ABSTRACT
DNA extraction of plants with high quality is very important to researches in molecular biology. Several extraction protocols have been used to obtain soybean DNA; however, there is a lack of papers about extraction protocols optimization and the best developmental stage of the plant to collect them. Therefore, the main purpose of the study was to extract high quantity and quality of DNA from fresh or frozen soybean samples, using different protocols. Moreover, we analyzed the best developmental stage of the plant to do the extraction. Fresh leaves or leaves kept for two years in the ultra-freezer were submitted to the DNA extraction protocols: Haberer et al., 1996 (modified); second modification from Haberer et al., 1996; Murray & Thompson, 1980 (modified) e Doyle & Doyle, 1990 (modified). Modified protocol of Doyle & Doyle was used to value the best stage to collect the leaves to do the DNA extraction. The samples were collected in the stages of development VC, V1, V2, V3, V4 and R5. The experiments were conducted in completely randomized design with 10 samples per treatment. The data underwent variance analysis and the averages were compared by the Tukey test (p<0.05). Through Doyle & Doyle, 1990 and Haberer et al., 1996 modified protocols, for both fresh and frozen samples, it was possible to obtain a higher total DNA concentration if compared to the other tested protocols. However, the quality of DNAs extracted by the protocol Doyle & Doyle, 1990 (modified) was superior, due to a minor molecular degradation. Besides that, the extractions made with these protocols have shown to be more efficient using frozen leaves' tissue. Higher DNA concentrations were obtained analyzing VC samples; however, there were no statistical differences between the stages VC, V2 and V3. It is suggested thereby to use modified of Doyle & Doyle for DNA extraction from soybean leaves in V2 and V3 stages of development from frozen samples, providing the collect of a large number of samples and its storage until the analysis.
A extração de DNA de plantas com alta qualidade é de suma importância para pesquisas em biologia molecular. Diversos protocolos de extração vêm sendo utilizados para a obtenção de DNA de soja; contudo, há uma carência de trabalhos de otimização de protocolos de extração e de escolha do melhor estádio de desenvolvimento da planta para a coleta. Desta forma, o objetivo do estudo foi extrair DNA com alta quantidade e qualidade a partir de amostras frescas ou congeladas de soja, utilizando diferentes protocolos de extração. Além disso, foi analisado o melhor estádio de desenvolvimento da planta para a extração. Folhas frescas e armazenadas por cerca de dois anos em ultrafreezer foram submetidas aos protocolos de extração de DNA: Haberer et al., 1996 (modificado); segunda modificação de Haberer et al., 1996; Murray & Thompson, 1980 (modificado) e Doyle & Doyle, 1990 (modificado). Para a avaliação do melhor estádio de coleta das folhas para a extração de DNA foi utilizado o protocolo de Doyle & Doyle modificado. As coletas de amostras foram realizadas nos estádios de desenvolvimento VC, V1, V2, V3, V4 e R5. Os experimentos foram conduzidos em delineamento inteiramente casualizado com 10 amostras por tratamento. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste de Turkey (p<0,05). Através dos protocolos modificados de Doyle & Doyle, 1990 e Haberer et al., 1996, tanto para amostras frescas como para congeladas, foi possível obter uma maior concentração de DNA total se comparado aos demais protocolos testados. Porém, a qualidade dos DNAs extraídos pelo protocolo Doyle & Doyle, 1990 (modificado) foi superior, devido a menor degradação da molécula. Além disso, as extrações efetuadas com estes protocolos se mostraram mais eficientes quando foram utilizados tecidos foliares congelados. Maiores concentrações de DNA foram obtidas quando amostras em VC foram analisadas; porém, não houve diferença estatística entre os estádios VC, V2 e V3. Assim, sugere-se a utilização do protocolo modificado de Doyle & Doyle para extração de DNA de folhas de soja nos estádios de desenvolvimento V2 e V3 a partir de amostras congeladas, viabilizando a coleta de um grande número de amostras e o seu armazenamento até a análise.
Subject(s)
Glycine max , Specimen Handling , DNA, Plant , Process Optimization , Molecular BiologyABSTRACT
Natural selection acts to select better adapted individuals or alleles in segregating population and help plant breeding. The objective of this work was to verify the effect of natural selection on microsatellite alleles as indicators of better adaptation and identification of quantitative trait loci (QTLs) for grain yield. This study evaluated 107 progenies from the F8 and 107 from the F24 generation derived from crossing Carioca MG and ESAL 686 lines, carried out by the bulk method, and evaluated in three different seasons: winter 2001; rainy 2001 and dry 2002. It was utilized 22 polymorphic markers and the natural selection acted in all of them. The frequency of the alleles of the parent Carioca MG, the most adapted, was increased in all of the 22 loci in F8 and 19 loci in F24. Selection affected each locus with different intensities in different generations. All of the selected alleles can be important for breeding program. QTLs were identified in generation F8 and F24 at varied magnitudes. The best marker PVttc002 explained 11.76 percent of variation in grain yield. However, an elevated interaction between QTLs and the environments was observed, showing the great difficulty in assisted selection.