An optimized DNA extraction protocol for wood DNA barcoding of Pterocarpus erinaceus

摘要

The isolation of wood DNA is a crucial step in the process of genetic identification of wood tissues and the current wood DNA extraction method is a limiting factor. For some valuable wood samples sent for forensic identification, the size of allowable sample is limited. Additionally, the identification process is so lengthy that it often cannot meet the needs of law enforcement. This study describes an optimized protocol that minimizes the sample size and duration of DNA extraction without decreasing the yield of DNA. Experiments on sample mass per extraction, sample lysis time and DNA precipitation time were carried out by a series of gradient tests. The amounts of DNA extracted were evaluated by the copy numbers of target DNA barcodes (rbel, and matK) from droplet digital PCR (ddPCR). It is the first to apply ddPCR technology to quantify and evaluate accurately DNA extracted from wood. The results indicated that 300 mg is an optimal sample mass when keeping the volume of DNA lysis buffer constant, which reduces the sample usage by 40%. Five hours is the optimal sample lysis time. Extending the duration of DNA precipitation does not significantly increase the amplicon yield from wood specimens of Pterocarpus erinaceus. The protocol developed in this study shortens the period of DNA extraction from wood tissues by approximately 58%. The amplicon yields obtained using the optimized method in this study indicate good extraction efficiency, and the wood samples sent for certification were identified as Pterocarpus erinaceus using the barcode combination matK+ndhF-rpl32+ITSz. This method will be suitable for the broad applicability of DNA identification and conservation of global wood resources.