DNA条形码技术鉴定贝母属植物

Identification of plants in Fritillariae L.by DNA barcoding technology

目的采用DNA条形码技术,比较了条形码序列ITS1和ITS2在贝母属植物中的鉴定效果。方法从8个贝母属21个样品中提取了基因组DNA,进行ITS1、ITS2序列的PCR扩增和测序,并比较了各样品DNA提取率、PCR扩增率及测序成功率。测序数据采用BLAST搜索法评价2种序列的鉴定能力,采用SPSS软件进行遗传距离分析,采用MEGA软件进行系统发育树的构建。结果对比不同物种ITS1、ITS2序列的一级结构差异,发现其ITS1序列长度为205-229 bp,有信息位点27个（所占比例11.8%）,鉴定成功率为72.2%;ITS2序列长度为236-244 bp,有信息位点20个（所占比例8.2%）,鉴定成功率为100%。ITS1序列的种内及种间遗传距离分别为0.001 8和0.066 1,而ITS2序列的种内及种间遗传距离分别为0.000 5和0.046 8,且种内高度保守;系统发育树构建结果显示,2种ITS序列能准确区分出浙贝母类群、川贝母类群和北方贝母类群,而ITS2序列所构建的系统发育树准确度更高。结论 ITS2序列能够更准确鉴定不同物种贝母,适合作为贝母属植物的通用条形码序列。

Objective DNA barcoding technology was employed to compare the identification efficiency of ITS1and ITS2 sequences in plants of Fritillariae L.Methods Twenty-one samples from eight species of Fritillariae L.were collected.Genomic DNAs from these samples were extracted,ITS1 and ITS2 sequences were amplified by PCR,and the PCR products were sequenced.The DNA extraction rate,PCR amplification rate,and sequencing achievement ration were compared.The identification efficiency of two sequences was evaluated by BLAST-Search,the distribution of genetic distance was analyzed by SPSS software,and the phylogenetic trees were constructed by MEGA software.Results Comparing the primary structures of ITS1 and ITS2 sequences amplified from different species,the results showed that the sequence length of IT1 was 205—229,informative sites were 27 and occupied 11.8%,and the identification achievement rate was 72.2%; The sequence length of IT2 was 236—244,informative sites were 20 and occupied 8.2%,and the identification achievement rate was 100%.Intra-specific and inter-specific genetic distances of ITS1 were 0.001 8 and 0.066 1,and those of ITS2 were 0.000 5 and 0.046 8,respectively; The results also showed that ITS2 was relatively conservative in intra-species; The NJ phylogenetic trees showed that these two sequences could accurately identify Fritillaria thunbergii,F.cirrhosa,and north-Fritillaria,but phylogenetic trees based on ITS2 had a higher degree of accuracy.Conclusion ITS2 sequence is optimal as barcoding sequence for Fritillaria L.genus and is more suitable for identifying the different species of Fritillaria L.