苦水玫瑰ITS及ITS2序列分析与鉴别

Analysis and Identification of ITS and ITS2 Sequences in Rosa rugose‘Kushui'

目的:对苦水玫瑰、平阴玫瑰、月季、金边玫瑰、法兰西玫瑰进行DNA条形码序列比对,对苦水玫瑰予以鉴别。方法:对样本的细胞核基因内转录间隔区(ITS)和ITS2片段进行聚合酶链式反应(PCR)扩增并双向测序,所得序列经CodonCodeAligner 3.7.1软件拼接后,用MEGA 5.0软件进行序列的分析比对,采用邻接法(NJ)构建系统聚类树。结果:ITS引物扩增产物中大部分样品的测序结果双峰严重,无法有效拼接,可用数据量低,含不确定性碱基比例高,因此未对该部分数据进行分析;平阴玫瑰与对照药材均可获得高质量的ITS2序列,且序列完全一致,苦水玫瑰的ITS2序列(序列6和8)与平阴玫瑰及玫瑰对照药材相比存在1~2个变异位点,月季、法兰西玫瑰与平阴玫瑰及玫瑰对照药材相比存在≥2个变异位点。结论:利用ITS2序列能够有效区分苦水玫瑰及其他同属物种样品。

Objective:To compare the DNA barcodes of Rosa rugosa‘Kushui',R.rugosa Thunb.,R.chinensis Jacq.,R.Jinbian,and R.gallica L.for the identification of R.rugosa‘Kushui'.Methods:The internal transcribed spacer(ITS)and ITS2 regions of the nuclear genes of the samples were amplified by polymerase chain reaction(PCR)and PCR products were sequenced in both directions.The resulting sequences were assembled by Codon Code Aligner 3.7.1 and then aligned with MEGA 5.0.Afterwards,a phylogenetic tree was established with the Neighbor-Joining(NJ)algorithm.Results:The sequence peak maps of the products of most samples amplified with ITS primers showed severe double peaks at different loci and sequences failed to be effectively assembled.In consideration of the loss of effective data and large proportion of uncertain bases,the data were not analyzed.High-quality ITS2 sequences were obtained from R.rugosa and the control herb,and their sequences were completely consistent.The ITS2 sequences(sequence 6 and 8)of R.rugosa‘Kushui'had 1-2 mutation sites and R.chinensis Jacq.and R.gallica L.had≥mutation sites compared with R.rugosa and the rose control.Conclusion:ITS2 sequence can be used to effectively distinguish R.rugosa‘Kushui'from other varieties of R.rugosa and congerners.