 3,835,373 reads of leaves). De novo assembly and digital gene expression analyses was performed to generate a collection of 35,462 contigs from C.arabica cv. Mundo Novo (average length of 691 bp) and 36,935 contigs of C.eugenioides (average length of 701 bp). Funnctional annotation were performed by sequence comparison with public databases (NCBI-nr, Swiss-prot and PlantCyc). Our results showed the presence of prevalent genes and species-specific either in C.arabica or C.eugenioides that could explain particular characteristics of these two species. Characterization of Coffea spp. transcriptome provides an effective tool for better understanding for differential gene expression in Coffea spp., providing important clues of C.arabica gene evolution, as well as valuable information for coffee breeding. (Résumé d'auteur

Organização estratégica de plataforma para caracterização espacial de riscos no setor agropecuário e agroflorestal.

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MR MAQ : algorisme de Read Mapping utilitzant la plataforma Hadoop

L'èxit del Projecte Genoma Humà (PGH) l'any 2000 va fer de la "medicina personalitzada" una realitat més propera. Els descobriments del PGH han simplificat les tècniques de seqüenciació de tal manera que actualment qualsevol persona pot aconseguir la seva seqüència d'ADN complerta. La tecnologia de Read Mapping destaca en aquest tipus de tècniques i es caracteritza per manegar una gran quantitat de dades. Hadoop, el framework d'Apache per aplicacions intensives de dades sota el paradigma Map Reduce, resulta un aliat perfecte per aquest tipus de tecnologia i ha sigut l'opció escollida per a realitzar aquest projecte. Durant tot el treball es realitza l'estudi, l'anàlisi i les experimentacions necessàries per aconseguir un Algorisme Genètic innovador que utilitzi tot el potencial de Hadoop.El éxito del Proyecto Genoma Humano (PGH) en el año 2.000 hizo de la "medicina personalizada" una relidad más cercana. Los descubrimientos del PGH han simplificado las técnicas de secuenciación de tal manera que actualmente cualquier persona puede conseguir su secuencia de ADN completa. La tecnología de Read Mapping destaca en este tipo de técnicas y se caracteriza por manejar una gran cantidad de datos. Hadoop, el Framework de Apache para aplicaciones intensivas de datos bajo el paradigma Map Reduce, resulta un aliado perfecto para este tipo de tecnología y ha sido la opción escogida para realizar este proyecto. A lo largo del trabajo se realiza el estudio, el análisis y las experimentaciones necesarias para conseguir un Algoritmo Genómico novedoso que utilice todo el potencial de Hadoop.In the 2000th the Human Genome Project (PGH) was accomplished successfully and it made "personalized medicine" a closer reality. The PGH has simplified the sequencing techniques in a high way so nowadays anyone can get his full ADN sequence. Read Mapping technology is one of most important sequencing techniques and it is characterized to work with lots of data. Hadoop is the Framework of Apache for data intensive applications under Map Reduce paradigm and it becomes a perfect tool for this kind of technology. For this reason it has been selected for this project. Along this entire project we will realize the study, the analysis and the experimentations to get a new Genetic Algorithm with all Hadoop potential

Phenotypic analysis of Coffea arabica accessions from Ethiopia: Contribution to the undestanding of Coffea arabica diversity

As a consequence of Coffea arabica domestication process, the genetic diversity of cultivated genotypes of this species is extremely restricted. According to this observation, FAO organized, in 1964-1965, surveys of spontaneous and subspontaneous genotypes in the main center of origin (Ethiopia). Various studies were performed regarding the phenotypic variability of these accessions. However a clear understanding of the structure of the phenotypic diversity in Ethiopia remains to be reached. We present here the phenotypic analysis of 130 Ethiopians accessions with 37 phenotypic traits. Two main goals were pursued: to evaluate the interest of Ethiopian accessions for Coffea arabica breeding, and to test relationships between phenotypic variability and geographic origins. (Résumé d'auteur

Analise In silico e in vivo da via de isoprenoides em café

The diterpenes khaweol and cafestol, which are present in the coffee lipid fraction, are originated from the isoprenoid pathway. Despite their diversity in functions and structures, all isoprenoids derive from the common-five carbon building unit isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In higher plants, there are two independent pathways located in the cytosol (mevalonic acid or MVA pathway) and in the plastids (methylerythritol phosphate - MEP pathway). Through the data mining of the Brazilian Coffee Genome Project we studied the genes that code for the enzymes 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR) and mevalonate diphosphate decarboxylase (MPDC) for the MVA pathway, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and isopentenyl diphosphate synthase (IDS) for the MEP pathway. The search for HMGR resulted in 13 ESTs that originated three incomplete contigs, forming two isoforms. For MPDC 7 ESTs formed only one isoform, differently in A. thaliana that has two isoforms. The search for the genes in the MEP pathway resulted in 22 ESTs for DXR and 47 ESTs for IDS. In each case only one contig was formed, similar to found in A. thaliana as well. Southern blots of genes HMGR and DXR demonstrated the presence two isoforms for HMGR and one for DXR. Northern blot analysis detected transcripts of DXR at the beginning of the perisperm development and at the late stages of endosperm and pulp development. Transcripts of the isoform HMGR2 were detected in pulp, perisperm and endosperm, in all the stages of development of the fruit. On the other hand, HMGRI transcripts were observed only in pulp and initial stage of the development of the perisperm and endosperm. (Résumé d'auteur

Plataforma de logística.

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Genetic and molecular determinism of diterpenes metabolism in Coffea spp

Cafestol and kahweol are two coffee-specific diterpenes. Although various effects of these compounds have been shown or hypothesized on human health, little information is currently available on the genetic and molecular bases of their metabolism. This work presents data on diterpenes accumulation during fruit development and analysis of gene expression for three key enzymes of the diterpenes biosynthesis pathway. (Résumé d'auteur

Identificação e caraterização molecular de genes envolvidos no metabolismo de diterpenos especificos do cafeeiro

The coffee lipids are important components of aromatic and flavor characteristics of the coffee cup. In the lipid fraction, the diterpens cafestol, kahweol and derived compounds (16-O-methylcafestol, 16-O-methylkahweol) are diterpens specific Coffea spp. compounds. Studies have revealed their influence on human health, showing effect on cholesterol level and their action as chemoprotector against toxins with carcinogenic action. In order to increase our knowledge about the genes involved on the coffee diterpens biosynthesis a in silico and in vivo study of the genes involved in the first stages of their pathway (i.e copalyl diphosphate synthase (CPS), kaurene synthase (KS) and kaurene oxidase (KO)) was performed. For gene expression analysis fruits of Coffea arabica cv. IAPAR 59 and Coffea canephora cv. Apoatã at different stages of maturation was used. In silico studies allowed the identification of one gene for CPS and KS and two for KO. It also revealed different expression patterns specific to each gene in terms of level and timing of expression. Results of expression in vivo were slightly different from the ones obtained in silico, and the comparison of C. canephora and C. arabica also revealed some differences. These data will be confirmed by RT-PCR and a whole transcriptome approach using macroarrays based on the Genoma Café Project will be initiated to identify other genes involved in this bisoynthesis pathway. (Résumé d'auteur

Identificação de polimorfismos em genótipos de Coffea arabica de uma coleção da Etiópia : [n.142]

Os marcadores moleculares são ferramentas importantes para acelerar os programas de melhoramento. Para o cafeeiro, uma espécie perene, o uso de marcadores é particularmente desejável devido ao tempo e recursos gastos para o lançamento de uma nova cultivar. Duas espécies do gênero Coffea são responsáveis por quase toda a produção de café: Coffea arabica e C. canephora. Contudo, para C. arabica, o número de marcadores polimórficos é relativamente baixo comparado a C. canephora e outras culturas, uma vez que a espécie apresenta baixa diversidade genética. Muitos estudos com marcadores genéticos foram feitos para analisar a diversidade da C. arabica, mas os resultados não foram eficientes para a discriminação genotípica detalhada e mapeamento genético. O Instituto Agronômico do Paraná (IAPAR) possui uma coleção de 132 acessos de C. arabica originários da Etiópia, que apresentam variabilidade fenotípica com potencial para serem utilizados para exploração da diversidade. Neste sentido, este estudo buscou analisar a diversidade nucleotídica pela identificação de polimorfismos, SNPs e INDELs, de uma população do centro de origem de C. arabica, associado com o sequenciamento de nova geração. O RNA-seq de dois tecidos, frutos e folhas, de quatro genótipos de C. arabica de uma população da Etiópia, C. arabica cv. Mundo Novo e de um dos seus ancestrais de C. arabica - C. eugenioides, foram sequenciados pela metodologia Illumina HiSeq2000. Os reads obtidos foram processados e posteriormente as sequências foram mapeadas em uma referência de C. canephora para identificação dos polimorfismos. Foram feitas duas estratégias: i) na primeira estratégia, foi utilizado uma ferramenta chamada SNiPloid com critérios de cobertura para o polimorfismo identificado e ii) uma segunda estratégia que considera os polimorfismos encontrados diretamente dos arquivos de detecção dos polimorfismos. Os resultados identificaram um número grande de polimorfismos. Na primeira estratégia, foram encontrados pelo menos 5.500 SNPs potenciais para a genotipagem e na segunda, 103.791 SNPs potenciais. Para essa última, ainda é necessário estabelecer critérios e filtros para escolher os polimorfismos que serão inicialmente genotipados. Os dados também mostraram a importância de utilizar um grupo mais diverso de genótipos associado com o sequenciamento de nova geração para detecção de SNPs. Este trabalho será importante para direcionar futuros trabalhos na caracterização da diversidade genética em C. arabica, além de estudos de mapeamento genético por associação. (Résumé d'auteur

Polimorfismos nucleotidicos de genes envolvidos nas caracteristicas quimicas do grão de café, complementaridade das estratégias In silico e in vivo

The understanding of the genetic bases of coffee bean chemical composition is a requirement for breeding programs, which aim to improve coffee beverage quality. Nowadays, the development of the genomic toolkit allows the identification of candidate genes potentially controlling these traits. However, the direct utilization of these new tools for breeding relies on the ability to identify within this set of candidates the ones that are responsible for the variability observed between genotypes, i.e. the ones whose polymorphisms affect the variability of the traits of interest. Evaluation of the nucleotide diversity of the genes, which is a pre-requisite to test these associations, can be done in two ways: through the analysis of the EST libraries available (in silico strategy) or through direct sequencing of genotypes of interest (in vivo strategy). The purpose of this study was to evaluate the relevance of the in silico polymorphism discovery strategy in Coffea based on the EST libraries currently available. Genes for the biosynthetic pathways leading to sucrose and coffee specific diterpens (cafestol and kawheol) were analysed in silico. This strategy yielded the identification of 1.1 polymorphism per 100 bp in average for the 14 analysed genes, this result underlying the feasibility of this method for Coffee. Analysis of the nucleotide diversity in vivo for a few genes of the sucrose biosynthesis pathway allowed a comparison of the two strategies. The in silico discovery strategy is complementary to the in vivo one providing in a cost effective manner a first evaluation of nucleotide diversity at the whole genome level. (Résumé d'auteur