Transcriptome analysis of leaves, flowers and fruits perisperm of Coffea arabica L. reveals the differential expression of genes involved in raffinose biosynthesis

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages. (Résumé d'auteur

Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis.

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a largescale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages

Transcriptome Analysis Of Leaves, Flowers And Fruits Perisperm Of Coffea Arabica L. Reveals The Differential Expression Of Genes Involved In Raffinose Biosynthesis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified similar to 24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages.121Brazilian Coffee Research Consortium, National Institute for Coffee Science and Technology (INCT-Cafe)Coordination for the Improvement of Higher Education Personnel (CAPES)National Council of Technological and Scientific Development (CNPq)Brazilian Innovation Agency (FINEP)Center for Computational Engineering and Sciences at Unicamp/SP-BrazilCAPESFundacao Araucaria (FA)CNPqCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Diterpenes biochemical profile and transcriptional analysis of cytochrome P450s genes in leaves, roots, flowers, and during Coffea arabica L. fruit development.

Lipids are among the major chemical compounds present in coffee beans, and they affect the flavor and aroma of the coffee beverage. Coffee oil is rich in kaurene diterpene compounds, mainly cafestol (CAF) and kahweol (KAH), which are related to plant defense mechanisms and to nutraceutical and sensorial beverage characteristics. Despite their importance, the final steps of coffee diterpenes biosynthesis remain unknown. To understand the molecular basis of coffee diterpenes biosynthesis, we report the content dynamics of CAF and KAH in several Coffea arabica tissues and the transcriptional analysis of cytochrome P450 genes (P450). We measured CAF and KAH concentrations in leaves, roots, flower buds, flowers and fruit tissues at seven developmental stages (30e240 days after flowering - DAF) using HPLC. Higher CAF levels were detected in flower buds and flowers when compared to fruits. In contrast, KAH concentration increased along fruit development, peaking at 120 DAF. We did not detect CAF or KAH in leaves, and higher amounts of KAH than CAF were detected in roots. Using P450 candidate genes from a coffee EST database, we performed RT-qPCR transcriptional analysis of leaves, flowers and fruits at three developmental stages (90, 120 and 150 DAF). Three P450 genes (CaCYP76C4, CaCYP82C2 and CaCYP74A1) had transcriptional patterns similar to CAF concentration and two P450 genes (CaCYP71A25 and CaCYP701A3) have transcript accumulation similar to KAH concentration. These data warrant further investigation of these P450s as potential candidate genes involved in the final stages of the CAF and KAH biosynthetic pathways

In silico identification of coffee genome expressed sequences potentially associated with resistance to diseases

Sequences potentially associated with coffee resistance to diseases were identified by in silico analyses using the database of the Brazilian Coffee Genome Project (BCGP). Keywords corresponding to plant resistance mechanisms to pathogens identified in the literature were used as baits for data mining. Expressed sequence tags (ESTs) related to each of these keywords were identified with tools available in the BCGP bioinformatics platform. A total of 11,300 ESTs were mined. These ESTs were clustered and formed 979 EST-contigs with similarities to chitinases, kinases, cytochrome P450 and nucleotide binding site-leucine rich repeat (NBS-LRR) proteins, as well as with proteins related to disease resistance, pathogenesis, hypersensitivity response (HR) and plant defense responses to diseases. The 140 EST-contigs identified through the keyword NBS-LRR were classified according to function. This classification allowed association of the predicted products of EST-contigs with biological processes, including host defense and apoptosis, and with molecular functions such as nucleotide binding and signal transducer activity. Fisher's exact test was used to examine the significance of differences in contig expression between libraries representing the responses to biotic stress challenges and other libraries from the BCGP. This analysis revealed seven contigs highly similar to catalase, chitinase, protein with a BURP domain and unknown proteins. The involvement of these coffee proteins in plant responses to disease is discussed

II Workshop Interação Molecular Planta-Praga.

bitstream/CENARGEN/29684/1/doc229.pdfEditores: Maria Fátima Grossi de Sá (Coordenação Geral), Embrapa Recursos Genéticos e Biotecnologia; Maria Cristina Mattar da Silva (Vice-coordenação), Embrapa Recursos Genéticos e Biotecnologia; Alexandre Augusto Pereira Firmino; bolsista, Embrapa Recursos Genéticos e Biotecnologia; Osmundo Brilhante de Oliveira Neto, bolsista, Embrapa Recursos Genéticos e Biotecnologia; Thales Lima Rocha, Embrapa Recursos Genéticos e Biotecnologia; Arnúbio Valencia Jimenez, bolsista, Embrapa Recursos Genéticos e Biotecnologia; Érico Augusto Rosas de Vasconcelos, bolsista, Embrapa Recursos Genéticos e Biotecnologia

Study of the MADS-box genes expression during floral development in Coffea arabica L.

Orientador: Marcelo Carnier DornelasDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A família dos genes MADS-box codifica fatores de transcrição que atuam como reguladores importantes em muitas etapas no desenvolvimento de diversos organismos. Em plantas, estes genes estão envolvidos na determinação da identidade dos meristemas reprodutivos e dos órgãos florais, bem como no controle de diversos processos durante o desenvolvimento. O presente trabalho teve como objetivo estudar o padrão de expressão dos prováveis ortólogos dos genes do modelo ABC (APETALA1, APETALA3, PISTILATA e AGAMOUS de Arabidopsis thaliana, e do gene TM6 de Solanum lycopersicum) em Coffea arabica L. Estes genes pertencem à família MADS-box e estão relacionados à determinação da identidade dos órgãos florais na planta-modelo A. thaliana. A partir do banco de dados de sequências expressas de cafeeiro (CAFEST), foram identificados 23 possíveis homólogos de genes MADS-box em cafeeiro. Perfis de expressão por RT-PCR indicaram que a maioria destes genes são expressos em flor e fruto. A análise dos dados gerados pelo uso de microscopia óptica e de varredura permitiu estabelecer uma sequência de desenvolvimento para estabelecimento dos órgãos florais em cafeeiro, facilitando a identificação dos locais de expressão dos ortólogos do modelo ABC pela técnica de hibridização in situ. Sendo C. arabica uma espécie relativamente recente e com características peculiares, foi proposto um mecanismo de atuação dos genes do modelo ABC. Dessa forma, os resultados obtidos contribuem para a compreensão do estabelecimento dos órgãos florais em C. arabica. Adicionalmente, pela caracterização de um número elevado de genes da família MADS, foram identificados outros genes potencialmente envolvidos em outros processos de desenvolvimento, que futuramente poderão ser utilizados para incremento da indústria cafeeira.Abstract: The MADS-box gene family encodes transcription factors that act as key regulators in many steps in the development of various organisms. In plants, these genes are involved in determining the identity of reproductive meristems and floral organs as well as in controlling several processes during development. This work aimed to study the expression patterns of putative orthologs of the ABC model genes (APETALA1, APETALA3, AGAMOUS and PISTILATA from Arabidopsis thaliana, and TM6 from Solanum Lycopersicum) in Coffea arabica L. These genes belong to the MADS-box family and are related to the determination of floral organ identity in the model plant A. thaliana. From the CAFEST database of expressed sequence tags, 23 MADS-box gene sequences were identified in coffee. Expression profiles of these genes, determined by RT-PCR, indicated that most of these genes are expressed in flowers and fruits. The analysis of data from optical microscopy and scanning electron microscopy allowed the establishment of a developmental sequence for the establishment of floral organ, facilitating the characterization of the spatial expression patterns of orthologs of the ABC genes by in situ hybridization. A diversified role of conserved genes of the ABC model was proposed for the relatively recent and peculiar specie that is C. arabica. The obtained results aid the understanding of the establishment of floral organs in C. Arabica. Additionally, as many other coffee MADS-box genes were also characterized, other genes, potentially involved in other developmental processes that could be of interest to the industry in the future were also identified.MestradoBiologia VegetalMestre em Biologia Vegeta

Genotipagem por sequenciamento para identificação de SNPs e associação com características agronômicas em Coffea canephora

A genotipagem por sequenciamento (GBS) é capaz de identificar e genotipar milhares de polimorfismos do tipo SNPs de forma simultânea. Objetiva-se contribuir para o melhoramento genético do cafeeiro Conilon através da caracterização da ocorrência de SNPs no genoma de Coffea canephora e de associações destes com características de interesse agronômico. Os145 indivíduos de duas famílias de irmãos completos (clones 109x120/120x109 e 76x48) do programa de melhoramento do Instituto Capixaba de Pesquisa, Assistência Técnica e Extensão Rural (Incaper)foram fenotipados e os DNAs foram multiplexados em sequenciador Ilumina na Universidade de Cornell. Detectaram-se 91.105 SNPs antes de aplicar os parâmetros de filtragem, sendo que após os filtros houve redução de 64%. Ampla distribuição dos SNPs foi encontrada, sendo que foram detectados em média 1330 SNPs gênicos e 2955 intergênicos, por pseudocromossomo. Verificou-se que o padrão de distribuição dos SNPs nas regiões do genoma difere. A menor ocorrência de SNPs detectada em regiões gênicas é esperada como consequência da pressão de seleção, que limita as alterações de aminoácidos nas sequências proteicas. Os estudos de associação permitiram encontrar 18 SNPs associados a características fenotípicas de Coffea canephora (S2_9329731, S2_4579518, S2_41329025, S2_17821870, S2_20934616, S3_23227842, S4_22978689, S5_10964474, S6_9949547, S7_13991105, S7_13991086, S7_13991077, S9_4618814, S9_18527411, S10_24840747, S11_30063996, S11_23828233). Localizam-se em regiões intergênicas 33% dos SNPs, sendo que os demais se distribuem em região de íntrons, éxons e 3UTR. Os SNPs em região codificadora são responsáveis por alterações não sinônimas em 82% das ocorrências. Os resultados encontrados são importantes para a cafeicultura e podem contribuir para a seleção assistida por marcadores. Palavras-chave: cafeeiro, sequenciamento, SNP, genotipagem, estudos de associação