African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12294

Full Length Research Paper

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

Rogério Mercês Ferreira Santos
  • Rogério Mercês Ferreira Santos
  • Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brazil.
  • Google Scholar
Uilson Vanderlei Lopes
  • Uilson Vanderlei Lopes
  • Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brazil.
  • Google Scholar
Didier Clement
  • Didier Clement
  • Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brazil; CIRAD, UMR AGAP, Avenue Agropolis TA96/03, 34398 Montpellier cedex 5, France.
  • Google Scholar
Jose Luis Pires
  • Jose Luis Pires
  • Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brazil.
  • Google Scholar
Eline Matos Lima
  • Eline Matos Lima
  • Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brazil; Universidade Estadual de Santa Cruz (UESC), 45662-900 Ilhéus-BA, Brazil.
  • Google Scholar
Tamiles Batista Messias
  • Tamiles Batista Messias
  • Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brazil; Universidade Estadual de Santa Cruz (UESC), 45662-900 Ilhéus-BA, Brazil.
  • Google Scholar
Karina Peres Gramacho
  • Karina Peres Gramacho
  • Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brazil.
  • Google Scholar


  •  Accepted: 05 January 2014
  •  Published: 12 February 2014

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.