基于DNA条形码技术的知母种子基原鉴定

目的:建立基于中药材DNA条形码技术的知母种子基原鉴定方法,保障知母种子基原的真实性。方法:收集知母的30份基原植物样品和9份生药材样品,获取其参考DNA条形码序列,采用克隆测序方法验证参考序列的准确性。收集51份待检测知母种子样品,获取其DNA条形码序列,基于BLAST法、遗传距离法和邻接(NJ)系统发育树法进行物种鉴定。结果:对于基原植物和生药材样品,由于基因组内存在异质性,核糖体DNA第2内部转录间隔区(ITS2)序列无法稳定获得,但可成功获得psbA-trnH序列。知母psbA-trnH序列分为6个单倍型,有3个变异位点,2处插入/缺失,种内变异最大值0.003 5,种间变异最小值0.1,在NJ系统发育树上聚为独立的支。共获得153条知母种子的psbA-trnH序列,经BLAST法、遗传距离法和NJ系统发育树法鉴定均为百合科植物知母Anemarrhena asphodeloides。结论:psbA-trnH是知母种子鉴定的适合条形码序列,可用于知母种子的真实性鉴定。所收集51份知母种子样品的基原均符合2015年版《中国药典》(一部)相关项下规定,未发现伪品。

Species Identification of Anemarrhenae Rhizoma Seeds by DNA Barcoding Technique

Objective: To establish a DNA barcoding based method for guarantee the species authenticity of Anemarrhenae Rhizoma seeds. Method: Thirty original plant materials and nine raw materials have been collected for obtaining the reference DNA barcode sequences.The clone sequencing technique was employed to confirm the reliability of reference sequences.Fifty-one samples of Anemarrhenae Rhizoma seeds pending to test have been collected,and DNA barcodes of these samples were obtained for taxonomic assignment by BLAST method,genetic distance method and neighbor-joining(NJ) phylogenetic tree method. Result: For the original plants and raw materials,their internal transcribed spacer 2(ITS2) sequences could not be obtained stably as a result of intra-genomic heterogeneity.Meanwhile,their psbA-trnH sequences could be successfully obtained.Their psbA-trnH sequences could be divide into six haplotypes,including three variation sites and two insertions/deletions.The maximum intra-specific distance was 0.003 5,whereas the minimum inter-specific distance was 0.1.Their psbA-trnH sequences were composed of one clade at the NJ dendrogram.There were 153 psbA-trnH sequences have been obtained and all can be assigned to be Anemarrhena asphodeloides with BLAST method,genetic distance method and NJ phylogenetic tree method. Conclusion: The DNA barcoding technology is a reliable method for species identification of Anemarrhenae Rhizoma seeds.All the samples of Anemarrhenae Rhizoma seeds pending to test are in accordance with the origin of that ruled on the 2015 edition of Chinese Pharmacopoeia and no adulterant has been detected.