DNA Extraction from Heartwood and Quick Species Authentication Using Real-Time PCR: A Case Study of the Rosewood (<i>Pterocarpus Indicus</i>)

Abstract

Illegal logging, felling and timber trade have continued to increase over the past two decades, leading to a decline in forest biodiversity and the extinction of some wood species. <i>Pterocarpus indicus</i> Willd., listed in the National Standards of the People’s Republic of China for Hongmu (GB/T 18107-2017), is widely used in production of high-end furniture, decorative flooring and musical instruments due to its high-quality timber. For molecular species identification, the quality and quantity of DNA extracted from wood samples should first be ensured. However, extracting DNA from dried, aged timber heartwood is difficult, as heartwood contains little fragmented DNA, along with lots of phenolic compounds known to impede sequence amplification. In order to protect <i>P. indicus</i> from over-exploitation and to achieve accurate species-level identification, we established a particular extraction method for obtaining amplifiable DNA from heartwood samples and the real-time PCR assay for species discrimination of <i>P. indicus</i> in this study. The quantity and quality of DNA extracted from dry heartwood samples using the modified CTAB method were 2.40-37.70 ng/μL and 1.55-2.12 demonstrated by OD_260/280, respectively. Primer set P9, targeting <i>P. indicus</i> specific microsatellite Pin2-20 sequence, was amplifiable in newly established real-time PCR. Through analysis, this real time PCR was shown to be specific and sensitive with a detection limit around 0.17 ng/μL. Hopefully, this study will contribute to heartwood DNA extraction and species identification of timber logs for forensic discrimination, law enforcement and natural resource conservation.