野生人参RAPD指纹的研究

目的：分析山参遗传多样性及其遗传特性。方法：用随机扩增多态DNA(RAPD)标记方法对7个来源地不同的山参和1个园参样品进行遗传多样性检测和遗传分析。结果和结论：用14个10-mer寡聚核苷酸引物共检测111个位点,其中多态位点76个,占67.6%,远大于园参内的遗传变异,因此山参在人参育种上有很大利用价值。聚类分析表明,山参之间及其与园参之间的遗传变异,没有超出与近缘种西洋参之间的遗传差异;遗传因素在人参形态变异上的作用小于环境因素,这一结果为“山参”的培育提供了理论依据。     

铁皮石斛野生居群遗传多样性的RAPD分析与鉴别

目的采用RAPD分子标记技术，对铁皮石斛8个野生居群的遗传多样性、亲缘关系以及分子鉴别等进行研究。方法筛选随机引物进行RAPD分析，通过UPGMA聚类，研究铁皮石斛各居群间的遗传关系，构建居群亲缘关系的分子系统树；利用特异性条带对铁皮石斛野生居群进行指纹分析鉴别。结果共筛选出10个有效引物，在8个野生居群材料的RAPD扩增中共得到439个位点，平均每个引物扩增出43.9个位点，每个居群扩增出54.9个位点；在所获得的104条可重现谱带中，9条是单态的，95条是多态的，多态性程度达91.35%，遗传距离在0.590～0.727之间，平均为0.686。结论铁皮石斛居群间遗传差异明显，具有较丰富的遗传多样性；RAPD可以作为铁皮石斛野生居群遗传多态性、居群亲缘关系和分子鉴别研究的有效手段；引物S412可以有效鉴别铁皮石斛的8个野生居群。     

Molecular authentication of Panax ginseng species by RAPD analysis and PCR-RFLP

In order to develop convenient and reproducible methods for the identification of ginseng drugs at a DNA level, randomly amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses were applied within Panax species. To authenticate Panax ginseng among ginseng populations, RAPD analysis was carried out using a 20 mer-random primer. The similarity coefficients among the DNA of ginseng plants analyzed were low, ranging from 0.197 to 0.491. In addition, by using PCR-RFLP analysis, very different fingerprints were obtained within Korean ginseng plants. These results suggest that these methods are able to authenticate the concerned Panax species. Broader application of this approach to authenticate other morphologically similar medicinal materials is rationalized.     

AFLP法构建人参、西洋参基因组DNA指纹图谱

目的　采用扩增片段长度多态性DNA(AFLP)分子遗传标志技术,分析人参、西洋参基因组DNA多态性。方法　人参、西洋参干燥根基因组DNA,经EcoRI/MseI酶切并与其相应的人工接头连接后,使用选择性引物进行PCR扩增。结果　经变性聚丙烯酰胺凝胶电泳检测,成功构建出多态性丰富和重复性好的人参、西洋参DNA指纹图谱。结论　AFLP法有望成为一种独立的切实可行的手段,将在人参、西洋参等药用植物的鉴定、生物进化、系统发育研究及指导道地性药材的科学栽培等方面发挥重要作用。     

不同居群白芍的DNA随机扩增多态性研究

目的分析6个不同居群白芍的遗传多样性,为白芍的种质鉴定及遗传多样性分析提供依据。方法运用随机扩增多态性DNA(RAPD)技术,对浙江磐安、四川中江、安徽亳州、上海崇明、江苏宿迁和山东荷泽居群白芍的基因组DNA进行随机扩增,利用NTsys2.10e软件计算遗传相似性,运用UPGMA法进行聚类分析并构建树状图。结果共筛选了70个随机引物,从中挑选出8条多态性强、重复性好的引物,共检测出215个位点,多态性位点137个,多态位点比率为63.7%,UPGMA聚类可以将不同来源的白芍很好地区分开。结论不同产地间的白芍存在丰富的遗传多样性,RAPD分子标记方法可以用来鉴定不同产地的白芍。     

鱼腥草种质资源的RAPD分析

目的分析鱼腥草种质资源在分子水平上的遗传多样性。方法应用RAPD技术对92份鱼腥草种质资源进行检测。结果34个随机引物中有32个引物(94.1%)扩增产物具多态性。34个引物共得到200条扩增DNA片段,其中93.5%的片段具多态性。每个多态性引物平均可扩增出5.8个多态性片段。峨眉蕺菜和蕺菜种内平均遗传相似系数(genetic similarity,GS)分别为0.521和0.572,二者种间GS值为0.517。峨眉蕺菜与蕺菜中染色体数目为36的细胞型间相似程度最高,其平均GS值达0.530。栽培蕺菜类群比其野生类群遗传多样性相对较高。聚类分析表明,利用RAPD技术可将全部供试材料区分开,所有材料共划分为14类。其中,绝大多数(62个)聚为一类,且根据RAPD遗传相似系数划分的类群同地理分布有一定关系。结论(1) 鱼腥草种质资源在分子水平上确实存在较大遗传差异。(2) RAPD标记可作为构建鱼腥草DNA指纹图谱的有效工具。(3) 鱼腥草药材道地性与环境因素有关,但更大程度上由其遗传因素所决定。     

西洋参与籽播参的傅里叶变换红外光谱法鉴别

目的 为了建立西洋参与籽播参有效的鉴别方法。方法 采用傅里叶变换红外光谱（FTIR）仪并借助OMNI采样器直接测定了样品的FTIR。结果 西洋参及籽播参外表皮及木质部的傅里叶变换红外光谱吸收差别较大。结论 可以采用FTIR直接测定法鉴别籽播参与西洋参，本法简便、快速、准确，而且不需制备样品。     

中药材分子鉴别新方法:锚定引物扩增多态性DNA的研究

为了寻找稳定性好、可操作性强的分子鉴定新方法，在充分吸取RAPD优势的基础上，对其引物和退火温度进行了改进。本文以人参、西洋参为例进行了方法的探索和各种验证，并推广应用到天花粉以及白芷类药材的鉴别。结果显示引物Pg-q36F得到人参、西洋参及其9种伪品的多态性条带。对于人参、西洋参的鉴别结果与文献鉴别方法结果一致，并且具有更高的稳定性。引物TkS1-64F得到了天花粉及其11种伪品的多态性条带，引物AfS1-100F得到白芷及其3种伪品的多态性条带，均能准确鉴别各种药材。实验结果证明本方法具有简单易行、稳定性和重复性好、提供的信息量大等优点，是一种极具前途的中药材分子鉴定新方法，被命名为锚定引物扩增多态性DNA(anchored primer amplification polymorphism DNA，APAPD)。     

Genetic ecotoxicology II: population genetic structure in mosquitofish exposed in situ to radionuclides

In 1977, approximately 250 mosquitofish (Gambusia affinis) from a relatively uncontaminated site (Crystal Springs) were transplanted into a small pond on the Department of Energy Oak Ridge Reservation which is heavily contaminated with radionuclides (Pond 3513). Starting in 1992, DNA polymorphism was evaluated using the RAPD (Randomly Amplified Polymorphic DNA) and allozyme genotype techniques to determine if genetic differentiation had occurred between the two populations. Fish from a second radionuclide-contaminated population (White Oak Lake) and another unrelated non-contaminated population (Wolf Creek) were also examined. For the RAPD analyes, 15 RAPD primers (from a total of 40) were found to produce polymorphic banding patterns in at least two of the four populations and subsequently were used to produce a total of 142 bands. Data generated by these RAPD primers indicated an increased genetic diversity in radionuclide-contaminated sites relative to reference sites. Furthermore, the patterns from six RAPD primers produced a higher average number of bands when using DNA from radionuclide- contaminated populations than from non-contaminated, and for three RAPD primers the average number of bands from radionuclide- contaminated populations was lower. In addition, 17 bands occurred at a higher frequency in the radionuclide-contaminated compared to the non-contaminated populations. For the allozyme analyses, it was found that there was a higher percentage of polymorphism and heterozygosity in the radionuclide-contaminated relative to non-contaminated sites. These findings contribute to our understanding of the evolutionary effects of contaminant exposure as well as to the development of population-level biomarkers     

铁皮石斛野生居群基于RAMP标记的遗传多样性评价

本文运用RAMP标记16对引物组合,对铁皮石斛9个野生居群的112个样本进行了检测,共得到123条扩增带,其中86条(占69.92%)具有多态性,每对引物组合可扩增出3～8条多态性带,平均5条,表明铁皮石斛不同居群间存在较丰富的遗传多样性;铁皮石斛居群间遗传相似系数的变化范围为0.250～0.813,平均值为0.550。利用RAMP扩增条带数据进行聚类分析,可将9个铁皮石斛野生居群划分为3个类群,聚类结果表现出较好的地域相关性,显示RAMP标记可以有效地评价铁皮石斛野生居群的遗传多样性及遗传结构。     

A random amplified polymorphic DNA (RAPD) primer to assist the identification of a selected strain, aizu K-111 of Panax ginseng and the sequence amplified

Panax ginseng is widely used as a Chinese medicine, but it takes a long time to reach harvest and to establish its qualified strains. In the course of searching high quality Panax ginseng, we found a useful random amplified polymorphic DNA (RAPD) primer, which showed a 725 base pair band for a selected elite strain Aizu K-111 (now called Kaishusan) including its cultured tissues, while the other strains did not necessarily show this band. We sequenced the DNA fragment amplified and designed primers to improve electrophoretic profiles, based on the sequence.     

人参和其他中草药的遗传学鉴定(英文)

The main objective of this paper is to review the chemical and genetic methods used in authentication of ginseng, especially the recent advances in microsatellite genotyping and its application to the authentication of other traditional Chinese medicines (TCM). The standardization and modernization of TCM hinge on the authentication of their botanical identities. Analysis of well-characterized marker compounds is now the most popular method for identifying the herbal materials and quality control of TCM, eg, ginsenoside profiling for authentication of Panax species. However, in many herbal species the chemical composition of the plant changes with the external environment and processing conditions, which lowers the reliability of these authentication methods. In the light of the advances in molecular biotechnology in the past few decades, genetic tools are now considered to provide more standardized and reliable methods for authentication of herbal materials at the DNA level. These genetic tools include     

Intersimple sequence repeats (ISSR) molecular fingerprinting markers for authenticating populations of Dendrobium officinale Kimura et Migo

Intersimple sequence repeats (ISSR) molecular fingerprinting markers have been employed to authenticate eight populations of Dendrobium officinale using 10 primers selected from 76 ISSR primers. A total of 127 DNA fragments were amplified, of which 115 were polymorphic (90.5% of all bands). Sixteen specific authentication markers have been found. To enhance the efficiency of authentication, ISSR fingerprinting codes have been constructed using six polymorphic bands for authenticating D. officinale populations. Eight wild populations of D. officinale have been authenticated accurately using ISSR.     

HPLC 法鉴别西洋参和人参

用ＨＰＬＣ法测定了２４种西洋参根皂苷、５种西洋参茎叶皂苷、７种人参根皂苷、一种人参茎叶皂苷及８种单体皂苷（Ｒｏ，Ｒｅ，Ｒｇ１，Ｒｆ，Ｒｂ１，Ｒｇ２，Ｒｃ，Ｒｂ２）．结果表明，西洋参与人参的明显区别在于：人参含有Ｒｆ及Ｒｇ１；而西洋参不含Ｒｆ及Ｒｇ１     

不同产地白及遗传多态性的RAPD分析

目的 研究国内不同产地白及的遗传多样性和亲缘关系。方法 利用随机扩增多态DNA （random amplified polymorphic DNA,RAPD）引物,RAPD分子标记技术分析国内50个不同产地的白及。结果 RAPD技术扩增产物经琼脂凝胶电泳检测,从50条引物中筛选出10条（S8,S9,S14,S19,S23,S25,S28,S29,S30,S31）条带清晰的引物,10条引物共扩增出88条DNA条带,其中多态性的DNA条带数目为81条,占总数的92.05%。每个引物能扩增出5~11条DNA条带,平均可扩增出8.8条;扩增最少的引物为S19,扩增出5条DNA条带;扩增最多的引物为S29,扩增出11条DNA条带。而每个引物能扩增的多态性DNA条带数为3~11条,平均可扩增出8.6条。结论 不同产地的白及具有较丰富的遗传多样性,RAPD可有效应用于白及的遗传多样性研究。     

湖北麦冬及其近缘药用植物的随机扩增引物DNA分析

目的 探讨湖北麦冬及其近缘药用植物的亲缘关系和遗传多样性。方法 对不同栽培措施处理的湖北麦冬、湖北麦冬组培苗、山麦冬、恩施麦冬、杭麦冬和川麦冬的22种材料进行随机扩增引物DNA（RAPD）法分析,用SPSS软件中的Jaccd和Dice两种方法对扩增片断的相关性进行聚类分析。结果 从S系列100个随机引物中筛选出10个扩增条带清晰、稳定、重现性好和多态性明显的引物,10个引物共扩增出75个条带,其中多态性条带58个,占总扩增条带的77.3%。湖北麦冬不同栽培条件和组织培养植株的相关系数均接近1。湖北麦冬与山麦冬的相关系数在0.74～0.88之间。麦冬（杭麦冬和川麦冬）与湖北麦冬和与山麦冬之间的相关系数分别为0.41～0.66和0.48～0.69。杭麦冬与川麦冬的相关系数为0.74,介于湖北麦冬与山麦冬之间。不同的山麦冬样品相关系数均>0.8。结论 22种麦冬类药用植物之间的亲缘关系与形态分类结果基本一致,组织培养和栽培措施对遗传关系影响不大。但产于不同地区的川、杭、恩施麦冬具有一定的遗传差异.     

Molecular Characterization of Contaminant-Indicative RAPD Markers

This paper describes genetic markers which can be used to study selection and genetic adaptation of organisms to radionuclide and other types of contaminant stress. Previous research using the randomly amplified polymorphic DNA (RAPD) technique has identified several markers which revealed genetic differences between contaminated and reference western mosquitofish (Gambusia affinis) populations. Experimental evidence suggested that these markers may be associated with loci involved in determining relative fitness in radionuclide-contaminated environments ("contaminant-indicative markers"). In the present study, Southern blot analyses show these markers to be highly conserved in DNA sequence and molecular length in sea urchins, mosquitofish, herring gulls and humans. Such conservation is thought to be rare among RAPD bands. Results of DNA sequencing efforts did not provide definitive evidence as to the identity of these loci, but indicated that short segments (<40 bp) of known DNA sequences were homologous to various regions of the RAPD sequences. Furthermore, the regions of homology seemed to be non-randomly distributed along the length of the RAPD markers. Although the identity of these bands is still unknown, the high degree of conservatism suggests that these loci might play an important role in molecular processes.     

Molecular differentiation ofPanax species by RAPD analysis

Traditional taxonomic methods used for the identification and differentiation of ginsengs rely primarily on morphological observations or physiochemical methods, which cannot be used efficiently when only powdered forms or shredded material is available. Randomly amplified polymorphic DNA (RAPD) was used to determine the unique DNA profiles that are characteristic not only of the genus Panax but also of various Panax subgroups collected from five different countries. RAPD results of OP-5A primer showed a specific single band that is characteristic of all ginseng samples. RAPD results of OP-13B primer demonstrated that OP-13B primer could be used as a unique RAPD marker to differentiate Panax species. These results support that this approach could be applied to distinguish Korean Ginseng (Panax ginseng) from others at the molecular level.     

Choice of Methodology for Assessing Genetic Impacts of Environmental Stressors: Polymorphism and Reproducibility of RAPD and AFLP Fingerprints

PCR-based multi-locus DNA fingerprints represent one of the most informative and cost-effective measures of genetic diversity and are useful population-level biomarkers of toxicologic and other anthropogenic impacts. However, concerns about reproducibility of DNA fingerprints have limited their wider use in environmental biology. We assessed polymorphism and reproducibility of two common fingerprinting techniques, RAPD (randomly amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), in pedigreed populations of rainbow trout (Oncorhynchus mykiss) to derive general rules for selective removal of problematic fingerprint bands. We found that by excluding bands that comprised less than 1% of total intensity, and by excluding the largest and smallest 10% of the bands, we could achieve nearly 100% reproducibility of AFLP fingerprints. Similar application of band exclusion criteria to RAPD fingerprints did not significantly enhance their reproducibility, and at least 15% of RAPD bands were not fully repeatable, heritable, or transmittable. The RAPD technique produced more polymorphic fingerprints than AFLP; however, considering that a substantial proportion of RAPD markers did not demonstrate Mendelian inheritance patterns, the AFLP methodology is to be preferred for future research.     

利用ISSR及代谢产物标记检测贵州金钱草遗传多样性

目的：结合ISSR分子标记与金钱草主要代谢产物标记对贵州省共21份野生金钱草资源的遗传多样性进行分析,为其驯化种植及遗传改良提供理论依据。方法：以金钱草基因组DNA为模板,以优化的PCR体系筛选出引物进行ISSR标记扩增,应用NTsys 2.10e软件,对所得数据进行分析处理,并采用高效液相色谱法测定了主要代谢产物槲皮素和山柰素的含量,使用SPSS 19.0软件对测定数据进行统计分析与绘制聚类图。结果：用筛选出的15条ISSR引物共扩增出105条多态性条带,多态性条带占比达98.1%,各金钱草种质资源之间的遗传相似性系数范围在0.598 1~0.953 3之间,地理位置相距越近,遗传相似性越高,不同资源的槲皮素和山柰素含量差异较大,ISSR标记的聚类将21份金钱草资源分为2大类群6个亚群,代谢产物标记的聚类将其分为2大类群5个亚群。结论：贵州野生金钱草具有丰富的遗传多样性,应用ISSR及代谢产物槲皮素和山柰素标记均能较好地对金钱草资源进行聚类,但聚类结果存在差异,其亲缘关系与地理位置呈现一定的关联性。