Avaliação de métodos de preservação de amostras de plantas de savanas neotropicais para a obtenção de DNA de alta qualidade para estudos moleculares

Abstract

-(Evaluation of methods of neotropical savanna plant samples preservation for yielding high quality DNA for molecular studies). Methods for the preservation of leaf sample from plants occurring in neotropical savannas were compared, with the aim to get high quality DNA for molecular studies. Different methods for DNA isolation of the same samples were also compared. The DNA quality was evaluated by two empirical methods: digestion of the DNA with three restriction enzymes and by amplification of the mitochondrial cox3 gene by PCR. The results show that, for the savanna plants investigated, the most commonly employed method for plant sample preservation (quick dehydratation using silica gel) is not the most efficient. Our results demonstrate the importance of testing different methods of plant tissue preservation for molecular studies to guarantee high quality DNA. For plants from neotropical savannas, the silica gel method might be often less efficient than other equally simple methods of sample preservation.Key words -DNA isolation, molecular analysis, molecular systematics, sample preservation, tropical savanna plants RESUMO -(Avaliação de métodos de preservação de amostras de plantas de savanas neotropicais para a obtenção de DNA de alta qualidade para estudos moleculares). Foram comparadas metodologias para preservação de amostras de folhas de plantas de savanas neotropicais, visando a posterior obtenção de DNA de alta qualidade para estudos moleculares. Foram também testadas diferentes metodologias para extração de DNA das amostras investigadas. A qualidade do DNA extraído foi avaliada através de dois métodos: por digestão, utilizando-se três enzimas de restrição e através da amplificação do gene mitocondrial cox3 (subunidade III da citocromo oxidase) por PCR. Os resultados revelaram que, para as plantas de savanas investigadas, a metodologia de preservação de amostras mais comumente empregada pelos botânicos (desidratação rápida usando sílica-gel) não é a mais eficiente. Os resultados obtidos demonstram a importância de testar diferentes métodos de preservação de amostras vegetais para estudos moleculares, para garantir a obtenção de DNA de alta qualidade. Para as plantas de savanas neotropicais, o método de preservação em sílica gel pode ser menos eficiente do que outros métodos igualmente simples de preservação de amostras.Palavras-chave -análises moleculares, extração de DNA, plantas de savanas tropicais, preservação de amostras, sistemática molecular

Preservation and maceration of amazon açai leaflet tissue to obtain genomic DNA

“…However, for Neotropical savanna species, the use of silica gel was not recommended for preservation of leaves prior to DNA extraction (FERES et al, 2005). However, these authors report that environmental conditions could have activated dehydration mechanisms in these plants and suggest that the silica gel preservation method may still be effective for other species (FERES et al, 2005). E. precatoria occurs in a different environment with high humidity.…”

“…The estimates of DNA concentrations for the different treatments were subjected to analysis of variance (ANOVA) and, when significant differences were observed, the means were grouped by the Scott-Knott test at the 1% significance level. The analysis was performed using the statistical package SISVAR (FERREIRA, 2011).…”

Research Article Improved methods for storing and extracting DNA from <i>Ilex</i> <i>paraguariensis</i> (Aquifoliaceae) tissue samples

“…These results with silica gel are consistent with those presented by Cascales et al (2014) and Gottlieb and Poggio (2014), for storing young mate leaves. In contrast, dehydration of Kielmeyera lathrophyton samples with silica gel was not adequate (Feres et al, 2005). Silica gel is commonly used to preserve plant samples as it is simple and conserves DNA integrity, even though moisture levels must be maintained after an extended period of time (Gostel et al, 2016;Funk et al, 2017).…”

“…As observed in previous studies, the CTAB extraction method described by Doyle and Doyle (1987) have been adapted for a large number of plant species and forest species, such as Apuleia leiocarpa (Lencina et al, 2016) and Plinia cauliflora (Danner et al, 2011). However, the method does not allow for DNA extraction for Kielmeyera lathrophyton and K. petiolaris (Feres et al, 2005). This methodology is characterized by the use of the cationic detergent CTAB in the extraction buffer, which is necessary for separating nucleic acids from polysaccharides, since polysaccharides have different solubilities in the presence of this detergent (Romano and Brasileiro, 1999).…”

Establishment of Simple and Efficient Methods for Plant Material Harvesting and Storage to Allow DNA Extraction from a Myrtaceae Species with Medicinal Potential

“…All molecular analyses, to be successful, depend on obtaining DNA samples of quality. Therefore, procedures are needed for efficient collection and storage of plant material, as well as isolation of the DNA molecule [19][20][21][22][23][24][25][26]. Experience shows that the use of fresh material is ideal for holding the DNA isolation [21].…”