Functional Genomics of Biotic and Abiotic Stresses in Phaseolus vulgaris

Common bean is the most important legume for human consumption in the world, being a crop extremely diverse in cultivation methods, uses, range of environments in which it is adapted, morphological variety, among others. Besides its high demand and production, this crop is threatened by a series of biotic and abiotic adversities during its life cycle, which leads to losses in yield of up to 100%. In this chapter, we explored the main constraints that affect common bean and the ways this plant reaches tolerance or resistance to them, highlighting studies at the molecular level that enabled to understand the mechanisms by which common bean perceives, responds, and adapts to a stress condition. Special focus has been given to the most recent findings in the understanding of the mechanisms underlying drought tolerance and anthracnose resistance. Thereby, we reviewed some genetic and functional genomic studies concerning the genes and pathways involved in each case. Furthermore, we outline important genetic resources of Phaseolus vulgaris, as well as the technologies and methods used toward these findings

Métodos CTAB de extração de DNA para a análise de microssatélites em batata-doce

Os marcadores microssatélites são úteis para a análise da diversidade genética de variedades tradicionais de batata-doce (Ipomoea batatas). Para estes estudos, métodos práticos de extração de DNA precisam ser estabelecidos para assegurar uma boa qualidade e quantidade de DNA extraído. Assim, foi comparada a eficiência de três metodologias para extração de DNA usando o tampão de extração CTAB, todas com modificações. Para verificar a quantidade e pureza na quantificação de DNA, bem como o padrão de bandas de microssatélites para as três metodologias utilizaram-se seis etnovariedades de batata-doce. Os testes mostraram que as três metodologias apresentaram resultados satisfatórios. Uma das metodologias baseada em tecido foliar macerado em nitrogênio líquido mostrou-se a mais adequada devido à simplicidade e menor custo. Entretanto, o método baseado em tecido foliar seco foi o mais vantajoso devido à praticidade na aquisição da planta e no processo de secagem, principalmente quando a coleção encontra-se em condições in situ, e pela possibilidade do armazenamento refrigerado das amostras secas e maceradas para futuras extrações de DNA.Microsatellite markers have proved to be useful in genetic diversity assessments of sweetpotato (Ipomoea batatas) but practical DNA extraction methods to ensure good quality and quantity DNA for these studies are yet to be established. This study compares the efficiency of three modified methodologies for DNA extraction of six sweetpotato landraces using the CTAB extraction buffer in regard to quantity and purity of DNA quantification and microsatellite band patterns. All methodologies yielded satisfactory results, but the method based in leaf tissue macerated in liquid nitrogen was deemed more adequate because of its simplicity and lower cost. However, the method based in dry leaf tissue was considered more advantageous, first because elicits practicability in the plant acquisition and drying process, especially when the collection is performed in situ, and also because its simplicity makes possible the cold storage of the dry, ground samples for future DNA extractions

Evolutionary processes and the origin of crop plants

A evolução das plantas cultivadas, que teve início há cerca de 13.000 anos, está sujeita aos mesmos processos evolutivos naturais, aliada à ação do homem de forma consciente ou inconsciente, levando à domesticação. Nesta revisão, são apresentados os principais fatores evolutivos, tais como mutação, hibridação, migração, seleção e deriva genética, que, de alguma maneira, estão envolvidos com a origem, evolução e domesticação de plantas cultivadas. São apresentados também exemplos de como esses processos influenciaram na diversidade intra e interespecífica de plantas cultivadas, com o aparecimento de novas variedades ou mesmo de novas espécies. De modo geral, tais processos atuaram na ampliação, na manutenção, bem como na redução da variabilidade genética das plantas cultivadas.The evolution of crop plants, which began at about 13,000 years ago, is subject to the same natural evolutionary processes, coupled with the action of man, consciously or unconsciously, leading to domestication. This review presents the main evolutionary factors such as mutation, hybridization, migration, selection and genetic drift, which somehow are involved in the origin, evolution and domestication of crop plants. Examples of how these processes influenced in the intra and interespecific diversity of crop plants, with the uprise of new varieties or even of new species, are also presented. In general, these processes have worked well in the increase, maintenance, as well as in the reduction of genetic diversity of crop plants

Differential gene expression analysis induced by the interaction between common beans (Phaseolus vulgaris L.) and arbuscular mycorrhizal fungi under drought

A seca é um dos principais problemas que afetam a produção do feijoeiro. A despeito da importância de caracteres fenotípicos radiculares, muitos dos esforços de melhoramento genético da cultura tem focado na seleção de cultivares com maior produção de grãos. A simbiose estabelecida entre plantas e FMA aumentam o potencial de captação de água no solo através das extensas redes formadas pelas hifas e alteram vias metabólicas vitais para a manutenção das relações hídricas da planta. O modelo de interação feijoeiro (BAT 477) colonizado por uma mistura de FMA (Glomus clarum, Acaulospora scrobiculata e Gigaspora rosea) foi submetido a um déficit hídrico de 96 h durante o pré-florescimento. O transcritoma global de raízes inoculadas e não-inoculadas, sujeitas ou não à seca, foi comparado por RNA-Seq. Um conjunto de 71 transcritos foram induzidos por FMA durante a seca. Comparando-se os tratamentos estresse e controle, 12.086 unigenes foram regulados em plantas inoculadas e 11.938 em não-inoculadas, refletindo o alto potencial de tolerância da linhagem BAT 477 e indicando que a presença de FMA produz uma regulação fina no perfil de expressão de genes regularmente envolvidos na resposta da planta ao estresse. Foram selecionados 15 fatores de transcrição e seus perfis de expressão foram caracterizados por RT-qPCR tomando-se três períodos, 48, 72 e 96 h de déficit hídrico. Plantas inoculadas ativaram a expressão destes genes mais tardiamente (após 72 h), refletindo melhorias nas condições hídricas da planta que adiam a percepção do estresse. Adicionalmente, a expressão de 23 transcritos foi avaliada em três amostras teciduais diferentes obtidas por microscopia de microdissecção a laser. Glucan 1,3 ?-Glucosidase e PIP2,3, foram detectados somente em células do córtex radicular contendo arbúsculos indicando uma possível indução tecido específica dependente da presença dos fungos. Análises complementares apontaram a regulação de 171 unigenes envolvidos na resposta das FMA ao estresse. Estes resultados validam a hipótese inicial de que a inoculação com FMA altera os perfis de expressão de genes vitais para a resposta da planta ao déficit hídricoDrought is one of the main problems that affect common bean\'s production. Despite the importance of root fenological characters, breeding efforts for the culture have focused on the selection of cultivars for grain yield. The symbiosis stablished between AMF and plants enhances the potential of water absorption from the soil through an extensive net formed by hyphae and alters vital metabolic pathways involved in the maintenance of the water relations in plants. The interaction model common bean (BAT 477) colonized by a mixture of AMF (Glomus clarum, Acaulospora scrobiculata and Gigaspora rosea) was exposed to a water deficit regime of 96 h during pre-flowering. Global transcriptome from inoculated and non-inoculated roots, exposed or not to drought, were compared through RNA-Seq. A set of 71 transcripts was induced by AMF during drought. Comparing both stress and control treatments, 12,086 unigenes were regulated in inoculated plants, and 11,938 in non-inoculated, reflecting the great tolerance potencial of the lineage BAT 477 and indicating that the presence of AMF produces a fine tune regulation on the expression of genes regularly involved on the drought response of the plant. It was selected 15 transcription factors and their expression profiles were characterized through RT-qPCR taking 3 periods, 48, 72 and 96 h of water deficit. AM plants activated earlier (after 72 h) the expression of these genes, reflecting improvements on the water conditions of the plant that delay the stress perception. Additionally, the expression of 23 transcripts was evaluated on three different tissue samples obtained through laser microdissection microscopy. Glucan 1,3 ?-Glucosidase and PIP2,3, were detected only in cortical cells containing arbuscules, pointing to a possible tissue specific induction dependent of the presence of the fungus. Additional analysis point to the regulation of 171 unigenes involved on the response of the AMF to drought. These results corroborate the initial hypothesis that the inoculation with AMF alters the gene expression profiles of genes that are vital for water deficit response in plant

Identification of differentially expressed genes in common bean involved in drought stress resistance

O Brasil é o segundo maior produtor de feijão, sendo a espécie mais cultivada o Phaseolus vulgaris L. Entre as três possíveis safras exploradas no Brasil, aquela que gera a maior produção é a da seca. Por outro lado, como a maioria das lavouras emprega pouca tecnologia, um dos problemas desta cultura é o estresse hídrico, que leva a uma redução na produtividade. Dessa forma, a identificação de genes que controlam os mecanismos de defesa e adaptação do feijoeiro à falta de água seria de grande utilidade. Nos últimos anos, muitas informações ômicas do feijoeiro foram geradas, criando uma visão integrada deste organismo e oferecendo uma complexa rede de interações entre genes e seus produtos. Este trabalho teve como objetivo central à identificação de genes diferencialmente expressos no sistema radicular de um genótipo de feijoeiro resistente ao estresse hídrico (BAT 477), quando submetido a uma interrupção de irrigação durante seu desenvolvimento. Foi construída uma biblioteca subtrativa de cDNA (SSH), que representou os genes diferencialmente expressos no genótipo resistente, utilizando-se como driver o genótipo Carioca 80SH (suscetível a seca). Foram obtidos 1572 reads válidos, sendo 931 destes singletons e 189 contigs com uma média de seis reads por cluster. A anotação das sequências foi conduzida via BLASTX, sendo consideradas para anotação somente os melhores resultados dos produtos gênicos similares com E- Value \'<OU=\' 10-5. A classificação funcional foi feita tendo-se como base modelos descritos para plantas (modelo CS e MIPS) e os resultados foram agrupados em seis classes funcionais distintas. As análises de bioinformática ajudaram na identificação de genes descritos como envolvidos na resposta da planta ao estresse hídrico. Entre eles: proteínas do grupo LEA; fatores de transcrição como DREB, NAC e proteínas ricas em leucina; enzimas sintetizadoras de carboidratos incluindo trehalose, sacarose e rhamnose; proteínas ricas em prolina; receptores de hormônios (ABA, etileno); aquaporinas; chaperonas; ubiquitinas; nodulinas; e proteínas associadas à fotossíntese e à respiração. A fim de se obter a validação das ESTs anotadas, foi conduzido um experimento de PCR em tempo real confrontando os padrões de expressão de 15 genes sob quatro tratamentos: ambos os genótipos sob estresse e respectivos controles. Três replicatas biológicas foram adotadas e dois genes de referência (act e skip2) foram escolhidos para normalização interna dos dados. Os padrões de expressão gênica obtidos confirmam a hipótese de que tais genes são mesmo mais expressos no genótipo resistente, embora não sejam exclusivos já que uma quantidade menor de tais transcritos também foi detectada no genótipo suscetívelBrazil is the second biggest producer of common bean, being Phaseolus vulgaris L. the most cultivated species. Among the three possible harvests exploited in Brazil, drought is the one which generates the greatest production. On the other hand, as the majority of the households employees low technology, one of the problems of this culture is drought stress that leads to a reduction in the productivity. So, the identification of gene that controls the mechanisms of defense and adaptation of common bean to the lack of water would be very useful. In the past years, many omics information of common bean have been generated, creating an integrated view of this organism and providing a complex network between genes and its products. The main goal of this work was the identification of differentially expressed genes in a genotype of common bean resistant to drought stress (BAT 477), when submitted to a interruption of irrigation during its development. It was build a cDNA suppression subtractive hybridization library (SSH), which represented the differentially expressed genes, on the resistant genotype, having as driver the genotype Carioca 80SH (susceptible to drought). It was obtained 1572 valid reads, being 931 singletons and 189 contigs, with the average of 6 reads per cluster. The sequences annotation was conducted via BLAST X, considering only the best similarity results with E value \'<OU=\' 10-5. The functional classification was done adopting models described for plants (CS and MIPS) and the results were grouped into six different functional classes. Bioinformatic analyses contribuited to the identification of genes described as involved on plants response to drought stress. Among them: LEA proteins; transcription factors like DREB, NAC and leucine-rich proteins; carbohydrates synthesizers enzymes like the ones for trehalose, sucrose and rhamnose; proline-rich proteins; hormone receptors (ABA and ethylene); aquaporins; chaperones; ubiquitins; nodulins; and proteins associated with photosynthesis and respiration. In order to validate the ESTs annotated, a RT-qPCR experiment was conducted comparing the expression patterns of 15 genes under four treatments: both genotypes under stress and their respective controls. Three technical replicates were used and two reference genes (act and skip2) were chosen for intern data normalization. The gene expression patterns obtained confirm the hypothesis that such genes are more expressed on the resistant genotype although they are not exclusive since a lower levels of these transcripts were also detected in the susceptible genotyp

Transcriptional Analysis of Drought-Induced Genes in the Roots of a Tolerant Genotype of the Common Bean (Phaseolus vulgaris L.)

In Brazil, common bean (Phaseolus vulgaris L.) productivity is severely affected by drought stress due to low technology cultivation systems. Our purpose was to identify differentially expressed genes in roots of a genotype tolerant to water deficit (BAT 477) when submitted to an interruption of irrigation during its development. A SSH library was constructed taking as “driver” the genotype Carioca 80SH (susceptible to drought). After clustering and data mining, 1572 valid reads were obtained, resulting in 1120 ESTs (expressed sequence tags). We found sequences for transcription factors, carbohydrates metabolism, proline-rich proteins, aquaporins, chaperones and ubiquitins, all of them organized according to their biological processes. Our suppressive subtractive hybridization (SSH) library was validated through RT-qPCR experiment by assessing the expression patterns of 10 selected genes in both genotypes under stressed and control conditions. Finally, the expression patterns of 31 ESTs, putatively related to drought responses, were analyzed in a time-course experiment. Our results confirmed that such genes are more expressed in the tolerant genotype during stress; however, they are not exclusive, since different levels of these transcripts were also detected in the susceptible genotype. In addition, we observed a fluctuation in gene regulation over time for both the genotypes, which seem to adopt and adapt different strategies in order to develop tolerance against this stress

CTAB methods for DNA extraction of sweetpotato for microsatellite analysis

Os marcadores microssatélites são úteis para a análise da diversidade genética de variedades tradicionais de batata-doce (Ipomoea batatas). Para estes estudos, métodos práticos de extração de DNA precisam ser estabelecidos para assegurar uma boa qualidade e quantidade de DNA extraído. Assim, foi comparada a eficiência de três metodologias para extração de DNA usando o tampão de extração CTAB, todas com modificações. Para verificar a quantidade e pureza na quantificação de DNA, bem como o padrão de bandas de microssatélites para as três metodologias utilizaram-se seis etnovariedades de batata-doce. Os testes mostraram que as três metodologias apresentaram resultados satisfatórios. Uma das metodologias baseada em tecido foliar macerado em nitrogênio líquido mostrou-se a mais adequada devido à simplicidade e menor custo. Entretanto, o método baseado em tecido foliar seco foi o mais vantajoso devido à praticidade na aquisição da planta e no processo de secagem, principalmente quando a coleção encontra-se em condições in situ, e pela possibilidade do armazenamento refrigerado das amostras secas e maceradas para futuras extrações de DNA.Microsatellite markers have proved to be useful in genetic diversity assessments of sweetpotato (Ipomoea batatas) but practical DNA extraction methods to ensure good quality and quantity DNA for these studies are yet to be established. This study compares the efficiency of three modified methodologies for DNA extraction of six sweetpotato landraces using the CTAB extraction buffer in regard to quantity and purity of DNA quantification and microsatellite band patterns. All methodologies yielded satisfactory results, but the method based in leaf tissue macerated in liquid nitrogen was deemed more adequate because of its simplicity and lower cost. However, the method based in dry leaf tissue was considered more advantageous, first because elicits practicability in the plant acquisition and drying process, especially when the collection is performed in situ, and also because its simplicity makes possible the cold storage of the dry, ground samples for future DNA extractions.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

DREB Genes from Common Bean (Phaseolus vulgaris L.) Show Broad to Specific Abiotic Stress Responses and Distinct Levels of Nucleotide Diversity.

We analyzed the nucleotide variability and the expression profile of DREB genes from common bean, a crop of high economic and nutritional value throughout the world but constantly affected by abiotic stresses in cultivation areas. As DREB genes have been constantly associated with abiotic stress tolerance, we systematically categorized 54 putative PvDREB genes distributed in the common bean genome. It involved from AP2 domain location and amino acid conservation analysis (valine at the 14th position) to the identification of conserved motifs within peptide sequences representing six subgroups (A-1 to A-6) of PvDREB proteins. Four genes (PvDREB1F, PvDREB2A, PvDREB5A, and PvDREB6B) were cloned and analyzed for their expression profiles under abiotic stresses and their nucleotide and amino acid diversity in genotypes of Andean and Mesoamerican origin, showing distinct patterns of expression and nucleotide variability. PvDREB1F and PvDREB5A showed high relative inducibilities when genotypes of common bean were submitted to stresses by drought, salt, cold, and ABA. PvDREB2A inducibility was predominantly localized to the stem under drought. PvDREB6B was previously described as an A-2 (DREB2) gene, but a detailed phylogenetic analysis and its expression profile clearly indicated it belongs to group A-6. PvDREB6B was found as a cold- and dehydration-responsive gene, mainly in leaves. Interestingly, PvDREB6B also showed a high nucleotide and amino acid diversity within its coding region, in comparison to the others, implicating in several nonsynonymous amino acid substitutions between Andean and Mesoamerican genotypes. The expression patterns and nucleotide diversity of each DREB found in this study revealed fundamental characteristics for further research aimed at understanding the molecular mechanisms associated with drought, salt, and cold tolerance in common bean, which could be performed based on association mapping and functional analyses