Full Length Research Paper

Field collection, preservation and large scale DNA extraction procedures for cassava (Manihot esculenta Crantz.)

Ranjana Bhattacharjee¹*, Morag Ferguson², Melaku Gedil¹, Dominique Dumet¹ and Ivan Ingelbrecht¹

¹International Institute of Tropical Agriculture, Ibadan, P. M. B. 5320, Nigeria.

²International Institute of Tropical Agriculture, ^c/_o International Livestock Research Institute, P. O. Box 30709, Nairobi 00100, Kenya.

*Corresponding author. E-mail: r.bhattacharjee@cgiar.org. Tel.: +234 2 7517472. Fax: INMARSAT: 873761798636

Abbreviations: CTAB, Cetyltrimethylammonium bromide; EDTA, hexadecyltri-methylammoniumbromide; PVPP, polyvinyl polypyrrolidone; PEG, polyethylene glycol; PCR, polymerase chain reaction; MAS, marker-assisted selection.

Accepted 12 may, 2009

   Abstract

Some genetic studies using molecular methods such as diversity assessment or marker-assisted selection require collection of a large number of samples from fields located in the vicinity or in remote areas, followed by isolation of good quality DNA in a short time span. In the present study, different tissue preservation methods were compared for subsequent DNA extraction using a modified CTAB method in two 96-well plates, following grinding of leaf tissues with a GenoGrinder 2000. We found that preservation of leaf tissues in NaCl-CTAB-azide buffer (as described in Rogstad, 1992) at 4°C is a better storage procedure than preservation at -20°C to obtain good quality DNA. Comparison of DNA extraction with or without use of phenol revealed that the quality of DNA was not drastically affected when non-phenol extraction protocol was used and did not affect PCR amplification. Thus, the recommended DNA extraction procedure allowed us to process 192 samples per day at a cost of $0.80 per sample, with an average yield of 1.8 μg, suitable for both PCR and genotyping.

Key words: Cassava, NaCl-CTAB-azide solution, phenol, genogrinder 2000.