钩藤属植物分子鉴定的DNA条形码筛选

Screening of DNA barcoding sequences for molecular identification of Uncaria genus

目的应用分子生物学鉴定技术,筛选出应用于鉴定钩藤属物种的最佳DNA条形码,建立快速、准确、便捷的钩藤属植物鉴定方法。方法从广东、广西等地收集了8个钩藤属物种的叶片、茎枝作为材料,共计44份样品。提取样品总DNA,分别对条形码ITS、matK、psbA-trnH、rbcL、trnL-trnF序列进行扩增、测序、拼接、校对;利用MEGA 7.0分析比对序列特征;基于Taxon DNA计算种内、种间的遗传距离以分析barcoding gap及Best Match、Best Close Match评估DNA条形码的鉴别能力;使用MEGA7.0、Phylosuite等软件构建单条形码及组合条形码的最大似然法(maximum likelihood,ML)、最大简约法(maximum parsimony,MP)、贝叶斯推断法(bayesian inference,BI)系统发育树。结果条形码ITS、matK、psbA-trnH、rbcL、trnL-trn F序列均扩增成功且具有较高的测序成功率,其中psb A-trn H具有最多的变异位点,ITS次之,rbc L最少;Best Match、Best Close Match与Barcoding gap分析结果显示:5个单一条形码中,ITS鉴定钩藤属物种效果突出且具有明显的Barcoding gap,而组合条形码ITS+rbcL序列表现更为优异。基于所有单一条形码构建的系统发育树,ITS序列的物种鉴别成功率最高,能够准确鉴定8个钩藤属物种;基于组合条形码构建的系统发育树,ITS+rbcL序列具有最高的平均节点支持率,且该序列集包含的11个钩藤属物种均能单独聚为一支。结论应用ITS+rbc L的序列组合能够实现钩藤属不同物种的准确鉴定。

Objective In order to establish a rapid,accurate,and convenient approach for identifying species in the Uncaria genus,DNA barcoding analysis and molecular biology techniques were performed to screen the best DNA barcode sequences for Uncaria species authentication.Methods Genomic DNA was extracted from a total of 44 leaf and stem specimens collected from different regions throughout China.The barcodes for ITS,matK,psbA-trnH,rbcl,and trnL-trnF were amplified,sequenced,assembled,and refined.The sequences were aligned and analyzed using MEGA 7.0.Intraspecific and interspecific genetic distances were calculated to analyze barcoding gaps using TaxonDNA software.The ability of the DNA barcodes to identify species was evaluated using the “Best Match” and “Best Close Match” functions in TaxonDNA.MEGA 7.0 and Phylosuite were used to design phylogenetic trees from single and combinations of barcodes based on Maximum Parsimony(MP),Maximum Likelihood(ML),and Bayesian Inference(BI) methods.Results The ITS,matK,psbA-trnH,rbcL,and trnL-trnF sequences were successfully amplified and had a high sequencing success rate.The psbA-trnH sequence had the highest number of variation sites,followed by ITS and rbcL,which had the lowest number of variation sites.Best Match,Best Close Match,and Barcoding gap analysis showed that ITS had the most prominent species discriminatory effect among the five single barcodes,while the combination of ITS + rbcL with even better discriminatory performance than any single barcodes.Based on the phylogenetic tree constructed using all of the single barcodes,ITS accurately identified eight Uncaria species and had the highest identification success rate when compared to the other barcodes.The combination of ITS + rbcL had the highest average bootstrap support rate based on the phylogenetic tree.All the 11 Uncaria species clustered separately into monophyletic clades.Conclusion The combination barcodes of ITS + rbcL achieve the accurate identification of Uncaria species when the DNA barcoding technology is applied to identify Uncaria species.