Abstract
Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the preservation of tissue samples for future use in cacao genome studies. The present study proposes a method for the preservation of sample tissues for DNA extraction and for manual extraction of large number of samples using spheres. The integrity and concentration of the DNA by these methods were assessed and compared with conventional method using mortar. The best parameters in order to obtain a fine powder using spheres was the use of 4 lyophilized leaf disks (50 mg), a single steel ball of 6 mm in diameter, followed by 30 s of manual maceration. The quantity of DNA obtained was four times higher than the conventional method. The purity of the DNA obtained was satisfactory and proved to be amplifiable by PCR using SSR primers. The present approach is a reliable, rapid, simple and consistent DNA isolation method for cacao, compared to the conventional methods. The protocol greatly increases the efficiency of extraction and suggests an inexpensive and practical way of DNA isolation of cacao for large scale. Key words: DNA extraction, cacao, spheres, lyophilized.
Highlights
Cacao (Theobroma cacao L.), the chocolate tree, is an important tropical species that provides sustainable economic and environmental benefits to some of the poorest and most ecologically sensitive areas of the world
The present study proposes a method for the preservation of sample tissues for DNA extraction and for manual extraction of large number of samples using spheres
The sampling of the cacao leaves used in this study allowed for efficient storing and normalization of the plant material obtained from lyophilized cacao tissues
Summary
Cacao (Theobroma cacao L.), the chocolate tree, is an important tropical species that provides sustainable economic and environmental benefits to some of the poorest and most ecologically sensitive areas of the world. Since dry tissue can be stored for several years with little loss in DNA quality (Murray and Thompson, 1980), it can be a perfect association with methods for DNA extraction suitable for genotyping and genetic studies The optimization of this initial stage of collection and storage of plant material depend on the success of subsequent steps of any molecular study. Another major bottleneck encountered in most genomic and molecular markers laboratories, besides the preservation of sample tissue, is the slowness in the maceration of samples. The integrity and concentration of the DNA by these methods were assessed and compared with conventional methods
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