A Sample Preparation Method for Direct and Non-direct PCR in Woody Plants

Abstract

Despite recent advances in DNA analysis techniques, the process of acquiring purified DNA is particularly costly and time-consuming in woody plants. The ability to obtain purified DNA is frequently hindered by the presence of secondary compounds, including polyphenols and polysaccharides. In this study, we developed a sample preparation method for direct and non-direct PCR that is highly reproducible and cost-effective. This method entails pricking a leaf sample with a toothpick and dissolving the collected DNA in a PCR mixture or TE0.2 buffer solution. We confirmed the utility of the method using seedlings derived from backcrossed hybrids of Citrus hassaku hort. ex Y. Tanaka × Poncirus trifoliata (Linn.) Raf. Using a direct PCR method, we obtained 99% amplification success rate for the 1200 bp CTg12A region. PCR amplification from DNA extracts, which was also produced by our method, succeeded in 96–100% of samples for five different DNA markers, except for CTg12A, where it was only 86%. We were able to process and commence PCR amplification of 95 leaf samples in 1.5–2.0 h, in contrast to 23 h using conventional methods. We also demonstrated the broader applicability of this method by extending our study to 18 other fruit trees and ornamental woody plants. PCR products were generated for all 18 tested species using chloroplast accD and rbcL markers, and for Japanese chestnut, mango, and Japanese pear using their species-specific SSR markers. Our new method might be suitable for analyses which handle large number of samples, such as marker-assisted selection and cultivar discrimination, and holds promise for use in DNA marker typing in various kinds of woody plants.