| It is wood that can not be replaced during the living and development of humanbeings. Wood protection and proper utilization are common concerned issues throughoutthe living, producing. With the development of the world and increasing living standard,people prefer the applications of the woody material, especially the high level of woodyproducts ranking among luxuries which become the symbol of high living quality peoplepursuing. However, the huge gap existing between rare&precious wood species and thenormal wood species, which result in disturbing markets of rosewood, archaize wooden,classical furniture, and precious musical instruments by using ordinary wood which cannot be distinguished from the real rare wood species during to the similar morphologicalcharacters, at last, the huge loss and damage would be produced to consumers on both theeconomic and mental sides. So the scientific rapid and accurate method identifying woodspecies is imperative and urgent in need during wood processing, utilization and trading.DNA analysis is an effective method comparing with traditional means mainly based onanatomical characteristics.The wood of Dalbergia odorifera T. Chen and Dalbergia rimosa Roxb both originatedfrom different sites of plantations were sampled as materials in this dissertation. Threemethods, i.e., modified CTAB, PTB (N-phenacylthiazolium bromide), and Plant Mini Kit(Qiagen) were employed to extract DNA from the two woods originated from differentsites, in different parts of the tree or with different drying treatments. The obtained DNAextracts were purified, and the5.8S rDNA sequences were amplified then sequenced. Theeffects of different drying treatments on DNA extraction and PCR for target sequenceswere analyzed. The divergences of Dalbergia odorifera T. Chen originated from differentsites and the divergence between Dalbergia odorifera T. Chen and Dalbergia rimosa Roxbwere revealed based on the sequences, which provide some evidences for identifying woodof Dalbergia odorifera T. Chen from the adulterants. The results are as following:1. The concentration ranges of DNA extracts from heartwood and sapwood were:75.95-937.38ng/μL，4.46-806.56ng/μL respectively using three methods (modified CTABmethod, PTB and kit method). The PTB method preformed best in the three methodsshowing the highest level of DNAconcentration both in heartwood and sapwood, while thelowest DNA concentration indicated Kit preformed worst. However, DNA extracts fromheartwood in the three methods were subjected to PCR for target sequences after rawextracts purified. DNA from wood tissue can be extracted successfully using the three methods, while PTB would be more suitable for heartwood.2. The effects of different heat-treatments on DNA from heartwood and sapwood fromDalbergia odorifera T. Chen and PCR for ITS sequences revealed that the genome fromheartwood and sapwood appeared diffusion in different extents and the fragments were allsmaller than15000bp after the heat-treatment of25℃and65℃, meanwhile, DNA weredegraded to smaller than250bp after heat-treatment in105℃during gel electrophoresistests. As for heartwood, only did PCR for ITS successful.Using DNA extract as template with heat-treatment of25℃, residue treated with65℃or105℃were all failed.This indicates that genome DNA degraded increasingly with thetemperature of heat-treatments rising leads to the DNA fragmented much smaller whenusing them as templates could not satisfied PCR. The effects would be more significantwhen the temperature of heat-treatment rising.3. The specific primer pair (JT1-JT2) were designed according to the database of18S,26S Dalbergia in GenBank, the primer pair can totally avoid interference of entophyteseffectively and eventually lead to successful PCR. The PCR system and parameters weredetermined after modified and optimized experiments. The key parameters andrequirements:1.5μM primer;2.0mM Mg2+, annealing temperature56.4℃.4. The ITS sequences of Dalbergia odorifera T. Chen from different sites andDalbergia rimosa Roxb were compared. The results showed that the length of ITSsequences(including5.8S) were603bp, and the length of5.8S of Dalbergia odorifera T.Chen and Dalbergia rimosa Roxb were164bp, what is more, no variation were observedrepresented much conserved coding5.8S section. The divergences of ITS amongDalbergia odorifera T. Chen originated form different sites were low. Variations in ITS2section were more than in ITS1section between Dalbergia odorifera T. Chen andDalbergia rimosa Roxb.6variations sites existing during ITS sequences betweenDalbergia odorifera T. Chen and Dalbergia rimosa Roxb, and the6variations sites wouldprovide specific sites for the identification of the two wood species. The phylogeny tree ofITS sequences shows that the steady divergences exist. The divergences reflect in variedbases, which also indicate the related genetic relationship of the two species while they donot belong to a plant group. |
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