Wood Identification For Phoebe Zhennan、phoebe Hui、phoebe Sheareri By DNA Molecular Markers

It’s difficult to distinguish Phoebe zhennan、Phoebe hui、Phoebe sheareri by the traditional morphological identification method.Therefore,This study used molecular marker technology to identify them.In this research,in order to obtain high quality genomic DNA and construct the DNA extraction system of Phoebe,the extraction method was improved by increasing the volume of DNA lysate,prolonging the time of water bath,increasing the number of extraction times and the times of elution of DNA,and then,comparing the quality and concentration of DNA obtained by the CTAB method,CTAB-SDS method,modified QIAGEN kit method,modified TAKARA kit method and modified MAGEN kit method to select a better method for extracting DNA from wood.By using RAD-seq technique,the high quality DNA was used to simplify genome sequencing and the SNPs were developed.At the same time,the cluster analysis and the specific SNPs of Phoebe zhennan、Phoebe hui、Phoebe sheareri were set up.And specific primers were selected to identify Phoebe zhennan、Phoebe hui、Phoebe sheareri.The main results of this study are as follows::（1）The difference of the quality of DNA obtained by the five DNA extraction improved methods was obvious.The worst effect was the CTAB method and no obvious strip;while the CTAB-SDS method had a strip,but it did not meet the requirement of PCR.In the other three improved kit methods,The quality of DNA obtained from the improved MAGEN kit was best,but the improved TAKARA kit was the worst.And the DNA A260/280 from the improved MAGEN kit method was range from1.72 to1.85 with the average of 1.78.and the concentration range was range from 63.7 to 89.9 ng·μL^-1with the average of 71.7 ng·μL^-1,which was in line with the requirement of PCR and RAD-seq.（2）The 29 samples were sequenced by Illumina sequencing platform with the endonuclease（Eco RI）,and the sequence data were counted,cut and filtered.The number of reads and bases was rich,the base error rate was very low,and 31777 high quality SNPs were obtained.（3）The results of cluster analysis showed that the three tree species of Phoebe zhennan、Phoebe hui、Phoebe sheareri were able to form independent branches,which were consistent with the traditional anatomical classification results,indicating that this cluster analysis can accurately distinguish the three species,and the relationship of Phoebe zhennan and Phoebe hui is closer.PCA analysis also showed the same result as cluster analysis.All three species could be clustered together independently,but the Phoebe zhennan close to the Phoebe hui.The Dujiangyan Phoebe zhennan and Ya’an Phoebe zhennan were divided and clustered together respectively.（4）The candidate sequences of Phoebe zhennan,Phoebe hui and Phoebe sheareri were compared with blast,and the primers with the highest overall quality were designed for subsequent validation.The specific sequence number of Phoebe zhennan,Phoebe hui and Phoebe sheareri were 4,21 and 44respectively.The temperature range of Tm specific primers Phoebe zhennan was 50℃to 62℃.When the intermediate temperature was range from 57.7 to 54.6,the amplification effect is good,and the target strip has high specificity and good abundance,so the annealing temperature is 55℃.（5）The poor specificity of the primers of Phoebe zhennan and Phoebe sheareri could not distinguish the three species.But the specific primer XYNP02（（F:5’-TGAATGCCCCTAACTC AGCT-3’;R:5’-TTCTCACTCCCACTCCCACT-3’））designed by the heterosexual fragment of Phoebe hui could distinguish Phoebe sheareri from other two species,but it could not distinguish Phoebe zhennan and Phoebe hui.