Wood Identification Technology Of Endangered Species Based On DNA Barcodes

With sharp increase of the global forest resources trade, timber especially tropical timber wood species has rapidly become a focus of attention for Convention on International Trade in Endangered Species of Wild Fauna and Flora(CITES) in recent years. Hence, identifying wood on the species level accurately is especially essential and crucial in the protection of endangered and precious wood species. However, it is often difficult or even impossible for traditional wood identification method based on anatomical features to identify timbers on a species level alone. To overcome such limitations, the newly developed DNA barcoding technology might provide effective information with high resolution. DNA extraction from fresh plant materials is a matter of routine in molecular biology. Nevertheless, DNA extraction from harvested wood tissues becomes more problematic and complicated, especially when it comes to dried wood treated at high temperature, as well as wood stored for a long term. The aim of the study is to distinguish wood on the species level, by breaking through the technical bottleneck of efficient DNA extraction from dried or long-term stored wood tissues, and screening out the suitable DNA barcodes for identification.In the study, a series of technical parameters of DNA extraction protocol, incubating time, ratio of wood powder to lysis buffer volume, and the chemical composition of lysis buffer, were modified to improve wood DNA extraction efficiency. Moreover, two DNA extraction technologies, modified CTAB method and Qiagen kit protocol, were compared to select and determine the optimized wood DNA isolation protocol. Subsquenly, a series of short DNA fragments were amplified by Polymerase Chain Reaction(PCR). Suitable DNA barcodes for wood species identification were evaluated and determined by sequence alignment with BLAST and phylogenetic trees analyses. Additionally, the factors, drying condition and storage time, were discussed to explore the influence for wood DNA. In brief, it was feasible to identify wood on the species level based on DNA barcoding technology, which could promote the development of wood identification technology, and also provide key technical support for improving the performance capabilities of CITES in our country.The main results and conclusions were summarized as follows:(1) Wood DNA extraction technical system based on modified Qiagen kit protocol was preliminarily established. Several improved measures were carried out. Firstly, wood DNA was fully released by means of extending incubating time and decreasing the ratio of wood powder to lysis buffer volume. Secondly, the quantity and quality of DNA was improved by modifying chemical composition of lysis buffer. Moreover, final concentration of wood DNA was increased by making several copies of DNA extraction buffer flow through the same spin column. Hence, optimized DNA isolation method was ultimately established. Meanwhile, two extraction method, modified CTAB method and modified Qiagen kit protocol, were compared to obtain more optimal DNA isolation technology. The results showed that Qiagen kit protocol was more efficient for wood DNA extraction when comparing the yield and purity of extracted DNA. The study provided foundation for further work of wood identification based on DNA method.(2) Influence of different radial positions of wood tissue for wood DNA isolation was investigated. The study proves that DNA content decreases when going from the sapwood to the heartwood. Additionally, the purity of DNA extracts from the sapwood and transition wood is greater than that from the heartwood.(3) In order to determine the influence of drying condition for wood DNA, Aquilaria sinensis(Lour.) Gilg wood dried at different temperature 80 °C and 120 °C were selected for DNA extraction. Results showed that both chloroplast and nuclear ribosomal DNA fragments, 300 to 500 bp in length, from sapwood and heartwood dried at 80 °C could be amplified successfully. When dried at 120 °C, the chloroplast and nuclear ribosomal DNA fragments from sapwood were successfully amplified, while the fragments from heartwood were failed to amplify. It indicated that wood DNA was degraded seriously at 120 °C drying condition. A high temperature causes genome DNA hydrolyze and oxidize, typically causes random DNA degradation into small fragments.(4) A series of different storage time of Populus euphratica Oliv. wood, the year of 30, 57, 80 and 3600, were chosen to discuss the influence of storage time for wood DNA by PCR amplification. The results indicate that as the extension of storage time, wood DNA will gradually degrade into small fragments due to the hydrolysis reaction, oxidation reaction and microbial decomposition. DNA fragments of 100 to 800 bp in length from wood stored for 30 years could be successfully amplified. As for the 57-year-old wood xylarium specimens, only chloroplast DNA fragments of longer than 200 bp could be successfully amplified. Furthermore, the chloroplast DNA fragment, with only a length of about 100 bp, could be successfully amplified for DNA of xylarium specimen stored for more than 80 years. Neverthless, no PCR products were obtained from DNA extracts of the 3600-year-old wood specimen.(5) The type and size of feasible and suitable DNA barcodes for wood identification was selected and determined. In the study, trnL-trnF and ITS1 could be used as DNA barcodes for distinguishing Aquilaria sinensis wood species. With the aid of phylogenetic analysis, based on barcode rbcL, ITS and several short DNA fragments combination, it was possible to differentiate Populus euphratica wood from other species of the same genus. DNA barcode trnL is more suitable than matK and psbA-trnH for identifying three endangered Dalbergia spp. wood, i.e. Dalbergia retusa Hemsl., Dalbergia tucurensis Donn. Sm. and Dalbergia cochinchinensis Pierre. In addition, taking into consideration of various factors including degradation of wood DNA and the number of informative characters in DNA fragments, it might be recommended that the length of DNA barcodes is between 200 and 500 bp.(6) It is feasible to identify wood species based on DNA barcoding technology. In the study, endangered Aquilaria sinensis wood, long-term stored Populus euphratica wood and three endangered Dalbergia spp. wood were distinguished on the species level based on phylogenetic trees constructed by selected DNA barcodes.