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. Key words: Ipomoea batatas, SSR, DNA isolation, landraces, protocol MÉTODOS CTAB DE EXTRAÇÃO DE DNA PARA A ANÁLISE DE MICROSSATÉLITES EM BATATA-DOCERESUMO: 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 mostrouse 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.

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.

Arbuscular mycorrhizal fungi (AMF) colonization in plants promotes both local and systemic changes in the gene expression profiles of the host that might be relevant for drought-stress perception and response. Drought-tolerant common bean plants (cv. BAT 477), colonized by a mixture of AMF (Glomus clarum, Acaulospora scrobiculata, and Gigaspora rosea), were exposed to a water deprivation regime of 96 h during pre-flowering. Root transcriptomes were accessed through RNA-Seq revealing a set of 9,965 transcripts with significant differential regulation in inoculated plants during a water deficit event, and 10,569 in non-inoculated. These data include 1,589 transcripts that are exclusively regulated by AMF-inoculation, and 2,313 under non-inoculation conditions. Relative gene expression analyses of nine aquaporin-related transcripts were performed in roots and leaves of plants harvested at initial stages of treatment. Significant shifts in gene expression were detected in AM water deficit-treated roots, in relation to non-inoculated, between 48 and 72 h. Leaves also showed significant mycorrhizal influence in gene expression, especially after 96 h. Root cortical cells, harboring or not arbuscules, were collected from both inoculation treatments through a laser microdissection-based technique. This allowed the identification of transcripts, such as the aquaporin PvPIP2;3 and Glucan 1,3 β-Glucosidase, that are unique to arbuscule-containing cells. During the water deficit treatment, AMF colonization exerted a fine-tune regulation in the expression of genes in the host. That seemed to initiate in arbuscule-containing cells and, as the stressful condition persisted, propagated to the whole-plant through secondary signaling events. Collectively, these results demonstrate that arbuscular mycorrhization leads to shifts in common bean’s transcriptome that could potentially impact its adaptation capacity during water deficit events.

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.

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