知母特异性DNA条形码筛选、种质资源鉴定与遗传多样性分析  

DNA barcode screening, identification of germplasm resources, and analysis of genetic diversity of Anemarrhena asphodeloides

作　　者：窦西铭 张靖晗 王馨 刘珊瑚 石玥[3] 黄钰莹 张晓芹[1] 何高洁 安克露 戚源 王晓晖 魏胜利[1] 张媛[1] DOU Xi-ming;ZHANG Jing-han;WANG Xin;LIU Shan-hu;SHI Yue;HUANG Yu-ying;ZHANG Xiao-qin;HE Gao-jie;AN Ke-lu;QI Yuan;WANG Xiao-hui;WEI Sheng-li;ZHANG Yuan(School of Chinese Materia Medica,Bejing University of Chinese Medicine,Bejing 102488,China;Institute of Chinese Pharmacy,Bejing University of Chinese Medicine,Bejing 102488,China;School of Life Sciences,Beijing University of Chinese Medicine,Beijing 102488,China;Hebei Xutong Seed Industry Group Co.,Ltd.,Shijiazhuang 050501,China)

机构地区：[1]北京中医药大学中药学院,北京102488 [2]北京中医药大学北京中医药研究院,北京102488 [3]北京中医药大学生命科学院,北京102488 [4]河北序彤种业集团有限公司,河北石家庄050501

出　　处：《中国中药杂志》2024年第1期88-99,共12页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2022YFC3501505);北京中医药大学横向课题(2020110031007533)。

摘　　要：知母Anemarrhena asphodeloides是临床方剂和中成药中常用药材。该研究利用Illumina平台测序获得7份不同产地的知母叶绿体基因组序列,通过比较基因组学分析筛选特异性DNA条形码,并利用DNA条形码对全国不同产地知母样品进行种质资源鉴定与遗传多样性分析。7份知母叶绿体基因组均具有环状结构,全长为156801~156930 bp,均注释113个基因,其中蛋白编码基因79个、tRNA基因30个、rRNA基因4个;比较基因组学分析显示rps16、trnG-GCC、atpF、rpoB、ycf3、rpl16、ndhF、trnS-GCU_trnG-GCC、petN-psbM、ndhF-rpl32可作为潜在的知母特异性DNA条形码。该研究选取序列长度在700~800 bp,且易于扩增的ycf3(第2内含子区)和atpF(内含子区)序列对26个产地594份知母样品进行PCR扩增和测序,序列分析结果显示ycf3和atpF序列分别能鉴定6、8个单倍型,2个序列联合分析能鉴别17个单倍型,命名为Hap1~Hap17。26个知母居群单倍型多样性(Hd)为0.68;核苷酸多样性(Pi)为0.93×10^(-3);遗传距离为0~0.0031,表明知母种内遗传多样性较丰富。中介邻接网络分析显示Hap5为最古老单倍型,主要分布在河北保定易县、山西忻州河曲县和山西临汾襄汾县。该研究对知母品种的鉴定、种质资源保护和开发及产地鉴别具有重要指导意义,为进一步揭示知母的遗传进化规律提供了研究基础。Anemarrhena asphodeloides is a common medicinal material used in clinical prescriptions and Chinese patent medicine. In this study, the Illumina platform was used to obtain the chloroplast genome sequences of seven kinds of A. asphodeloides from different areas. The specific DNA barcodes were screened by comparative genomics analysis, and the DNA barcodes were used to identify the germplasm resources and analyze the genetic diversity of A. asphodeloides samples from different areas in China. All the seven chloroplast genomes had a ring structure. The total length was 156 801-156 930 bp, and 113 genes were annotated, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. The comparative genomics analysis showed that rps16, trnG-GCC, atpF, rpoB, ycf3, rpl16, ndhF, trnS-GCU_trnG-GCC, petN-psbM, and ndhF-rpl32 were potential candidates for specific DNA barcodes of A. asphodeloides. In this study, the second intron of ycf3 and atpF intron sequences with a sequence length of 700-800 bp and easy amplification were selected for polymerase chain reaction(PCR) amplification and sequencing of 594 samples from 26 areas. The sequence analysis showed that six and eight haplotypes of ycf3 and atpF sequences could be identified, respectively, and 17 haplotypes could be identified by combined analysis of the two sequences, which were named Hap1-Hap17. The haplotype diversity(H_d), nucleotide diversity(P_i), and genetic distance of A. asphodeloides in 26 populations were 0.68, 0.93×10^(-3), and 0-0.003 1, respectively, indicating that the genetic diversity within the species of A. asphodeloides is rich. The intermediary adjacent network analysis showed that Hap5 was the oldest haplotype, which was mainly distributed in Yixian county of Baoding, Hebei province, Hequ county of Xinzhou, Shanxi province, and Xiangfen county of Linfen, Shanxi province. This study has important guiding significance for the identification of A. asphodeloides species, the protection and development of germplasm resources, and the identifi

关 键 词：知母 叶绿体基因组 种质资源 单倍型 遗传多样性 

分 类 号：S567.239[农业科学—中草药栽培]