The Preliminary Study On Wood Identification Methods Based On DNA Sequences

The difference among species is resulted from DNA sequence fundamentally. After logging and processing, the DNA remained in wood tissues could provide a new approach for wood identification. But DNA in wood cells depredates gradually during PCD, storage and processing, which makes DNA extraction and molecular identification very difficult. In order(i) to understand the degradation degree of xylem nuclei and plastids under different storage and processing condition, distribution and content, nuclei stained with aceto-carmine and DAPI and plastids stained with I2-KI and DAPI were analyzed.(ii) to improve PTB methods for DNA extraction and(iii) to establish SNPs and phylogenetic tree based on rbc L, mat K, trnH-psb A intergenic spacer region, trnL-F intergenic spacer region, ITS1 were analyzed. The suitable DNA barcoding and phylogenetic tree methods would be chosen.(iv) all sequences in this paper were tested using DNA Barcode of Rare and Endangered Plant(BREP) for identification.The content and distribution of DNANuclei and plastids are cellular organs which contain DNA. Compared with the control sample, nuclei stained with aceto-carmine presented red, stained with DAPI show blue. Amyloplasts stained with I2-KI presented dark blue, stained with crystal violet solution presented violet, and stained with DAPI did not present fluorescence.Most of nuclei and plaids presented in ray cells and axial parenchyma cells. The number of nuclei and plastids of sapwood was more than that in the heartwood. Meanwhile, nuclei and plaids content of fresh wood are greater than that of processed and stored wood. Estimation of nuclei quantity with staining methods could provide a direct basis for appropriate protocol selection of nuclei extraction.It was indicated that the concentration of remained DNA is mainly affected by PCD, heartwood formation, storage time and drying temperature. The nuclei content of sapwood is higher than that of heartwood.DNA extraction methodsThe DNA content of the fresh wood was greater than that of the processed and stored wood. The DNA content of the sap wood was greater than that of the heart wood. the variation in the DNA content from heartwood that were treated by dried at 80℃ and dried at 120℃ were insignificantly different. However, in sapwood they were a little bit significantly. The ratio of PCR amplification successful of cp DNA was more than the nuclear DNA region. Fresh wood could be amplified successfully more than dried wood, sapwood could be amplified successfully more than heartwood.DNA extraction for xylem should be optimized by(i) samples should be ground into powder adequately in low temperature.(ii) the volume radio of reagent and samples should be adjusted.(iii) DNA concentration should be increased using normal butanol to make it enough to PCR or sequencing. PTB made extraction more effectively than kit, both methods did not change bases of sequence.Wood identification using DNA barcodingAs statistics of SNPs for rbcL、mat K、trnL-F、trnH-psb A and ITS1, polymorphic percentage revealed intraspecific variation, trnL-F> ITS1>rbcL> mat K> trnH-psbA. Based on results from phylogenetic analysis, mat K, trnH-psbA and ITS1 sequences had better differential degree for wood identification between A. sinensis and other species, lower values were shown for rbcL and trn L-F.The validity of the sequences obtained from this paper were verified through BREP, so, they can be utilized in wood identification practically.Identification results influenced with MP method, ML and NJ were limited. Therefore, special barcoding with smaller intraspecific variation relatively is the most important part in wood identification.