A protocol for large scale genomic DNA isolation for cacao genetics analysis

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.

Aislamiento de ADN de alta calidad en Psidium guajava L. para estudios genómicos

“…De acuerdo con lo que se observó durante cada procedimiento de extracción, la disminución en la oxidación de los tejidos y soluciones intermedias, fue más dependiente de la condición del sustrato, ya que con el tejido liofilizado se logró disminuir considerablemente los problemas de oxidación en los tres tratamientos. Además, hay una mejora en el rendimiento y calidad de las extracciones de ADN al utilizar muestras liofilizadas (Nunes et al, 2011;Betancurt et al, 2018), ya que al disminuir la cantidad de agua dentro del tejido se disminuye el proceso catabólico dentro de la célula, la fluidez del ambiente y se disminuye la actividad catalítica de las nucleasas y proteasas, lo que evita que se degraden los ácidos nucleicos (Santos et al, 2014).…”

Exploration-Characterization Morphology of Local Cocoa On Bangka Island

“…Upaya mendapatkan materi genetik dapat dilakukan melalui pencarian atau eksplorasi serta evaluasi tanaman kakao yang telah dibudidayakan masyarakat. Pengayaan materi genetik dapat dilakukan melalui kegiatan eksplorasi dan identifikasi baik karakter fenotipik maupun genotipik terhadap genetik lokal yang ditanam petani atau pekebun (koleksi in situ) (Santos et.al 2012;Santos et al 2014;Susilo 2015) terutama di daerah-daerah produksi kakao. Penelitian bertujuan untuk inventarisasi materi genetik aksesi kakao yang dibudidayakan rakyat Bangka.…”

A Combined RNA Preservation and Extraction Protocol for Gene Expression Studies in Cacao Beans

“…RNA integrity levels were relatively low for FD samples kept at RT. This was unexpected since the decreased water content should inactivate proteolytic enzymes and nucleases, thereby reducing cellular component degradation (Santos et al, 2014;Garcıábaldenegro et al, 2015). We assume that this could originate from suboptimal drying and inadequate closing of the sample vials, resulting in the uptake of water from the humid air during transport (Garcıá-baldenegro et al, 2015).…”