目的 本实验旨在从常用DNA条形码序列中筛选出有效的序列,从而实现沉香属内常见物种的快速鉴别。方法 本实验以8个不同国家5种常见的沉香属物种为材料,对其核基因ITS2,叶绿体基因matK、rbcL和trnH-psbA的序列特征进行评估,并通过构建系统进化树分析不同序列及组合对沉香属物种的鉴别效率。结果 4种序列均具有较优的PCR扩增成功率及测序成功率,其中matK序列变异位点和SNPs最多,trnH-psbA最少,Barcoding gap检验结果表明,鉴定效果最优序列依次是matK>ITS2>rbcL>trnH-psbA,matK片段具有明显的遗传变异间隔区域;通过计算平均节点支持率及同质性检验, matK+ITS2+rbcL组合构建进化树可将多个沉香属物种分别聚为一支,且具有最高的节点支持率;基于Taxon DNA的BBA方法验证,matK序列与其他序列组合的鉴定效果最好。结论 使用DNA条形码技术鉴定沉香属物种,单一片段中鉴别成功率最高的为matK序列,应用matK+ITS2+rbcL的序列组合可以实现沉香属不同物种的准确鉴别。
Abstract
OBJECTIVE To screen out efficient sequences from DNA barcoding sequences in order to rapidly identify multiple species of Aquilaria. METHODS Five species of Aquilaria were collected from eight countries as test materials. The ITS2 sequence in nuclear genome as well as matK, rbcL and trnH-psbA sequences from chloroplast genome were analyzed and evaluated. Phylogenetic trees based on different sequence combinations were built to evaluate the identification efficiency of Aquilaria species. RESULTS All four kinds of sequences exhibited relatively high success rate of PCR amplification and sequencing. matK had the most variation sites and SNPs while trnH-psbA had the fewest. Barcoding gap test demonstrated that the identification effect of sequences from high to low was matK>ITS2>rbcL>trnH-psbA. According to the homogeneity test and average node supporting rate, phylogenetic trees based on the combination of matK+ITS2+rbcL could clearly divide different species into separated clusters with high node supporting rate. The matK showed the best identification effect when combined with other sequences as shown by the BBA method of Taxon DNA. CONCLUSION When using DNA barcoding technique to identify Aquilaria species, matK has the highest identification rate among single barcoding candidate sequences. And the combination of matK+ITS2+rbcL can precisely distinguish multiple Aquilaria species.
关键词
DNA条形码 /
沉香属 /
进化树 /
物种鉴别
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Key words
DNA barcoding /
Aquilaria /
phylogenetic tree /
species identification
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中图分类号:
R284
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基金
国家重点研发计划项目资助(2018YFC1706400);中组部“万人计划”项目资助(99950534);国家自然科学基金项目资助(81703660);国家中药材产业技术体系资助(CARS-21);海南省自然科学基金项目资助(319QN343);中国医学科学院医学与健康科技创新工程资助(2016-I2M-2-003)
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