Selection And Identification Of DNA Barcodes For Wood Of Dalbergia Odorifera T.Chen And Dalbergia Tonkinensis Prain

The traditional method of wood identification does not only rely on microscopic characters distinctly, but also require the identifier to possess rich professional knowledge and experience. Because macroscopic and microscopic characters of wood are quite similar among species when they belong to one family or are close with each other genetically, it is very difficult to identify them accurately at a species level using the traditional methods. Therefore, it is urgent and important to found a new scientific and accurate method for identifying wood for wood processing, utilization and trading. DNA barcoding is an effective method comparing with traditional means mainly based on anatomical characteristics. The wood of Dalbergia odorifera T. Chen and Dalbergia tonkinensis Prain both originated from different sites of plantations were sampled as materials in this dissertation. Genomic DNA was extracted from the sapwood and heartwood of D. odorifera with different heat treatments, The effects of different heat treatments on DNA extraction and PCR for target sequences were analyzed. The intraspecific and interspecific variability of DNA barcoding sequences were analyzed from wood of D. odorifera and D. tonkinensis from different sites. The suitable DNA Barcodes were evaluated and selected for wood of Dalbergia odorifera T. Chen and Dalbergia tonkinensis Prain. This research would provide evidences for identifying wood of Dalbergia odorifera T. Chen from Dalbergia tonkinensis Prain. The main results are as follows:1. The wood structure characteristics of Dalbergia odorifera T. Chen and Dalbergia tonkinensis Prain were compared. The result showed that the macroscopically structure characteristics was not significantly different between D. odorifera and D. tonkinensis. The differences were mainly the color depth and the strength of the odors between them. The differences of microscopic structure were mainly size of vessels and the number of heterogeneous rays of type III. Therefore, the wood of D. odorifera and D. tonkinensis cannot be differentiated based on wood anatomy.2. Genomic DNA was extracted from the sapwood and heartwood of D. odorifera with different heat treatments using PTB merhod and PTB-Kit combination method, respectively. The effects of different heat treatments on DNA extraction and PCR for target sequences were analyzed. The effects of DNA extraction were evaluated with different heat treatments using PTB merhod and PTB-Kit combination method, respectively. The results indicates that the effects would be more significant when the temperature of heat treatment rising. The temperature of heat treatment above 65℃ has a more great effect for DNA extracts from wood on the follow-up study. The efficacy of PCR of sapwood was higher than that of heartwood. The efficacy of DNA extraction PTB-Kit combination method was higher than PTB merhod from the sapwood and heartwood with different heat treatments.3. Genomic DNA was extracted from wood of D. odorifera and D. tonkinensis from different sites. Six potential DNA barcode sequences, namely ITS, ITS2, rpoC1, rbcL, trnH-psbA and trnL-trnF, were amplified from the genomic DNA by PCR and sequenced. The results revealed that the genome from heartwood apperaed diffusion and the fragment was smaller than 2000 bp, moreover, the quality of DNA extracts from sapwood and twigs was higher heartwood, furthermore, the DNA extracts from heartwood was invisible in agarose gels after electophoresis. Therefore, PCR amplification was very important for the efficiency of DNA extraction testing. The PCR successrate of heartwood was lower than that of sapwood and twigs. The PCR success rate for long fragments was lower than short fragments, and it is more diffcult to amplify DNA from the chloroplast DNA than the chloroplast intergentic spacer.4. The intraspecific and interspecific variability of DNA barcoding sequences were analyzed from wood of D. odorifera and D. tonkinensis from different sites. The results indicates that the interspecific distance for three loci, rpoC1, rbc L and trnH-psbA, was greater than other three loci. The results demonstrated that barcoding gap is observed in the distribution of inter- versus intraspecific variability of the chloroplast trnH-psbA intergenic spacer.5. The six proposed DNA barcodes, ie., the nuclear ribosomal DNA internal transcribed spacer(ITS and ITS2), the chloroplast DNA region(rpoC1 and rbcL) and chloroplast intergenic spacer(trnH-psbA and trnL-trnF), were employed to test the feasibility of wood identification between D. odorifera and D. tonkinensis using different evaluation methods such as tree-based method, sequence character-based method, TAXONDNA, Wilcoxon signed-rank test. The results indicates that the PCR success rate and sequencing success rate of trnH-psbA were higher than other five loci, moreover, the feasibility of wood identification of trnH-psbA were higher than other five loci using different evaluation methods. Therefore, we suggest that the chloroplast trnH-psbA intergenic spacer is a suitable DNA barcode for wood identification between D. odorifera and D. tonkinensis.