药学学报, 2019, 54(12): 2326-2334
引用本文:
郑辉, 邓楷煜, 陈安琪, 付绍兵, 周德, 王威威, 倪典墨, 任瑶瑶, 周嘉裕, 廖海. 基于DNA条形码的川贝母及其近缘种的分子鉴定与亲缘关系研究[J]. 药学学报, 2019, 54(12): 2326-2334.
ZHENG Hui, DENG Kai-yu, CHEN An-qi, FU Shao-bing, ZHOU De, WANG Wei-wei, NI Dian-mo, REN Yao-yao, ZHOU Jia-yu, LIAO Hai. Molecular identification and genetic relationship of Fritillaria cirrhosa and related species based on DNA barcode[J]. Acta Pharmaceutica Sinica, 2019, 54(12): 2326-2334.

基于DNA条形码的川贝母及其近缘种的分子鉴定与亲缘关系研究
郑辉1, 邓楷煜1, 陈安琪1, 付绍兵2, 周德3, 王威威1, 倪典墨1, 任瑶瑶1, 周嘉裕1, 廖海1
1. 西南交通大学生命科学与工程学院, 四川 成都 610031;
2. 青海绿康生物开发有限公司, 青海 西宁 810003;
3. 阿坝州食品药品检验所, 四川 阿坝 624000
摘要:
基于ITS2与psbA-trnH条形码序列,对川贝母及其近缘种开展分子生物学鉴定与亲缘关系研究。本文在采用《中国药典》规定的PCR-RFLP鉴定方法对川贝母及其近缘种的鉴定基础上,扩增所有样品的ITS2及psbA-trnH条形码序列,对其遗传距离进行分析,构建进化树,评价ITS2及psbA-trnH条形码的鉴定效果,探讨样品的亲缘关系。结果显示,PCR-RFLP方法仅能将样品分为非酶切组(包括平贝母、浙贝母及伊犁贝母)与酶切组(包括浓蜜贝母、中华贝母、康定贝母、长腺贝母与川贝母)。进一步获得川贝母与其近缘种的ITS2与psbA-trnH序列,发现ITS2序列长度为235~239 bp,种内和种间平均遗传距离为0.001和0.022,NJ树能够将样品分为"北方贝母群"(包括平贝母与伊犁贝母)与"南方贝母群"(包括浙贝母、浓蜜贝母、中华贝母、康定贝母、长腺贝母和川贝母)。后者又可细分为浙贝母与"川贝母复合群",其中"川贝母复合群"中的贝母品种具有相近的亲缘关系。psbA-trnH序列长度为337~373 bp,种内及种间的遗传距离为0.263和0.329,NJ树未能有效聚类。说明ITS2条形码序列不仅能够对川贝母及其部分近缘种进行快速、准确地鉴定,还能够清晰不同贝母种之间的亲缘关系,这为川贝母分子标记的开发、药用植物资源的有效保护和合理开发利用提供了重要的理论依据。
关键词:    川贝母      ITS2      psbA-trnH      DNA条形码      PCR-RFLP     
Molecular identification and genetic relationship of Fritillaria cirrhosa and related species based on DNA barcode
ZHENG Hui1, DENG Kai-yu1, CHEN An-qi1, FU Shao-bing2, ZHOU De3, WANG Wei-wei1, NI Dian-mo1, REN Yao-yao1, ZHOU Jia-yu1, LIAO Hai1
1. School of Life and Science, Southwest Jiaotong University, Chengdu 610031, China;
2. Qinghai Lukang Biological Development Co., Ltd., Xining 810003, China;
3. Aba Food and Drug Inspection, Aba 624000, China
Abstract:
Based on the ITS2 and psbA-trnH sequences, molecular biological identification and genetic relationship of Fritillaria cirrhosa with its relative species were carried out. In this paper, the PCR-RFLP method specified by the Chinese Pharmacopoeia was performed on all samples at first. Secondly, the ITS2 and psbA-trnH sequences of all samples were amplified. Then, the amplified products were used to analyze the genetic distance, construct the phylogenetic tree, assess the identification efficiency, and evaluate the genetic relationship as well. The result showed that all the samples were divided into two groups by PCR-RFLP method. The samples in the first group, including Fritillaria ussuriensis, Fritillaria thunbergii and Fritillaria pallidiflora, could not be digested by SmaI, while the other samples in the second group, including Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa, could be digested by SmaI. Then, ITS2 and psbA-trnH sequences of all samples were obtained. The length of various ITS2 sequences were distributed from 235 to 239 bp, and the average intra-and inter-specific genetic distance were 0.001 and 0.022, respectively. NJ tree showed that all samples were separated into "Northern Fritillaria" group (Fritillaria ussuriensis and Fritillaria pallidiflora) and "Southern Fritillaria" group (Fritillaria thunbergii, Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa). The latter group could be further divided into Fritillaria thunbergii and Fritillaria cirrhosa subgroup, and the species in Fritillaria cirrhosa subgroup had close phylogenetic relationships. The length of psbA-trnH sequences was distributed from 337 to 373 bp, and the intra-and inter-specific genetic distance were 0.263 and 0.329, respectively. The samples in this paper could not be clustered effectively by NJ tree. This indicated that the ITS2 sequences were not only able to identify Fritillaria cirrhosa with its partial relative species quickly and accurately, but also clarify the relationship between different Fritillaria species. Therefore, it provided an important theoretical foundation for the development of molecular markers, effective protection, and rational development and utilization of Fritillaria resources.
Key words:    Fritillaria cirrhosa    ITS2    psbA-trnH    DNA barcode    PCR-RFLP   
收稿日期: 2019-06-27
DOI: 10.16438/j.0513-4870.2019-0514
基金项目: 国家自然科学基金资助项目(31500276);四川省应用基础研究项目(2017JY0222);四川省重点研发项目(2018SZ0061);成都市科技技术研发项目(2016-HM01-00260-SF);四川省中医药管理局项目(2016Z013);国家级大学生创新创业计划(201910613082);西南交通大学个性化实验(GX201913084).
通讯作者: 周嘉裕,E-mail:spinezhou@home.swjtu.edu.cn;廖海,E-mail:ddliaohai@home.swjtu.edu.cn
Email: spinezhou@home.swjtu.edu.cn;ddliaohai@home.swjtu.edu.cn
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