Rapid, simple and potentially universal method for DNA extraction from Opuntia spp. fresh cladode tissues suitable for PCR amplification
In Opuntia spp., the cladode tissues contain many polysaccharides and secondary metabolites that interfere with obtaining high-quality deoxyribonucleic acid (DNA), using currently available methods. To circumvent this problem, three commercial kits, three modified versions of the conventional cetyltrimethylammonium bromide method (CTAB) method and one combined method were tested in Opuntia ficus-indica, O. robusta, O. dillenii and O. elata species. We obtained a rapid and simple protocol that allows the extraction of DNA from all the tested species with good DNA yield and purity, namely, the combined method. With this method (DNeasy® Plant Mini Kit combined with the CTAB method), DNA yields from 13.2 ± 7.8 to 15.9 ± 11.3 µg g−1 of fresh tissue were obtained in the four Opuntia species. The purity, evaluated by the ratio A260/A280 ratio, ranged from 1.67 ± 0.12 to 2.01 ± 0.25, revealing low levels of problematic metabolites. The extracted DNA quality was confirmed by amplifying a set of nuclear microsatellites obtained for the genus. Reliable reproducible bands and electropherogram profiles were obtained. The combined method has potential to be universal for good-quality DNA extraction in cacti, particularly in the Opuntia genus and other difficult-to-extract species.
KeywordsCactus pear CTAB DNA extraction Polysaccharides Viscosity
We acknowledge the Fundação para a Ciência e Tecnologia (Portugal) with grant UID/AGR/00239/2013, which funded MMR and grant UID/AMB/00681/2013, which funded MMR and CMGR, respectively. Our thanks are extended to Estelle Lerceteau-Köhler and Maria João Magalhães Gaspar for reviewing an earlier version of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Cladode sampling of Opuntia samples was non-destructive. No specific permission was required to sample Opuntia species in Portugal. Field studies did not involve endangered or protected species.
Human and animal rights
No humans or animals were involved in the experiments.
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