党参线粒体和叶绿体微卫星标记的开发及应用  

Development and Application of Mitochondrial and Chloroplast Microsatellite Markers for Codonopsis Plants

作　　者：张丹 杜晨晖 裴香萍 刘晓丽 詹海仙 ZHANG Dan;DU Chen-hui;PEI Xiang-ping;LIU Xiao-li;ZHAN Hai-xian(School of Chinese Medicine and Food Engineering,Shanxi University of Traditional Chinese Medicine,Jinzhong 030619,China)

机构地区：[1]山西中医药大学中药与食品工程学院,山西晋中030619

出　　处：《中国实验方剂学杂志》2021年第23期153-162,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：山西省重点研发计划项目(201903D221053);山西中医药大学博士科研启动基金项目(2020BK05)。

摘　　要：目的:由于不同基原和产地的党参有效成分差异较大,开发党参特异分子标记对于党参种质资源准确鉴定意义重大。方法:该研究应用生物信息学软件Primer 5.0,NTSYS-pc 2.10e,PopGene 32,对小党参Codonopsis minima叶绿体、秦岭党参C.tsinlingensis叶绿体和轮叶党参C.lanceolata线粒体基因组序列进行简单重复序列标记(SSR)位点搜索,利用Primer5.0软件设计出120对SSR引物,筛选效果好且多态性高的16对cpSSR引物和10对mtSSR引物对20份党参材料进行种间通用性分析。结果:结果显示,分别从基因组序列中搜索到66个cpSSR位点和26个mtSSR位点,其中小党参叶绿体中单核苷酸(86.20%),二核苷酸(6.9%),三核苷酸(6.9%);秦岭党参叶绿体中单核苷酸(83.78%),二核苷酸(13.51%),三核苷酸(2.71%);轮叶党参线粒体中单核苷酸(46.15%),二核苷酸(53.85%)。聚合酶链式反应(PCR)鉴定结果表明开发的26对SSR引物在党参属内具有很好的适用性。NTSYS-pc 2.10e软件分析显示20份党参材料的遗传相似系数在0.38~1.00,在阈值0.53处分为2个亚群。运用PopGene 32软件对20份党参进行多样性分析并筛选出4对多态性引物,分析发现观测等位基因数(Na)共为12个,有效等位基因数(Ne)为1.362 9~2.605 9个,各位点多态性位点百分率(PPL)均为100%,遗传学参数Ho,He,香浓指数(I)的平均值分别为0.555 8,0.444 2,0.753 2,说明各位点的多态性水平较高。以筛选出的4对引物对20份党参材料进行DNA指纹图谱绘制,发现DNA指纹图谱可将20份材料进行鉴别。结论:该研究可为党参属种间的亲缘关系和种内遗传分化研究提供分子依据。Objective:To develop the specific molecular markers of Codonopsis plants and better identify their germplasm resources considering the significant difference in active ingredients of Codonopsis Radix from various origins and producing areas.Method:Such bioinformatics software as Primer 5.0,NTSYS-pc 2.10e,and PopGene 32 were used for searching the simple sequence repeat(SSR)markers of C.minima chloroplast genome,C.tsinlingensis chloroplast,and C.lanceolata mitochondrial sequences,and 120 pairs of SSR primers were designed by Primer 5.0.Then 16 pairs of cpSSR primers and 10 pairs of mtSSR primers with good screening effect and high polymorphism were selected for analyzing the interspecific versatility of 20samples.Result:The results showed that 66 cpSSR primer sites and 26 mtSSR sites were identified from the genome sequences,with 86.20%of single nucleotide,6.9%of dinucleotide,and 6.9%of trinucleotide for C minima chloroplast,83.78%of single nucleotide,13.51%of dinucleotide,and 2.71%of trinucleotide for C tsinlingensis chloroplast,and 46.15%of single nucleotide and 53.85%of dinucleotide for C.lanceolata mitochondria.As demonstrated by polymerase chain reaction(PCR)identification results,the developed 26pairs of SSR primers had good applicability in the genus Codonopsis.The analysis by NTSYS-pc 2.10e revealed that the genetic similarity coefficients of 20 samples were within the range of 0.38-1.00,and they were divided into two subgroups at a threshold of 0.69.Four pairs of polymorphic primers were screened out in the diversity analysis of 20 samples using PopGene 32.The number of observed alleles(Na)was 12,and the effective number of alleles(Ne)ranged from 1.362 9 to 2.605 9.The percentage of polymorphic loci(PPL)at each site was 100%,and the average values of genetic parameters Ho,He,and I at each site were 0.555 8,0.444 2,and0.753 2,respectively,indicating high polymorphism at each site.The screened four pairs of primers were utilized for DNA fingerprinting of the 20 samples,and it was found that the DNA fingerpr

关 键 词：党参 叶绿体 线粒体 微卫星 开发及应用 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学]