防风及其近缘种的DNA指纹分析及质量相关研究
摘要
本实验应用RAPD及ISSR分子标记，对来源于黑龙江、河北、陕西、四川、甘肃的五个防风及其近缘种共38份样本进行了DNA指纹分析。从100个RAPD引物和32个ISSR引物中分别筛选出21、6个扩增结果稳定、扩增条带清晰的引物。其中，8个RAPD引物和3个ISSR引物扩增出的带可以将防风及其近缘种区分开。用筛选出的引物对所有样本进行PCR扩增，得到DNA指纹图，从DNA指纹图中可见不同品种之间仅有少数引物得到可以明确区分它们的不同条带。同时对电泳条带进行基于遗传相似度的聚类分析可将5类38份材料分为三类：第一类包括竹节防风、前胡，第二类包括硬苗防风、葛缕子，第三类为正品防风。前两类之间的遗传相似度范围为0.77～0.80，正品防风和这二者之间的遗传相似度范围为0.72～0.75。不同品种之间的遗传相似度范围显示了正品防风与其他防风近缘种或混伪品之间的遗传距离，以及防风近缘种之间的遗传相似性，但也说明正品防风与其他防风近缘种或混伪品遗传相似度比较低，具有比较远的亲缘关系。
在药物有效成分含量分析方面，本文采用《中华人民共和国药典》中规定的高效液相色谱法对所有样本进行了防风升麻苷的含量测定。同时，尝试使用了先进的毛细管电泳法。《中华人民共和国药典》中规定防风中色原酮类成分的含量不可低于0.308%，产地黑龙江富裕的正品防风的有效成分升麻苷的含量为0.842%～0.864%明显高于其他如硬苗防风（河北 0.159%～0.163%）、竹节防风（四川0.350%～ 0.411%）、前胡（陕西 0.473%～0.590%）、葛缕子（甘肃 0.456%～0.501%）等防风亲缘种中的升麻苷含量，结合DNA指纹分析，引物SBS A17对正品防风扩增出一条特异带，其它产地的防风近缘种无此带。
本研究筛选得到的RAPD及ISSR引物可用于防风类药材的分子鉴定。黑龙江富裕地区的防风质量最好，升麻苷含量高，是培育防风优良品种的首选引种之地。引物SBS A17扩增所得特异带可初步作为防风品质优良的特异性标记，在种苗的早期品质鉴定方面应用价值很高。
38 samples of five Species Fangfeng and Its Close Relative Species collected from Heilongjiagn, Hebei, Shanxi, Sichuan, and Gansu were analysed by RAPD and ISSR molecular markers. 21 RAPD primers and 9 ISSR primers were selected from 100 and 32 respectively, and could produced qualified fragments. Among these selected primers only 8 RAPD and 3 ISSR primers could identify the Fangfeng and Its Close Relative Species. DNA fingerprints were obtained by PCR with all selected primers and analyzed using cluster analysis. The fingerprints indicated that only few primers could get the specific fragments able to identify Fangfeng and Its Close Relative Species. The cluster analysis of cluster analysis based on the genetic similarity divide 38 samples of five Species Fangfeng to 3 groups: fisrt group included P.dielsianum Fedde ex wolff and Peucedanum ledebourieuoides K.T.Fu; second group included Curum Carvil and Friocycla albescens (Franch). Wolff; last group included Fangfeng. The genetic similarity of first two groups ranged between 0.77 to 0.80, the genetic similarity of first two groups and last group Fangfeng ranged between 0.72 to 0.75. The genetic similarity range of the different varieties show that The Genetic Distance of Fangfeng and Its Close Relative Species ,The genetic similarity of Its Close Relative Species,and also illustrate that The genetic similarity of Fangfeng and Its Close Relative Species were tiny, and relatively big between them.
Content of prim-O-glucosylcimifugin, the mainly effective compound was determined by high performance liquid chromatography, the method used in People’s Republic of China pharmacopeial convention. Also, the high performance capillary electrophoresis was used. People's Republic of china pharmacopeial convention prescript the content of prim-O-glucosylcimifugin in Fangfeng and Its Close Relative Species were aove 0.308. The Content of prim-O-glucosylcimifugin in Fangfeng selected from Heilongjiang Fuyu ranged from 0.842% to 0.864%,which obviously were much more than the Content in Its Close Relative Species,such as Friocycla albescens (Franch). Wolff(Hebei 0.159%～0.163%)、 P.dielsianum Fedde ex wolff (Sichuan 0.350%～ 0.411%)、Peucedanum ledebourieuoides K.T.Fu (Shanxi 0.473%～0.590%)、Curum Carvil(Gansu 0.456%～0.501%)。In concern of the DNA fingerprints, primer SBS A17 amplified a specific fragment within Fangfeng, while not in other samples.
The selected primers could be used in identifying Fangfeng selected form HeilongjianFuyu produced the best quality Fangfeng containing highest content of prim-O-glucosylcimifugin, and should be considered first while culturing new good variety. The specific fragment amplified by primer SBS A17 could be used as a potential DNA marker indicating good quality of Fangfeng, especially useful in the use of identifying good seedling in an early stage of growth.
在药物有效成分含量分析方面，本文采用《中华人民共和国药典》中规定的高效液相色谱法对所有样本进行了防风升麻苷的含量测定。同时，尝试使用了先进的毛细管电泳法。《中华人民共和国药典》中规定防风中色原酮类成分的含量不可低于0.308%，产地黑龙江富裕的正品防风的有效成分升麻苷的含量为0.842%～0.864%明显高于其他如硬苗防风（河北 0.159%～0.163%）、竹节防风（四川0.350%～ 0.411%）、前胡（陕西 0.473%～0.590%）、葛缕子（甘肃 0.456%～0.501%）等防风亲缘种中的升麻苷含量，结合DNA指纹分析，引物SBS A17对正品防风扩增出一条特异带，其它产地的防风近缘种无此带。
本研究筛选得到的RAPD及ISSR引物可用于防风类药材的分子鉴定。黑龙江富裕地区的防风质量最好，升麻苷含量高，是培育防风优良品种的首选引种之地。引物SBS A17扩增所得特异带可初步作为防风品质优良的特异性标记，在种苗的早期品质鉴定方面应用价值很高。
38 samples of five Species Fangfeng and Its Close Relative Species collected from Heilongjiagn, Hebei, Shanxi, Sichuan, and Gansu were analysed by RAPD and ISSR molecular markers. 21 RAPD primers and 9 ISSR primers were selected from 100 and 32 respectively, and could produced qualified fragments. Among these selected primers only 8 RAPD and 3 ISSR primers could identify the Fangfeng and Its Close Relative Species. DNA fingerprints were obtained by PCR with all selected primers and analyzed using cluster analysis. The fingerprints indicated that only few primers could get the specific fragments able to identify Fangfeng and Its Close Relative Species. The cluster analysis of cluster analysis based on the genetic similarity divide 38 samples of five Species Fangfeng to 3 groups: fisrt group included P.dielsianum Fedde ex wolff and Peucedanum ledebourieuoides K.T.Fu; second group included Curum Carvil and Friocycla albescens (Franch). Wolff; last group included Fangfeng. The genetic similarity of first two groups ranged between 0.77 to 0.80, the genetic similarity of first two groups and last group Fangfeng ranged between 0.72 to 0.75. The genetic similarity range of the different varieties show that The Genetic Distance of Fangfeng and Its Close Relative Species ,The genetic similarity of Its Close Relative Species,and also illustrate that The genetic similarity of Fangfeng and Its Close Relative Species were tiny, and relatively big between them.
Content of prim-O-glucosylcimifugin, the mainly effective compound was determined by high performance liquid chromatography, the method used in People’s Republic of China pharmacopeial convention. Also, the high performance capillary electrophoresis was used. People's Republic of china pharmacopeial convention prescript the content of prim-O-glucosylcimifugin in Fangfeng and Its Close Relative Species were aove 0.308. The Content of prim-O-glucosylcimifugin in Fangfeng selected from Heilongjiang Fuyu ranged from 0.842% to 0.864%,which obviously were much more than the Content in Its Close Relative Species,such as Friocycla albescens (Franch). Wolff(Hebei 0.159%～0.163%)、 P.dielsianum Fedde ex wolff (Sichuan 0.350%～ 0.411%)、Peucedanum ledebourieuoides K.T.Fu (Shanxi 0.473%～0.590%)、Curum Carvil(Gansu 0.456%～0.501%)。In concern of the DNA fingerprints, primer SBS A17 amplified a specific fragment within Fangfeng, while not in other samples.
The selected primers could be used in identifying Fangfeng selected form HeilongjianFuyu produced the best quality Fangfeng containing highest content of prim-O-glucosylcimifugin, and should be considered first while culturing new good variety. The specific fragment amplified by primer SBS A17 could be used as a potential DNA marker indicating good quality of Fangfeng, especially useful in the use of identifying good seedling in an early stage of growth.
引文
曹晖，刘玉萍，邵鹏柱，毕培曦.DNA分子指纹图谱与测序技术在中药品质研究中的现状及展望.中国药学杂志 .1998，23（11）：643-653
曹晖，刘玉萍.DNA 分析技术在中药质量研究中的应用.中国药学杂志.1998，38（5）： 112-115
曹进，饶毅，沈群，王义明，罗国安.中药指纹图谱及其建立原则.中药新药与临床药理.2001，12（3）：200-203
陈月琴，届良鹄，周惠.杜仲原植物25SrDNA5,端序列分析及其分子识别.中国中药杂志.1998，23(12)：707-710
陈永久，张亚平.随机扩增多态DNA影响因素的研究.动物学研究.1997，18（2）：221-227
方宣钧，吴为人，唐纪良.作物DNA标记辅助育种.科学出版社.2002：15
陈桂信，潘东明，吕柳新，赖钟雄，王凤华.DNA分子标记技术及其在热带亚热带果树上的应用.江西农业大学学报(自然科学版).2002，24（2）：176-182
窦忠健,杨勤福,李祎. 中药防风及其混伪品的鉴别.河南中医药学刊. 1999,14(4)：15-16
郭宝林，戴波.中药材DNA分子标记研究的技术问题.中草药.2002，33（2）：178-180
郭军，寿森炎，卢钢，吴韩英，黄锡志.DNA分子标记技术在品种鉴定和纯度分析上的应用.种子科技.2000，4：215-217
葛卫红. 荆芥、防风挥发油抗炎作用及其机理研究(博士论文).成都中医药大学.2002-04-01,2-835
胡宝忠，刘娣，胡国富，张阿英，姜述君.中国紫花苜蓿地方品种随机扩增多态DNA的研究.植物生态学报.2000，24（6）：697-701
胡国富.用微卫星及ISSR法对羊草（Aneurolepidium chinese (Trin)Kitag）遗传多样性的研究.东北农业大学硕士论文.2002年5月：8
黄中文，王莹，辛慧，刘明久.RFLP原理及在作物基因定位和育种中的应用.河南职技师院学报.2002，28（4）：24-27
黄璐琦.分子生药学.北京医科大学出版社.2000：29
赖小平,陈建南,刘中秋,周华. 高效毛细管电泳及其在中药质量分析的应用.中药新药与临床药理.1998，9（3）：186～187
罗国安，王义明.中药指纹图谱的分类和发展.中国新药杂志.2002，11（1）：46-51
李军,郭晏海,党琳.几种药用植物DNA提取与纯化方法的比较.陕西中医学院学报.2002，25(5)：72-73
孟祥才,江延辉,娄志红. 栽培防风质量分析.特产研究.2000,4：43
孙岳.防风DNA指纹分析及质量相关研究（博士论文）.东北农业大学.2003
王关林，方宏筠.植物基因工程原理与技术.科学出版社.1998：598
王培训，黄丰，周联，曹柳英，梁瑞燕.植物中药材总DNA提取方法的比较.中药新
药与临床药理.1999，10（1）：18-22
王天松.毛细管电泳与中草药指纹图谱.中成药.2000，22（6）：397-399
王志林，赵树进，吴新荣.分子标记技术及其发展.生命的化学.2002，21（4）：39-42
吴俊，魏钦平，束怀瑞，徐凯，钟家煌.分子标记及其在果树种质资源研究中的应用.安徽农业大学学报.2002，29（2）:158-162
吴田.中医药现代化与中药指纹图谱分析.中国中西医结合杂志.2002，22（9）：645-646
吴兴奎，吕继红.浅谈影响中药疗效的因素.河南中医药学刊.1999，14（6）：52-53
肖小河，刘峰群，袁海龙，贺承山，史成和.中药DNA分子标识鉴定研究进展.中草药.2000，31（8）：561-565
肖永庆,杨滨,姚三桃,李文,李丽,黄璐琦,薛宝云.?防风质量标准的研究.中国中药杂志.2001 26(3)：185～187
肖永庆,李丽,杨滨,黄璐琦.防风化学成分研究.中国实验方剂学杂志.2002 S1:117-119
薛宝云，李文，李丽，肖永庆.防风色原酮甙类成分的药理活性研究.中国实验方剂学杂志.2001,S1:297-299
邢俊波，刘云.高效毛细管电泳技术近年在中药分析中的应用.解放军药学学报.2002，18（4）：220-222
邢旺兴，宓鹤鸣，田倩.RAPD技术在生药学研究中的应用.解放军药学学报.1999，15（3）：25-28
杨滨,黄璐琦,姚三桃,肖永庆.防风类药材高效液相色谱鉴别.中国药学杂志. 1999,34(10):658-660
杨剑.影响中成药质量的因素及对策.中国医院药学杂志.1998，18（12）：561-562
尹茶，吴玉田.高效毛细管电泳技术及其在中药分析中的应用.药物分析杂志.1999，19（3）：209-217
尤勇.应用RAPD标记对杉木属两个种遗传多态性的研究.聊城师院学报（自然科学版）.1998，11（4）：72-74
于清峰，倪坤仪，王志群.药物中有关物质的色谱法分析.药学进展.2000，24（5）：270-274
张宇红，周常文.PCR-SSCP分析技术的研究进展及应用前景.中国优生与遗传杂志.2002，10（5）：126-127
张云武，陈永久，沈发荣，杨跃雄，杨大荣，张亚平.滇西北地区冬虫夏草和阔孢虫草的遗传分化研究.菌物系统.1999，18（2）：176-183
赵凌侠，李景富，唐克轩，许向阳，开国银.番茄属（Lycopersicon）RAPD标记遗传分析.复旦学报（自然科学版）.2002，41（2）：222-226
张贵君,张艳波,李影. 我国生药防风近10年的研究概况.时珍国医国药.1997,8(1): 73-75
张宇红，周常文.PCR-SSCP分析技术的研究进展及应用前景.中国优生与遗传杂志.2002，10（5）：126-127
张云武，陈永久，沈发荣，杨跃雄，杨大荣，张亚平.滇西北地区冬虫夏草和阔孢虫草的遗传分化研究.菌物系统.1999，18（2）：176-183
赵凌侠，李景富，唐克轩，许向阳，开国银.番茄属（Lycopersicon）RAPD标记遗传分析.复旦学报（自然科学版）.2002，41（2）：222-226
Anthony F, Combes C, Astorga C, Bertrand B, Graziosi G, Lashermes P. The Origin of Cultivated Coffea Arabica L.Varieties Revealed by AFLP and SSR Markers. Theor Appl Genet. 2002, 104(5):894-900
Blair M W.Panaud O, McCouch S R. Inter-Simple Sepuence Repeat (ISSR)Amplification for Analysis of Microsatellite Motif Frequency and Fingerprinting in Rice (Oryza sativaL.) Theor Appl Genet. 1999,98:780~792.
Cekic C,Battey N H, Wilkinson M J.The Potential of ISSR-PCR Primer-Pair Combinations for Genetic Llinkage Analysis Using the Seasonal Flowering Llocus in Fragaria as a Model. Theor Appl Genet.2001,103:540-546
Cheung KS, Kwan HS, But PP, Shaw PC. Phar-Macognostical Identification of American and Oriedtal Ginseng Roots by Genomic Fingerprinting Using Arbitrarily Primed Polymerase Chain Reaction ( AP- PCR) . J Ethnopharmacol. 1994,42:67
Devos K M, M D Gale. The Use of Random Amplified Polymorphis DNA Markers in Wheat. TAG.1992,82:567-572
Dirlewanger E, Pronier V, Parvery C. Genetic Linkage Map of Peach (Prunus persica L)Using Morphological and Molecular Markers. Theor Appl Genet. 1998,97:888-896
Ellsworth D L, Rittenhpuse K D, Honeycutt R L. Artifactual Variation in Randomly Amplified Ploymorphic DNA Banding Patterns. Biotechniques.1993, 14:214-217
Esselman E J, Li J Q, Crawford D J, et al. Clonal Diversity in the Rare Calamagrostis Porterissp. Insperata (Poaceae): Comparative Results for Allozymes and RAPD and ISSR Markers. Mol Ecol. 1999,8:443-451
Fushimi H, Komatsu K, Isobe M, Namba T. Application of PCR-RFLP and MASA Analyses on 18S Ribosomal RNA Gene Sequence for the Identification of Three Ginseing Drugs. Biol Pharm Bull. 1997 ,20 : 765
Gillan R, Cole MD, Linacre A, Thorpe JW, Watson ND. Comparison of Cannabis Sativa by Radom Amplification of Polymorphic DNA (RAPD) and HPLC of Cannabinoides:A preliminary Study. Sci Justice.1995,35(3):169-177
Gupta M, Chyi Y S, Romers-Severson J et al. Amplification of DNA Markers From Evolutionarily Diverse Fenomers Single Primers of Simple-Sequence Repeats. Theor Appl Genet.1994,89:998-1006
Kohjyouma M, Nakajima S, Namera A, Shimizu R, Mizukami H, Kohda H. Random Amplification Polymorphic DNA Analysis and Variation of Essential Oil Components of Atractylodes Plants.Biol Pharm Bull.1997,20: 502
Mizukami H, Shimizu R, Kohda H, Kohjyouma M, Kawanishi F, Hiraoka N. Restriction
Fragments Length Polymorphisms of rDNA and Variation of Esssential Oil Components in Atractylodes Plants. Biol Pharm Bull.1996, 19:577
Noumi T, Mosher M E, Natori S, et al. A Pheny Lalanine for Serine Substitution in the B Subunit of Escherichina Coli F, Atpase Affects Dependence of Its Activity Ondivalentcations. Journal of Biological chemistry.1984, 259:10071-10076
Ngan F, Shaw P, But P, Wang J. Molecular Authentication of Panax Species. Phytochemistry.1999,50:787
Panda S, Martin JP, Aguinagalde I. Chloroplast and Nuclear DNA Studies in a Few Members of The Brassica Oleracea L. Group Using PCR-RFLP and ISSR-PCR Markers: a Population Genetic Analysis. Theor Appl Genet. 2003, 106(6):1122-1128
Parant I, Michelmore RW.Develpoment of PCR Based Markers Linked to Downy Mildew Resistance in Lettuce. Theor Appl Genet.1993,85(8):985-993
Raina SN, Rani V, Kojima T, Ogihara Y, Singh KP, Devarumath RM. RAPD and ISSR Fingerprints as Useful Genetic Markers for Analysis of Genetic Diversity, Varietal Identification, and Phylogenetic Relationships in Peanut (Archis hypogaea) Cultivars and Wild Species. Genome.2001, 44:763-772
Romero C, Pedryc A, Munoz V, Llacer G, Badenes ML. Genetic Diversity of Different Apricot Geographical Groups Determined by SSR Markers. Genome.2003, 46(2):244-252
Welsh J, McClenlland M.Fingerprinting Genome Using PCR with Arbitrary Primers. Nucl Acids Res.1990, 18(24): 7213-7218
Wen J, Zimmer E A. Phylogeny and Biogeography of Panax L. (the Ginseng Genus, Araliaceae): Inferences from ITS Sequences of Nuclear Ribosomal DNA. Molec Phylogen Evol. 1996,6(2):167-177
Welsh J, McClelland M.. Fingerprinting Genomes Using PCR with Arbitrary Primers. Nucl.Acids Res., 1990,18(24): 7213-7218
Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA Polymorphisms Amplified by Arbitrary Primers are Useful as Genetic Markers. Nucl.Acids Res. 1990,18(22):6531-6535
Yamazaki M, Sato A, Saito K, et al. Molecular Phylogeny Based on RFLP and Its Relation with Alkaloid Patterns in Lupinus plants. Biol Pharm Bull.1993,16:1182
Zabeau M et al. European Patent. 0535 858A1, 1993-03-31
Zietkiewicz E, Rafalski A, Labuda D. Genome Fingerprinting by Simple Sequence Repeat (SSR)-Anchored Polymerase Chain Reaction Amplification. Genomica.1994, 18:
1524-1528
Zhebentyayeva N, Reighard L, Gorina M, Abbott G. Simple Sequence Repeat (SSR) Analysis for Assessment of Genetic Variability in Apricot Germplasm. Theor Appl Genet.2003, 106(3):435-444
曹晖，刘玉萍.DNA 分析技术在中药质量研究中的应用.中国药学杂志.1998，38（5）： 112-115
曹进，饶毅，沈群，王义明，罗国安.中药指纹图谱及其建立原则.中药新药与临床药理.2001，12（3）：200-203
陈月琴，届良鹄，周惠.杜仲原植物25SrDNA5,端序列分析及其分子识别.中国中药杂志.1998，23(12)：707-710
陈永久，张亚平.随机扩增多态DNA影响因素的研究.动物学研究.1997，18（2）：221-227
方宣钧，吴为人，唐纪良.作物DNA标记辅助育种.科学出版社.2002：15
陈桂信，潘东明，吕柳新，赖钟雄，王凤华.DNA分子标记技术及其在热带亚热带果树上的应用.江西农业大学学报(自然科学版).2002，24（2）：176-182
窦忠健,杨勤福,李祎. 中药防风及其混伪品的鉴别.河南中医药学刊. 1999,14(4)：15-16
郭宝林，戴波.中药材DNA分子标记研究的技术问题.中草药.2002，33（2）：178-180
郭军，寿森炎，卢钢，吴韩英，黄锡志.DNA分子标记技术在品种鉴定和纯度分析上的应用.种子科技.2000，4：215-217
葛卫红. 荆芥、防风挥发油抗炎作用及其机理研究(博士论文).成都中医药大学.2002-04-01,2-835
胡宝忠，刘娣，胡国富，张阿英，姜述君.中国紫花苜蓿地方品种随机扩增多态DNA的研究.植物生态学报.2000，24（6）：697-701
胡国富.用微卫星及ISSR法对羊草（Aneurolepidium chinese (Trin)Kitag）遗传多样性的研究.东北农业大学硕士论文.2002年5月：8
黄中文，王莹，辛慧，刘明久.RFLP原理及在作物基因定位和育种中的应用.河南职技师院学报.2002，28（4）：24-27
黄璐琦.分子生药学.北京医科大学出版社.2000：29
赖小平,陈建南,刘中秋,周华. 高效毛细管电泳及其在中药质量分析的应用.中药新药与临床药理.1998，9（3）：186～187
罗国安，王义明.中药指纹图谱的分类和发展.中国新药杂志.2002，11（1）：46-51
李军,郭晏海,党琳.几种药用植物DNA提取与纯化方法的比较.陕西中医学院学报.2002，25(5)：72-73
孟祥才,江延辉,娄志红. 栽培防风质量分析.特产研究.2000,4：43
孙岳.防风DNA指纹分析及质量相关研究（博士论文）.东北农业大学.2003
王关林，方宏筠.植物基因工程原理与技术.科学出版社.1998：598
王培训，黄丰，周联，曹柳英，梁瑞燕.植物中药材总DNA提取方法的比较.中药新
药与临床药理.1999，10（1）：18-22
王天松.毛细管电泳与中草药指纹图谱.中成药.2000，22（6）：397-399
王志林，赵树进，吴新荣.分子标记技术及其发展.生命的化学.2002，21（4）：39-42
吴俊，魏钦平，束怀瑞，徐凯，钟家煌.分子标记及其在果树种质资源研究中的应用.安徽农业大学学报.2002，29（2）:158-162
吴田.中医药现代化与中药指纹图谱分析.中国中西医结合杂志.2002，22（9）：645-646
吴兴奎，吕继红.浅谈影响中药疗效的因素.河南中医药学刊.1999，14（6）：52-53
肖小河，刘峰群，袁海龙，贺承山，史成和.中药DNA分子标识鉴定研究进展.中草药.2000，31（8）：561-565
肖永庆,杨滨,姚三桃,李文,李丽,黄璐琦,薛宝云.?防风质量标准的研究.中国中药杂志.2001 26(3)：185～187
肖永庆,李丽,杨滨,黄璐琦.防风化学成分研究.中国实验方剂学杂志.2002 S1:117-119
薛宝云，李文，李丽，肖永庆.防风色原酮甙类成分的药理活性研究.中国实验方剂学杂志.2001,S1:297-299
邢俊波，刘云.高效毛细管电泳技术近年在中药分析中的应用.解放军药学学报.2002，18（4）：220-222
邢旺兴，宓鹤鸣，田倩.RAPD技术在生药学研究中的应用.解放军药学学报.1999，15（3）：25-28
杨滨,黄璐琦,姚三桃,肖永庆.防风类药材高效液相色谱鉴别.中国药学杂志. 1999,34(10):658-660
杨剑.影响中成药质量的因素及对策.中国医院药学杂志.1998，18（12）：561-562
尹茶，吴玉田.高效毛细管电泳技术及其在中药分析中的应用.药物分析杂志.1999，19（3）：209-217
尤勇.应用RAPD标记对杉木属两个种遗传多态性的研究.聊城师院学报（自然科学版）.1998，11（4）：72-74
于清峰，倪坤仪，王志群.药物中有关物质的色谱法分析.药学进展.2000，24（5）：270-274
张宇红，周常文.PCR-SSCP分析技术的研究进展及应用前景.中国优生与遗传杂志.2002，10（5）：126-127
张云武，陈永久，沈发荣，杨跃雄，杨大荣，张亚平.滇西北地区冬虫夏草和阔孢虫草的遗传分化研究.菌物系统.1999，18（2）：176-183
赵凌侠，李景富，唐克轩，许向阳，开国银.番茄属（Lycopersicon）RAPD标记遗传分析.复旦学报（自然科学版）.2002，41（2）：222-226
张贵君,张艳波,李影. 我国生药防风近10年的研究概况.时珍国医国药.1997,8(1): 73-75
张宇红，周常文.PCR-SSCP分析技术的研究进展及应用前景.中国优生与遗传杂志.2002，10（5）：126-127
张云武，陈永久，沈发荣，杨跃雄，杨大荣，张亚平.滇西北地区冬虫夏草和阔孢虫草的遗传分化研究.菌物系统.1999，18（2）：176-183
赵凌侠，李景富，唐克轩，许向阳，开国银.番茄属（Lycopersicon）RAPD标记遗传分析.复旦学报（自然科学版）.2002，41（2）：222-226
Anthony F, Combes C, Astorga C, Bertrand B, Graziosi G, Lashermes P. The Origin of Cultivated Coffea Arabica L.Varieties Revealed by AFLP and SSR Markers. Theor Appl Genet. 2002, 104(5):894-900
Blair M W.Panaud O, McCouch S R. Inter-Simple Sepuence Repeat (ISSR)Amplification for Analysis of Microsatellite Motif Frequency and Fingerprinting in Rice (Oryza sativaL.) Theor Appl Genet. 1999,98:780~792.
Cekic C,Battey N H, Wilkinson M J.The Potential of ISSR-PCR Primer-Pair Combinations for Genetic Llinkage Analysis Using the Seasonal Flowering Llocus in Fragaria as a Model. Theor Appl Genet.2001,103:540-546
Cheung KS, Kwan HS, But PP, Shaw PC. Phar-Macognostical Identification of American and Oriedtal Ginseng Roots by Genomic Fingerprinting Using Arbitrarily Primed Polymerase Chain Reaction ( AP- PCR) . J Ethnopharmacol. 1994,42:67
Devos K M, M D Gale. The Use of Random Amplified Polymorphis DNA Markers in Wheat. TAG.1992,82:567-572
Dirlewanger E, Pronier V, Parvery C. Genetic Linkage Map of Peach (Prunus persica L)Using Morphological and Molecular Markers. Theor Appl Genet. 1998,97:888-896
Ellsworth D L, Rittenhpuse K D, Honeycutt R L. Artifactual Variation in Randomly Amplified Ploymorphic DNA Banding Patterns. Biotechniques.1993, 14:214-217
Esselman E J, Li J Q, Crawford D J, et al. Clonal Diversity in the Rare Calamagrostis Porterissp. Insperata (Poaceae): Comparative Results for Allozymes and RAPD and ISSR Markers. Mol Ecol. 1999,8:443-451
Fushimi H, Komatsu K, Isobe M, Namba T. Application of PCR-RFLP and MASA Analyses on 18S Ribosomal RNA Gene Sequence for the Identification of Three Ginseing Drugs. Biol Pharm Bull. 1997 ,20 : 765
Gillan R, Cole MD, Linacre A, Thorpe JW, Watson ND. Comparison of Cannabis Sativa by Radom Amplification of Polymorphic DNA (RAPD) and HPLC of Cannabinoides:A preliminary Study. Sci Justice.1995,35(3):169-177
Gupta M, Chyi Y S, Romers-Severson J et al. Amplification of DNA Markers From Evolutionarily Diverse Fenomers Single Primers of Simple-Sequence Repeats. Theor Appl Genet.1994,89:998-1006
Kohjyouma M, Nakajima S, Namera A, Shimizu R, Mizukami H, Kohda H. Random Amplification Polymorphic DNA Analysis and Variation of Essential Oil Components of Atractylodes Plants.Biol Pharm Bull.1997,20: 502
Mizukami H, Shimizu R, Kohda H, Kohjyouma M, Kawanishi F, Hiraoka N. Restriction
Fragments Length Polymorphisms of rDNA and Variation of Esssential Oil Components in Atractylodes Plants. Biol Pharm Bull.1996, 19:577
Noumi T, Mosher M E, Natori S, et al. A Pheny Lalanine for Serine Substitution in the B Subunit of Escherichina Coli F, Atpase Affects Dependence of Its Activity Ondivalentcations. Journal of Biological chemistry.1984, 259:10071-10076
Ngan F, Shaw P, But P, Wang J. Molecular Authentication of Panax Species. Phytochemistry.1999,50:787
Panda S, Martin JP, Aguinagalde I. Chloroplast and Nuclear DNA Studies in a Few Members of The Brassica Oleracea L. Group Using PCR-RFLP and ISSR-PCR Markers: a Population Genetic Analysis. Theor Appl Genet. 2003, 106(6):1122-1128
Parant I, Michelmore RW.Develpoment of PCR Based Markers Linked to Downy Mildew Resistance in Lettuce. Theor Appl Genet.1993,85(8):985-993
Raina SN, Rani V, Kojima T, Ogihara Y, Singh KP, Devarumath RM. RAPD and ISSR Fingerprints as Useful Genetic Markers for Analysis of Genetic Diversity, Varietal Identification, and Phylogenetic Relationships in Peanut (Archis hypogaea) Cultivars and Wild Species. Genome.2001, 44:763-772
Romero C, Pedryc A, Munoz V, Llacer G, Badenes ML. Genetic Diversity of Different Apricot Geographical Groups Determined by SSR Markers. Genome.2003, 46(2):244-252
Welsh J, McClenlland M.Fingerprinting Genome Using PCR with Arbitrary Primers. Nucl Acids Res.1990, 18(24): 7213-7218
Wen J, Zimmer E A. Phylogeny and Biogeography of Panax L. (the Ginseng Genus, Araliaceae): Inferences from ITS Sequences of Nuclear Ribosomal DNA. Molec Phylogen Evol. 1996,6(2):167-177
Welsh J, McClelland M.. Fingerprinting Genomes Using PCR with Arbitrary Primers. Nucl.Acids Res., 1990,18(24): 7213-7218
Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA Polymorphisms Amplified by Arbitrary Primers are Useful as Genetic Markers. Nucl.Acids Res. 1990,18(22):6531-6535
Yamazaki M, Sato A, Saito K, et al. Molecular Phylogeny Based on RFLP and Its Relation with Alkaloid Patterns in Lupinus plants. Biol Pharm Bull.1993,16:1182
Zabeau M et al. European Patent. 0535 858A1, 1993-03-31
Zietkiewicz E, Rafalski A, Labuda D. Genome Fingerprinting by Simple Sequence Repeat (SSR)-Anchored Polymerase Chain Reaction Amplification. Genomica.1994, 18:
1524-1528
Zhebentyayeva N, Reighard L, Gorina M, Abbott G. Simple Sequence Repeat (SSR) Analysis for Assessment of Genetic Variability in Apricot Germplasm. Theor Appl Genet.2003, 106(3):435-444