Optimization of DNA extraction and amplified fragment length polymorphism (AFLP) analysis of pomegranate (Punica granatum L.)

Abstract

Pomegranate (Punica granatum L.) is a plant rich in polysaccharides, polyphenols and secondary metabolites, which makes it difficult to obtain high quality DNA. The present study reports a quick, simple and inexpensive method to isolate genomic DNA suitable for amplified fragment length polymorphism (AFLP) analysis and other PCR-based applications. This method is a modification of a protocol described by Doyle and Doyle (1990). It is a cetyl trimethyl ammonium bromide (CTAB)-based protocol modified by the use of potassium acetate (KoAc) and polyvinylpyrrolidone (PVP) to remove polyphenols and polysaccharides and a high concentration of β-mercaptoethanol to reduce oxidation. Moreover, the final optimized protocol was then compared with three different methods, which are routinely used for many plant species. The results show that our modified CTAB protocol produced a high yield (>500 ng/μl) of good-quality DNA (A 260 /A 280 >1.8) compared to the other three methods. The DNA purity was further confirmed by complete digestion with EcoRI and MseI enzymes. The modified CTAB protocol used in this study could be a useful protocol for extraction of high quality DNA not only for pomegranate but also for other plants rich in polysaccharides, polyphenolices and secondary metabolites. Using this method, DNA was extracted from 67 accessions of pomegranate. The DNA was then used for AFLP analysis. To optimize the AFLP protocol, the effects of MgCl 2 concentration during selective amplification, the dilution level of pre-amplified DNA and the cycle number used in the preamplification were studied. After optimization of the reaction conditions, AFLP was used to study genetic diversity among Iranian pomegranate accessions.