内蒙古不同产地小秦艽ITS序列比较研究

Comparative Study on ITS Sequences of Xiaoqinjiao(Gentianae Xiaoqinjiao Radix)from Different Regions in Inner Mongolia

目的:研究内蒙古不同产地野生小秦艽内转录间隔区(ITS)序列的遗传变异情况并进行聚类分析,为保护及优化小秦艽种质资源提供参考。方法:对野外采集的内蒙古不同产地的62份小秦艽样品进行DNA提取,选取合适引物进行PCR扩增并测序,测序结果使用BioEdit、CLC Sequence Viewer 8及MEGA 11.0软件进行处理后,分析遗传变异并构建系统发育树。结果:聚类分析结果表明,62份小秦艽样品主要分为两支,编号为BT1(采自包头市白云鄂博巴润园区东南1 km处)和BY1(采自巴彦淖尔盟乌拉特前旗阿嘎如泰苏木大桦背)的样品聚为一个小分支,其他60份样品聚为一个大分支,说明各分支内存在较近的亲缘关系,遗传变异过程相似;在相似的遗传变异条件下,样品中龙胆苦苷含量相近,排除环境主要生态因子的影响,推测小秦艽样品的龙胆苦苷含量可能与遗传变异过程相关;样品BT1和HH4(呼和浩特市和林格尔县太平夭),BT1和AL1(阿拉善左旗巴润别立镇贺兰山雪岭子沟)的遗传距离最大,因其地理距离较大,推测地理环境因素在一定程度上影响了小秦艽的生物进化过程;序列中的变异位点为129个;各样品ITS序列的Kimura-2-parameter(K2P)遗传距离范围为0.0000~0.2632,平均值为0.0494。结论:DNA分子技术—ITS序列有助于研究药用植物小秦艽种质资源遗传多样性,了解种内遗传变异的大小及其与环境的关系,从而保护及发展小秦艽种源优势。

Objective:To study the genetic variation of the internal transcribed spacer(ITS)sequences in wild Xiaoqinjiao(Gentianae Xiaoqinjiao Radix)from different regions of Inner Mongolia and to perform cluster analysis,so as to provide reference for the protection and optimization of Xiaoqinjiao(Gentianae Xiaoqinjiao Radix)resources.Methods:62 samples of Xiaoqinjiao(Gentianae Xiaoqinjiao Radix)from different places in Inner Mongolia were collected in the field for DNA extraction,and suitable primers were selected for PCR amplification and sequencing.The sequencing results were processed by BioEdit,CLC Sequence Viewer 8 and MEGA 11.0 software,and the genetic variation was analyzed and the phylogenetic tree was constructed.Results:Cluster analysis showed that 62 samples of Xiaoqinjiao(Gentianae Xiaoqinjiao Radix)were mainly divided into two branches.The samples numbered BT1(collected from 1 km southeast of Bayan Obo Barun Garden,Baotou City)and BY1(collected from Dahuabei,Agarutai Sumu,Urad Front Banner,Bayan Nur City)were clustered into a small branch,and the other 60 samples were clustered into a large branch,which indicating that there were close genetic relationships among the branches and the genetic variation process was similar.Under the similar genetic variation conditions,the content of gentiopicroside in the samples was similar.Excluding the influence of major ecological factors in the environment,it was speculated that the gentiopicroside content in the samples of Xiaoqinjiao(Gentianae Xiaoqinjiao Radix)may be related to the process of genetic variation.The genetic distance of samples BT1 and HH4(Taipingyao,Horinger County,Hohhot City),BT1 and AL1(Xuelingzigou,Helan Mountains,Barenbeli Town,Alxa Left Banner)was the largest.Due to the large geographical distance,it was speculated that geographical environmental factors had affected the biological evolution of Xiaoqinjiao(Gentianae Xiaoqinjiao Radix)to some extent.There were 129 mutation sites in the sequence.The genetic distance of Kimura-2-parameter(K2P)of ITS sequence of each sample ranged from 0.0000 to 0.2632,with an average of 0.0494.Conclusion:DNA molecular technology-ITS sequence is helpful for studying the genetic diversity of medicinal plant Xiaoqinjiao(Gentianae Xiaoqinjiao Radix)germplasm resources,understanding the magnitude of intra species genetic variation and its relationship with the environment,and thus protecting and developing the advantages of Xiaoqinjiao(Gentianae Xiaoqinjiao Radix)germplasm.