An improved semiautomated rapid method of extracting genomic DNA for molecular marker analysis in cocoa,<Emphasis Type="Italic">Theobroma cacao</Emphasis> L |
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Authors: | Ranjana Bhattacharjee Maria Kolesnikova-Allen Peter Aikpokpodion Sunday Taiwo Ivan Ingelbrecht |
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Institution: | (1) Central Biotechnology Laboratory, International Institute of Tropical Agriculture, PMB 5034 Ibadan, Nigeria |
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Abstract: | DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the
total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such
as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf
tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory
for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution
(as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf
tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported
to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a
rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol
method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues.
The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR
reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively
high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits. |
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Keywords: | ball bearing cocoa DNA extraction high-throughput SSR |
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