丹参种质资源鉴定及遗传多样性研究
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摘要
鼠尾草属是唇形科中最大的属,全球约有1050种,在热带和温带都有分布,我有78种24变种8变型,各地均有分布,西南地区是最集中也是种类最丰富的地区。本属药用资源丰富,有记载的可作药用的有36种(含变种和变型),其中丹参已被《中华人民共和国药典》收载,是我国传统大宗中药材之一。随着丹参需求量的剧增,药材市场经常会以不同种类的植物来源充当药用丹参销售,严重影响丹参药材的临床应用与研究开发,建立科学可靠、快速简单的鉴定体系显得极为重要。近年来丹参品质退化严重,没有优良的种质就不可能有优良的品种,而了解物种的遗传基础及物种间的亲缘关系是合理有效利用种质资源的基础与前提。因此本研究利用丹参及其近缘野生种资源,从表观形态、显微形态、细胞染色体形态和分子生物学分析等方面开展丹参种质资源的鉴定与亲缘关系探讨,分析种内与种间遗传多样性,揭示其遗传基础丰富程度、各居群的遗传差异和遗传距离,为丹参种质资源的鉴定及合理利用提供依据,为进一步的深入研究和遗传改良奠定良好的基础。主要研究结果如下：
(1)通过野外资源的调查与收集,结合标本资料,对四川鼠尾草属植物31个分类群的18个主要生物性状进行比较研究,分析物种表观形态的遗传多样性水平。数量性状研究结果显示叶片部位变异较大,变异程度最大的是叶柄长/叶片长,其次是叶柄长,叶片宽和叶片长,而最小的是花冠长/花萼长。质量形状方面,各生物性状都具有多种类型。主成分分析结果表明叶片宽、花下药室可否育、叶柄长/叶片长和主根是否明显是鼠尾草属植物的特征性状,并在此基础上进行聚类分析,R型聚类结果表明所考察的性状独立遗传,可作为分类的标准；Q型聚类将31个物种分为了4大类,与传统分类学中对该属植物的亚属分类结果基本相符,但柔毛荞麦地鼠尾草的系统地位有待进一步论证。进一步分析了物种间的亲缘关系,为四川鼠尾草属植物的系统分类、亲缘关系鉴定及种质资源利用等提供依据。
(2)采用石蜡切片法在光学显微镜下对7个省的丹参及云南鼠尾草、短唇鼠尾草、峨眉鼠尾草、甘西鼠尾草、三叶鼠尾草、黄鼠狼花、血盆草等14份材料的根横切片显微特征进行比较研究。结果显示研究材料在根部显微结构上存在许多共性,如次生皮层均较宽广,韧皮部较狭窄,木质部很发达,形成层一般都比较明显等。但在落皮层、木栓层厚度及木栓化程度、木质部导管多少、孔径大小及排列方式上存在不同程度的差异,这些差异及建立的分类检索表可以为鱼龙混杂的药材市场提供鉴别正品丹参药材的证据,也可以为鼠尾草属植物的鉴定提供依据。
(3)通过对不同产地丹参及近缘种植物共12份材料的花粉母细胞减数分裂过程中染色体形态和行为的观察,显示研究的所有物种染色体基数均为x=8,存在二倍体与四倍体两种倍性,除了甘西鼠尾草和短唇鼠尾草为四倍体以外,其余物种均为二倍体。不同物种在染色体构型上存在差异,鼠尾草属物种细胞染色体中,棒状二价体构型比例相对较高,环状二价体比例最高的黄花鼠尾草,表明其交换频率最高,揭示其种内变异程度相对较大,而种内变异最低的为陕西丹参。在河南丹参及短唇鼠尾草中各发现了一条B染色体的存在,异常的细胞分裂行为可能是导致河南丹参出现不育花药的原因之一。
(4)从70个RAPD随机引物中筛选出条带清晰、多态性明显、重复好的27个引物进行PCR扩增,鼠尾草属植物扩增后呈现出较为丰富的多态性,平均多态性带为7.67条,多态性比率为83.47%,显示该属物种遗传变异较高。根据不同引物的RAPD指纹图谱中条带的有无计算遗传相似性系数,可以对各样品进行区别。聚类分析显示样品分为四类,所有的丹参材料及其变型白花丹参共8份材料聚为一类,表明遗传多样性主要存在于种间,种内如丹参遗传距离相对较小。RAPD对丹参种质资源材料进行聚类的结果与传统形态学分类结果不完全一致,主要表现在峨眉鼠尾草与黄鼠狼花的系统地位存在分歧。
(5)本研究选取四个叶绿体基因或间隔区序列(matK,rbcL, trnL-F和psbA-trnH)以及核基因组ITS区(包括ITS1和ITS2)为候选DNA条形码,探讨不同序列组合在鼠尾草属物种鉴定过程中的有效性,结果显示候选的条形码都达不到动物中COX I基因的理想效果,相对来说单一片段中核基因组ITS1的鉴定率最高,可以达到81.48%,其次为叶绿体trnL-F间隔区序列,鉴定率为77.77%。三种片段组合中,trnL-F+ITS1鉴定率最高,为74.74%,并未超过两个片段单独使用时的鉴定率。在所考察的片段中,ITS1是唯一一个可以将所有丹参样本区分开的序列,我们推荐ITS1作为丹参及鼠尾草属植物的DNA条形码。
The genus Salvia L., the largest member of the family Lamiaceae, includes approximately1050species distributed in tropical and temperate areas of both old and new world. There are78species,24variations,8forms in China, distributing in most areas especially the southwest regions. Among the large number of Salvia in China,36accessions (including variations and forms) have the medical value. As one of the famous medicinal materials, Salvia miltiorrhiza Bunge. has been recorded by "Pharmacopoeia of the Peoples Republic of China". With the rapid increasing of the demands in S. miltiorrhiza, different sources were sold as S. miltiorrhiza in the herbs markets, and which was affected seriously its clinical application and development. Therefore, it is extremely important to establish a scientific, reliable, quick and easy identification system for identification. In recent years, the quality of S. miltiorrhiza degraded severely. However, it is impossible to have a good variety without excellent germplasm, and the understanding of the genetic basis and genetic relationship among different species is the premise for exploiting these germplasm resources effectively. In this study, we studied the identification and relationship from superficial, micro, chromosome morphology and molecular biology by using S. miltiorrhiza and its wild relative resources as materials. The intraspecific and interspecific genetic diversity were analyzed to reveal the degree of genetic basis, genetic differences and genetic distance in different populations. The main aim of this research is to provide proofs for the identification and utilization of S. miltiorrhiza germplasm resources and lay a good foundation for further in-depth research and genetic improvement. The main results are shown as follows:
(1) Based on the investigation and collection of wild resources combined with specimen data,18morphological traits of the31taxa on the genus Salvia in Sichuan province were observed and measured. The genetic diversity was analyzed according to the apparent morphology. The results of the quantitative traits showed the varieties were significant in leaves and the most were petiole length/leaf length. The followed were petiole length, leaf width and leaf length. And the corolla length/calyx length was the least. About the quality characters, multiple types were appeared in each biological traits. Leaf length, fertility of anther chamber, petiole length/leaf length and number of years were the characteristic trait for Salvia based on the PCA analysis. And those traits were conducted by R cluster and Q cluster analysis. The result of R cluster analysis shows that18morphological traits are independent and could be taken as the standard for classification; the results of Q cluster analysis showed that the31taxa were gathered into4groups which are consistent with the traditional taxonomy on the genus. However, the system location of S. kiaoemetiemsis f. pubescens needs more new data to define. According to the results, the genetic relationships among these taxa are analyzed which can give evidences for system classification, identification of genetic relationships and utilization of germplasm resources on Salvia in Sichuan.
(2) The cross sections of14materials, containing S. miltiorrhiza from seven provinces, S. brevilabra, S. omeiana, S. cavaleriei var. simplicifolia, S. tricuspis, S. trijuga, S. yunnanensis and S. przewalskii were obtained by using paraffin sectioning and the optical microscope was used to characterize the structure. The results showed that there was much commonness in the materials studied on the microscopic structure of roots, such as wide secondary cortex, narrow phloem, well-developed xylem, obvious cambium and so on. But the differences exist in rhytidome, cork layer thickness and suberification degree, number and size of the canal in xylem and also the tactic mode. These differences and the build search of transverse section features on the roots could be as the anatomical evidences for the identification of the S. miltiorrhiza from the mixed medicinal markets, and also may be considered to be the main features for the classification of Salvia.
(3) Observations were made on chromosome morphology and behavior during meiosis of PMCs (pollen mother cells) for different origins of S. miltiorrhiza and its related species in the genus Salvia. Results show that the basic chromosome number is x=8and there were diploid and tetraploid in the genus Salvia. According to the chromosomes pairing in metaphase I, except that S. przewalskii and S. brevilabra are tetraploids, the others are diplonts. The chromosome configurations were different in each sample, and the rod bivalents occupied a relatively high proportion compared with the ring bivalents. With the highest proportion of ring bivalents, the switching frequency and intraspecific variation were the highest in S. flava, and the lowest was S. miltiorrhiza from Shanxi. There was a B chromosome in S. brevilabra and S. miltiorrhiza from Henan respectively. Additionally, the abnormal behaviors of the chromosomes in meiosis observed may have effects on the development of pollen grains in S. miltiorrhiza from Henan.
(4) Among70random primers examined,27randomly amplified polymorphic DNA (RAPD) primers produced248extending and repeatable bands, of which207bands (83.47%) were polymorphic and the average number of Polymorphic bands was7.67. All of the data revealed abundant genetic diversity in the genus of Salvia. According to the genetic similarity coefficient obtained from RAPD fingerprinting for each primer, we can distinguish these samples.18taxa were clustered into four groups based on RAPD markers and all S. miltiorrhiza and S. miltiorrhiza f. alba were in a cluster. The results showed that there were slighter genetic diversity and narrower genetic backgrounds within intra-species, and we can easily distinguish S. miltiorrhiza obviously from the plenty of Salvia species by RAPD. The clustering results based on RAPD were inconsistent with traditional morphological classification results, and the main differences were in the system status of S. omeiana and S. tricuspis.
(5) An effective DNA marker for authenticating the genus Salvia was screened using seven DNA regions (rbcL, matK, trnL-F, and psbA-trnH from the chloroplast genome, and ITS, ITS1, and ITS2from the nuclear genome) and three combinations (rbcL+matK, psbA-trnH+ITS1, and trnL-F+ITS1). However, the result did not show ideal target as COX I in animals. The discriminatory capabilities of these regions were evaluated in terms of identification efficiency via a tree-based method. ITS1was superior to the other marker for discriminating between species, with an accuracy of81.48%, and followed the trnL-F with an accuracy of77.77%. Among the three combinations, the highest was trnL-F+ITS1with an accuracy of74.74%and the three combinations did not increase species discrimination. In addition, in the examined fragments, ITS1was the only one sequence which can distinguish all S. miltiorrhiza samples. Finally, we recommend ITS1as the DNA barcoding for identifying Salvia species, especially S. miltiorrhiza.
(1)通过野外资源的调查与收集,结合标本资料,对四川鼠尾草属植物31个分类群的18个主要生物性状进行比较研究,分析物种表观形态的遗传多样性水平。数量性状研究结果显示叶片部位变异较大,变异程度最大的是叶柄长/叶片长,其次是叶柄长,叶片宽和叶片长,而最小的是花冠长/花萼长。质量形状方面,各生物性状都具有多种类型。主成分分析结果表明叶片宽、花下药室可否育、叶柄长/叶片长和主根是否明显是鼠尾草属植物的特征性状,并在此基础上进行聚类分析,R型聚类结果表明所考察的性状独立遗传,可作为分类的标准；Q型聚类将31个物种分为了4大类,与传统分类学中对该属植物的亚属分类结果基本相符,但柔毛荞麦地鼠尾草的系统地位有待进一步论证。进一步分析了物种间的亲缘关系,为四川鼠尾草属植物的系统分类、亲缘关系鉴定及种质资源利用等提供依据。
(2)采用石蜡切片法在光学显微镜下对7个省的丹参及云南鼠尾草、短唇鼠尾草、峨眉鼠尾草、甘西鼠尾草、三叶鼠尾草、黄鼠狼花、血盆草等14份材料的根横切片显微特征进行比较研究。结果显示研究材料在根部显微结构上存在许多共性,如次生皮层均较宽广,韧皮部较狭窄,木质部很发达,形成层一般都比较明显等。但在落皮层、木栓层厚度及木栓化程度、木质部导管多少、孔径大小及排列方式上存在不同程度的差异,这些差异及建立的分类检索表可以为鱼龙混杂的药材市场提供鉴别正品丹参药材的证据,也可以为鼠尾草属植物的鉴定提供依据。
(3)通过对不同产地丹参及近缘种植物共12份材料的花粉母细胞减数分裂过程中染色体形态和行为的观察,显示研究的所有物种染色体基数均为x=8,存在二倍体与四倍体两种倍性,除了甘西鼠尾草和短唇鼠尾草为四倍体以外,其余物种均为二倍体。不同物种在染色体构型上存在差异,鼠尾草属物种细胞染色体中,棒状二价体构型比例相对较高,环状二价体比例最高的黄花鼠尾草,表明其交换频率最高,揭示其种内变异程度相对较大,而种内变异最低的为陕西丹参。在河南丹参及短唇鼠尾草中各发现了一条B染色体的存在,异常的细胞分裂行为可能是导致河南丹参出现不育花药的原因之一。
(4)从70个RAPD随机引物中筛选出条带清晰、多态性明显、重复好的27个引物进行PCR扩增,鼠尾草属植物扩增后呈现出较为丰富的多态性,平均多态性带为7.67条,多态性比率为83.47%,显示该属物种遗传变异较高。根据不同引物的RAPD指纹图谱中条带的有无计算遗传相似性系数,可以对各样品进行区别。聚类分析显示样品分为四类,所有的丹参材料及其变型白花丹参共8份材料聚为一类,表明遗传多样性主要存在于种间,种内如丹参遗传距离相对较小。RAPD对丹参种质资源材料进行聚类的结果与传统形态学分类结果不完全一致,主要表现在峨眉鼠尾草与黄鼠狼花的系统地位存在分歧。
(5)本研究选取四个叶绿体基因或间隔区序列(matK,rbcL, trnL-F和psbA-trnH)以及核基因组ITS区(包括ITS1和ITS2)为候选DNA条形码,探讨不同序列组合在鼠尾草属物种鉴定过程中的有效性,结果显示候选的条形码都达不到动物中COX I基因的理想效果,相对来说单一片段中核基因组ITS1的鉴定率最高,可以达到81.48%,其次为叶绿体trnL-F间隔区序列,鉴定率为77.77%。三种片段组合中,trnL-F+ITS1鉴定率最高,为74.74%,并未超过两个片段单独使用时的鉴定率。在所考察的片段中,ITS1是唯一一个可以将所有丹参样本区分开的序列,我们推荐ITS1作为丹参及鼠尾草属植物的DNA条形码。
The genus Salvia L., the largest member of the family Lamiaceae, includes approximately1050species distributed in tropical and temperate areas of both old and new world. There are78species,24variations,8forms in China, distributing in most areas especially the southwest regions. Among the large number of Salvia in China,36accessions (including variations and forms) have the medical value. As one of the famous medicinal materials, Salvia miltiorrhiza Bunge. has been recorded by "Pharmacopoeia of the Peoples Republic of China". With the rapid increasing of the demands in S. miltiorrhiza, different sources were sold as S. miltiorrhiza in the herbs markets, and which was affected seriously its clinical application and development. Therefore, it is extremely important to establish a scientific, reliable, quick and easy identification system for identification. In recent years, the quality of S. miltiorrhiza degraded severely. However, it is impossible to have a good variety without excellent germplasm, and the understanding of the genetic basis and genetic relationship among different species is the premise for exploiting these germplasm resources effectively. In this study, we studied the identification and relationship from superficial, micro, chromosome morphology and molecular biology by using S. miltiorrhiza and its wild relative resources as materials. The intraspecific and interspecific genetic diversity were analyzed to reveal the degree of genetic basis, genetic differences and genetic distance in different populations. The main aim of this research is to provide proofs for the identification and utilization of S. miltiorrhiza germplasm resources and lay a good foundation for further in-depth research and genetic improvement. The main results are shown as follows:
(1) Based on the investigation and collection of wild resources combined with specimen data,18morphological traits of the31taxa on the genus Salvia in Sichuan province were observed and measured. The genetic diversity was analyzed according to the apparent morphology. The results of the quantitative traits showed the varieties were significant in leaves and the most were petiole length/leaf length. The followed were petiole length, leaf width and leaf length. And the corolla length/calyx length was the least. About the quality characters, multiple types were appeared in each biological traits. Leaf length, fertility of anther chamber, petiole length/leaf length and number of years were the characteristic trait for Salvia based on the PCA analysis. And those traits were conducted by R cluster and Q cluster analysis. The result of R cluster analysis shows that18morphological traits are independent and could be taken as the standard for classification; the results of Q cluster analysis showed that the31taxa were gathered into4groups which are consistent with the traditional taxonomy on the genus. However, the system location of S. kiaoemetiemsis f. pubescens needs more new data to define. According to the results, the genetic relationships among these taxa are analyzed which can give evidences for system classification, identification of genetic relationships and utilization of germplasm resources on Salvia in Sichuan.
(2) The cross sections of14materials, containing S. miltiorrhiza from seven provinces, S. brevilabra, S. omeiana, S. cavaleriei var. simplicifolia, S. tricuspis, S. trijuga, S. yunnanensis and S. przewalskii were obtained by using paraffin sectioning and the optical microscope was used to characterize the structure. The results showed that there was much commonness in the materials studied on the microscopic structure of roots, such as wide secondary cortex, narrow phloem, well-developed xylem, obvious cambium and so on. But the differences exist in rhytidome, cork layer thickness and suberification degree, number and size of the canal in xylem and also the tactic mode. These differences and the build search of transverse section features on the roots could be as the anatomical evidences for the identification of the S. miltiorrhiza from the mixed medicinal markets, and also may be considered to be the main features for the classification of Salvia.
(3) Observations were made on chromosome morphology and behavior during meiosis of PMCs (pollen mother cells) for different origins of S. miltiorrhiza and its related species in the genus Salvia. Results show that the basic chromosome number is x=8and there were diploid and tetraploid in the genus Salvia. According to the chromosomes pairing in metaphase I, except that S. przewalskii and S. brevilabra are tetraploids, the others are diplonts. The chromosome configurations were different in each sample, and the rod bivalents occupied a relatively high proportion compared with the ring bivalents. With the highest proportion of ring bivalents, the switching frequency and intraspecific variation were the highest in S. flava, and the lowest was S. miltiorrhiza from Shanxi. There was a B chromosome in S. brevilabra and S. miltiorrhiza from Henan respectively. Additionally, the abnormal behaviors of the chromosomes in meiosis observed may have effects on the development of pollen grains in S. miltiorrhiza from Henan.
(4) Among70random primers examined,27randomly amplified polymorphic DNA (RAPD) primers produced248extending and repeatable bands, of which207bands (83.47%) were polymorphic and the average number of Polymorphic bands was7.67. All of the data revealed abundant genetic diversity in the genus of Salvia. According to the genetic similarity coefficient obtained from RAPD fingerprinting for each primer, we can distinguish these samples.18taxa were clustered into four groups based on RAPD markers and all S. miltiorrhiza and S. miltiorrhiza f. alba were in a cluster. The results showed that there were slighter genetic diversity and narrower genetic backgrounds within intra-species, and we can easily distinguish S. miltiorrhiza obviously from the plenty of Salvia species by RAPD. The clustering results based on RAPD were inconsistent with traditional morphological classification results, and the main differences were in the system status of S. omeiana and S. tricuspis.
(5) An effective DNA marker for authenticating the genus Salvia was screened using seven DNA regions (rbcL, matK, trnL-F, and psbA-trnH from the chloroplast genome, and ITS, ITS1, and ITS2from the nuclear genome) and three combinations (rbcL+matK, psbA-trnH+ITS1, and trnL-F+ITS1). However, the result did not show ideal target as COX I in animals. The discriminatory capabilities of these regions were evaluated in terms of identification efficiency via a tree-based method. ITS1was superior to the other marker for discriminating between species, with an accuracy of81.48%, and followed the trnL-F with an accuracy of77.77%. Among the three combinations, the highest was trnL-F+ITS1with an accuracy of74.74%and the three combinations did not increase species discrimination. In addition, in the examined fragments, ITS1was the only one sequence which can distinguish all S. miltiorrhiza samples. Finally, we recommend ITS1as the DNA barcoding for identifying Salvia species, especially S. miltiorrhiza.
引文
[1]曹家树,秦岭.园艺植物种质资源学.北京：中国农业出版社.2005：1
[2]董玉琛.我国作物种质资源研究的现状与展望.中国农业科技导报,1999,2：36-40.
[3]王国山,顾恒琴,侯忠.对作物品种资源工作的认识与思考.国外农学-杂粮作物,1997,3：34-36.
[4]张华峰,季彪俊,植物种质资源研究概论.宜春学院学报,2005,27(2)：92-95.
[5]梁艳荣.大葱种质资源鉴定与其生理特性研究.博士学位论文.内蒙古农业大学,2008.
[6]Tautz D, Arctander P, Minelli A, et al. DNA points the way ahead in taxonomy. Nature,2002, 418(6897):479.
[7]Hebert PDN, Cywinska A, Ball SL. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B:Biological Sciences,2003, 270(1512):313-321.
[8]Hebert PDN, Penton EH., Burns JM, et al. Ten species in one:DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United States of America,2004,101(41):14812-14817.
[9]Hebert PDN, Stoeckle MY, Zemlak TS, et al. Identification of birds through DNA barcodes. PLoS biology,2004,2(10):e312.
[10]Ward RD, Zemlak TS, Innes BH, et al. DNA barcoding Australia's fish species. Philosophical Transactions of the Royal Society B:Biological Sciences,2005,360(1462): 1847-1857.
[11]Hajibabaei M, Janzen DH, Burns JM, et al. DNA barcodes distinguish species of tropical Lepidoptera. Proceedings of the National Academy of Sciences of the United States of America,2006,103(4):968-971.
[12]Kerr KCR, Stoeckle MY, Dove CJ, et al. Comprehensive DNA barcode coverage of North American birds. Molecular Ecology Notes,2007,7(4):535-543.
[13]Chase MW, Cowan RS, Hollingsworth PM, et al. A proposal for a standardised protocol to barcode all land plants. Taxon,2007,56(2):295-299.
[14]Kress WJ, Erickson DL. A two-locus global DNA barcode for land plants:the coding rbcL gene complements the non-coding trnH-psbA spacer region. PloS one,2007,2(6):e508.
[15]Kim KJ, Lee HL. Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants. DNA research,2004,11(4):247-261.
[16]Pennisi E. A barcode for plants. Science,2007,318:190-191.
[17]Hollingsworth ML, Clark AA, Forrest LL, et al. Selecting barcoding loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants. Molecular Ecology Resources,2009,9(2):439-457.
[18]刘宇婧,刘越,黄耀江,等.植物DNA条形码技术的发展及应用.植物资源与环境学报,2011,20(1)：74-82.
[19]闫化学,于杰.DNA条形码技术在植物中的研究现状.植物学报,2010,45(1)：102-108.
[20]CBOL Plant Working Group. A DNA barcode for land plants. Proceedings of the National Academy of Sciences,2009,106(31):12794-12797.
[21]陈士林,宋经元,姚辉,等.药用植物DNA条形码鉴定策略及关键技术分析.中国天然药物,2009,7(5)：322-327.
[22]Meier R, Zhang G, Ali F. The use of mean instead of smallest interspecific distances exaggerates the size of the "barcoding gap" and leads to misidentification. Systematic Biology,2008,57(5):809-813.
[23]宁淑萍,颜海飞,郝刚,等.植物DNA条形码研究进展.生物多样性,2008,16(5)：417-425.
[24]Wolfe KH, Li WH., Sharp PM. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proceedings of the National Academy of Sciences,1987,84(24):9054-9058.
[25]Chase MW, Salamin N, Wilkinson M, et al. Land plants and DNA barcodes:short-term and long-term goals. Philosophical Transactions of the Royal Society B:Biological Sciences, 2005,360(1462):1889-1895.
[26]Newmaster SG, Fazekas AJ, Ragupathy S. DNA barcoding in land plants:evaluation of rbcL in a multigene tiered approach. Botany,2006,84(3):335-341.
[27]Kress WJ, Wurdack KJ, Zimmer EA, et al. Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences of the United States of America,2005, 102(23):8369-8374.
[28]Presting GG. Identification of conserved regions in the plastid genome:implications for DNA barcoding and biological function. Botany,2006,84(9):1434-1443.
[29]Taberlet P, Coissac E, Pompanon F, et al. Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding. Nucleic Acids Research,2007,35(3):e14-e14.
[30]唐建阳,周先治.植物DNA条形码研究现状及应用前景.中国农学通报,2009,25(24)：35-43.
[31]张凤春,张文国.“生物多样性”释义(上)：L生物多样性的概念机现状.环境保护,2010.1(9)：45-48.
[32]32.王洪新,胡志昂.植物繁育系统、遗传结构和遗传多样性的保护.生物多样性,1996,4(2)：92-96.
[33]33.钱迎倩,马克平.生物多样性研究的原理与方法.北京：中国科学技术出版社.1994：13-36
[34]沈浩,刘登义.遗传多样性概述.生物学杂志,2001,18(3)：5-8.
[35]陈灵芝,中国的生物多样性-现状及其保护对策.北京：科学出版社.1993：99-113.
[36]冯夏莲,何承忠,张志毅,等.植物遗传多样性研究方法概述.西南林学院学报,2006,26(1)：69-74.
[37]国家药典委员会.中华人民共和国药典(第二部).北京：化学工业出版社.2005：52-53.
[38]李家实.中药鉴定学(第四版).上海：科学技术出版社.1995：169.
[39]李玉梅,陈立忠,李影.近年来丹参的药理研究进展.近黑龙江医药,1996,9(4)：234-235.
[40]柴瑞震.丹参的药理研究近况.中国中医药科技,2003,10(6)：390-392.
[41]郭宝林.丹参种质资源研究进展.中国中药,2002,27(7)：492-495.
[42]中国药材公司.中国中药资源.北京：科学出版社.1995：31-33.
[43]张力文.川丹参新品系区域试验及近缘种植物光合特性研究.硕士学位论文.四川农业大学,2011.
[44]中国科学院中国植物志编委会.中国植物志(第六十六卷).北京：科学出版社.1977：79-194.
[45]钟国成.四川省中江县丹参品系综合评价.硕士学位论文.四川农业大学,2010.
[46]杨在君,缪金城,张利,等.四川和重庆鼠尾草属植物的种类及药用资源.时珍国医国药,2008,19(4)：904-908.
[47]肖小河,方清茂,夏文娟,等.药用鼠尾草属数值分类与丹参药材道地性.植物资源与环境,1997,6(2)：17-21.
[48]倪梁红,赵志礼.华东地区鼠尾草属药用植物资源.时珍国医国药,2010,21(10)：2653-2655.
[49]Farkas A, Papp N, Horvath G, et al. Anatomical and genetic differences between Salvia taxa. European Journal of Pharmaceutical Sciences,2007,32(1):S20.
[50]Kandemir N. The morphological, anatomical and karyological properties of endemic Salvia hypargeia Fich & Mey. (Lamiaceae) in Turkey. Pakistan Journal of Botanly,2003,35(2): 219-236.
[51]Baran P, OZDEMlR C. The morphological and anatomical characters of Salvia napifolia Jacq. in Turkey. Bangladesh Journal of Botany,2006,35(1):77-84.
[52]Kaya A, Goger F, Baser KHC. Morphological, anatomical and palynological characteristics of Salvia halophila endemic toTurkey. Nordic Journal of Botany,2007,25(5):351-358.
[53]马志刚,杨永建,谢錾,等.甘肃鼠尾草属五种植物根的生药鉴定.兰州医学院学报,1993,19(4)：212.
[54]李素芬,李金芩,李小娇,等.中国民族民间医药.戟叶鼠尾草的生药学研究,2008,17(7)：18-20.
[55]唐丽萍,梁晓源,韦群辉,等.滇丹参的生药学研究.云南中医学院学报,2003,26(1)：11-14.
[56]张利,杨在君,黄霞,等.丹参及四川鼠尾草属植物叶表皮微形态研究.四川大学学报(自然科学版),2008,45(3)：674-680.
[57]Corsi G, Bottega S. Glandular Hairs of Salvia officinalis:New Data on Morphology, Localization and Histochemistry in Relation to Function. Annals of Botany,1999,84(5): 657-664.
[58]郑宝江,于丽杰,邢怡,等.兰花鼠尾草(Salvia farinacea Benth.)两类腺毛发育过程中超微结构的研究.植物研究,2002,22(1)：23-25.
[59]Werker E. Function of essential oil-secreting glandular hairs in aromatic plants of the Lamiaceae--a view. Flavour and Fragrance Journal,1993,8:249-255.
[60]杨德奎,孙京田,王丙全.山东鼠尾草属花粉形态的研究及其在分类上的意义.山东科学,2003,16(1)：14-16.
[61]Stewart WS. Chromosome numbers of Californian Salvias. American Journal of Botany, 1939,26(9):730-732.
[62]Gill LS. Chromosome studies in Salvia (Labiatae):West-himalayan species. Cellular and Molecular Life Sciences,1971,27(5):596-598.
[63]Alberto C M, Sanso A, Xifresa CC. Chromosomal studies in species of Salvia (Lamiaceae) from Argentina. Botanical Journal of the Linnean Society,2003,141(4):483-490.
[64]Ozkan M. Karyotype analysis on two endemic Salvia L.(Lamiaceae) species in Turkey. International Journal of botany,2006,2(3):333-335.
[65]Ozdemir C, Senel G The Morphological Anatomical and Karyological Properties of S. sclerea. Turkish Journal of Botany,1999,23(1):7-18.
[66]Palomino G, Mercado P, Ramamoorthy TP. Chromosomes of Salvia subgenus Calosphace (Lamiaceae), a preliminary report. Cytologia,1986,51(2):381-386.
[67]蔡朝晖,高山林,郭蓓.丹参组织培养材料的染色体显微鉴定技术.中国医科大学学报,1993,24(1)：49-52.
[68]张兴国,王义明,罗国安,等.丹参品种资源特性的研究.中草药,2002,33(8)：742-747.
[69]Yang ZY, Gong X, Pan Y. Cytological study of six Salvia species (lamiaceae) from the Hengduanshan Mountains region of China. Caryologia,2004,57(4):360-366.
[70]赵红霞,张利,凡星,等.丹参、黄花鼠尾和雪山鼠尾染色体数目的研究.中国中药杂志,2006,31(22)：1847-1849.
[71]Yang ZJ, Zhang L, Zhao HX, et al. Chromosome numbers of some species of Salvia (Lamiaceae) from the Sichuan Province, China. Nordic Journal of Botany,2009,27(4): 287-291.
[72]徐红,王燕燕,魏丹红,等.不同产地丹参药材的ISSR分析与鉴别.中药新药与临床药理,2007,18(6)：454-457.
[73]李廷春,樊洪泓,高正良,等.丹参遗传多样性的SRAP标记分析.核农学报,2008,22(5)：576-580.
[74]郝岗平,孙立彦,史仁玖,等.山东产丹参遗传多样性的扩增片段长度多态性指纹分析.时珍国医国药,2007,18(7)：51-55.
[75]汪红,王强.丹参及鼠尾草属植物的rDNA ITS序列分析.中草药,2005,36(9)：1381-1385.
[76]王迎,李大辉,张英涛.鼠尾草属药用植物及其近缘种的ITS序列分析.药学学报,2007,42(12)：1309-1314.
[77]Skoula M, Hilali I E, Makris A M. Evaluation of the genetic diversity of Salvia fruticosa Mill. clones using RAPD markers and comparison with the essential oil profiles. Biochemical Systematics and Ecology,1999.27(6):559-568.
[78]藤艳芳,王峥涛,余国奠.丹参的药用资源研究进展.中国野生植物资源,2003,20(2)：1-3.
[79]P史密斯, R.索卡尔.数值分类学—数值分类的原理与应用.赵铁桥译.北京：科学出版社.1984：97-98.
[80]四川植物志编委会.四川植物志(第十卷).成都：民族出版社.1992：.330-377.
[81]徐克学,生物数学.北京：科学出版社.1999：72-73.
[82]孔德政,管志涛.基于主成分和聚类分析的荷花品种生物学性状的比较研究.中国园林,2008,8：86-89.
[83]罗应婷,杨钰娟.SPSS统计分析从基础到实践(第2版).北京：电子工业出版社.2010：248-263.
[84]陈珊珊,周明芹.浅析遗传多样性的研究方法.长江大学学报(自然科学版),2010.7(3)：54-57.
[85]Lewin R. Limits to DNA fingerprinting. Science,1989.243:2549-2551.
[86]Nakipoglu M. Turkiye'nin Salvia turleri uzerinde karyolojik Arastirmalar I. S.frutiosa Mill., S. tomentosa Mill., S. officinalis L., S. smyrnaea Boiss. (Lamiaceae). Doga Turkish Journal of Botany,1993a.17:21-25.
[87]Nakipoglu M. Turkiye'nin Bazi Salvia L. turleri uzerinde karyolojik arastirmalar Ⅱ. S. viridis L., S. glutinosa L., S. virgata Jacq., S. verbenaca L., S. argentea L (Lamiaceae). Doga Turkish Journal of Botany,1993b.17:157-161.
[88]Mani T, Thoppil JE. Influence of B-chromosome on essential oil content and composition in Salvia coccinea Buc'hoz ex Etl. (Lamiaceae). Caryologia,2005,58:246-248.
[89]舒志明,梁宗锁,孙群,等.丹参不育系Sh-B的植物学特征.西北农林科技大学学报(自然科学版),2007,35(7)：175-179.
[90]Sikdar B, Bhattacharya M, Mukherjee A, et al. Genetic diversity in important members of Cucurbitaceae using isozyme, RAPD and ISSR markers. Biologia Plantarum,2005,54(1): 135-140.
[91]Ye YM, Zhang JW, Ning GG, et al. A comparative analysis of the genetic diversity between inbred lines of Zinnia elegans using morphological traits and RAPD and ISSR markers. Scientia Horticulturae,2008,118(1):1-7.
[92]Viana AJC, Souza MM, Araujo IS, et al. Genetic diversity in Passiflora species determined by morphological and molecular characteristics. Biologia Plantarum,2010,54(3):535-538.
[93]Zheng D, Liang Y, Liu G, et al. RAPD analysis of germplasm resources of Kudingcha species in Oleaceae. Agricultural Sciences in China,2009,8(7):784-792.
[94]郭宝林,林生,冯毓秀,等.丹参主要居群的遗传关系及药材道地性的初步研究.中草药,2002,33(12)：1113-1116.
[95]徐红,王燕燕,王峥涛,等.不同产地丹参遗传关系的DNA标记分析.时珍国医国药,2008,19(12)：2970-2972.
[96]Khalil A, Hassawi DS, Kharma A. Genetic relationship among Salvia species and antimicrobial activity of their crude extract against pathogenic bacteria. Asian Journal of Plant Sciences,2005,4(5):544-549.
[97]田晔林,刘克锋,石爱平,等.一串红品种(系)遗传多样性RADP分析.中国农学通报,2006,22(5)：76-78.
[98]侯艳霞,汤浩茹,董晓莉,等.鼠尾草属植物基因组DNA提取及RAPD反应条件优化. 中国农学通报,2008,24(3)：72-77.
[99]Doyle JJ. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull,1987,19:11-15.
[100]Curley J, Jung G. RAPD-Based Genetic Relationships in Kentucky Bluegrass. Crop science, 2004,44(4):1299-1306.
[101]Rohlf FJ. NTSYS-pc. Numerical taxonomy and multivariate analysis system. Version 1.80. Exeter software, Setauket, New York.1993.
[102]Nei M, Li WH. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences,1979,76(10):5269-5273.
[103]Kaundun SS, Zhyvoloup A, Park YG. Evaluation of the genetic diversity among elite tea (Camellia sinensis var. sinensis) accessions using RAPD markers. Euphytica,2000,115(1): 7-16.
[104]赵飞,樊军锋,杨培华,等.十二个油松种群遗传多样性的RAPD分析.北方园艺,2011,11：112-116.
[105]周延清.DNA分子标记技术在植物研究中的应用.北京：化学工业出版社.2005：56-57.
[106]Kim C, Na HR, Choi HK. Genetic diversity and population structure of endangered Isoetes coreana in South Korea based on RAPD analysis. Aquatic Botany,2008,89(1):43-49.
[107]Bhutta WM, Akhtar J, Ibrahim M, et al. Genetic variation between Pakistani wheat (Triticum aestivum L.) genotypes as revealed by Random Amplified Polymorphic DNA (RAPD) markers. South African Journal of Botany,2006,72(2):280-283.
[108]Uma S, Sudha S, Saraswathi MS, et al. Analysis of genetic diversity and phylogenetic relationships among indigenous and exotic Silk (AAB) group of bananas using RAPD markers. Journal of horticultural science & biotechnology,2004,79(4):523-527.
[109]Zamani Z, Sarkhosh A, Fatahi R, et al. Genetic relationships among pomegranate genotypes studied by fruit characteristics and RAPD markers. Journal of Horticultural Science and Biotechnology,2007,82(1):11-18.
[110]Ebrahimi R, Zamani Z, Kashi A. Genetic diversity evaluation of wild Persian shallot (Allium hirtifolium Boiss.) using morphological and RAPD markers. Scientia Horticulturae, 2009,119(4):345-351.
[111]陈云鹏,曹家树,缪颖,等.芸薹类蔬菜基因组DNA遗传多样性的RAPD分析.浙江大学学报：农业与生命科学版,2000,26(2)：131-136.
[112]Matkowski A, Zielinska S, Oszmianski J, et al. Antioxidant activity of extracts from leaves and roots of Salvia miltiorrhiza Bunge, S. przewalskii Maxim., and S. verticillata L. Bioresource technology,2008,99(16):7892-7896.
[113]刘美子,宋经元,罗焜,等.DNA条形码序列对9种蒿属药用植物的鉴定.中草药,2012,43(7)：1393-1397.
[114]韩建萍,宋经元,姚辉,等.中药材DNA条形码鉴定的基因序列比较.中国中药杂志,2012,37(8)：1056-1060.
[115]庞晓慧,徐海滨,韩建萍,等.中药材薄荷的DNA条形码鉴定研究.中国中药杂志,2012,37(8)：1114-1116.
[116]刘震,陈科力,罗煜,等.忍冬科药用植物DNA条形码通用序列的筛选.中国中药杂志,2010,35(19)：2527-2532.
[117]Fay MF, Bayer C, Alverson WS, et al. Plastid rbcL sequence data indicate a close affinity between Diegodendron and Bixa. Taxon,1998,47:43-50.
[118]Cuenoud P, Savolainen V, Chatrou LW, et al. Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB and matK DNA sequences. American Journal Botany,2002,89:132-144.
[119]Zhang C, Fan X, Yu HQ, et al. Different maternal genome donor to Kengyilia species inferred from chloroplast trnL-F sequences. Biologia Plantarum,2009,53(4):759-763.
[120]Newmaster SG, Ragupathy S, Testing plant barcoding in a sister species complex of pantropical Acacia (Mimosoideae, Fabaceae). Molecular Ecology Resources,2009,9(s1): 172-180.
[121]Zhang L, Zhao H X, Fan X, et al. Genetic diversity among Salvia miltiorrhiza Bunge and related species inferred from nrDNA ITS sequences. Turkish. Journal of Biology,2012, 36(3):319-326.
[122]Tamura K, Dudley J, Nei M, et al. MEGA4:molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular biology and evolution,2007,24(8):1596-1599.
[123]Lahaye R, van der Bank M, Bogarin D, et al. DNA barcoding the floras of biodiversity hotspots. Proceedings of the National Academy of Sciences,2008,105(8):2923-2928.
[124]Meyer CP, Paulay G DNA barcoding:error rates based on comprehensive sampling,2005, 3(12):. PLoS biology,2005,3(12):e422.
[125]Armstrong KF, Ball SL. DNA barcodes for biosecurity:invasive species identification. Philosophical Transactions of the Royal Society B:Biological Sciences,2005,360(1462): 1813-1823.
[126]Felsenstein J. Confidence limits on phylogenies:an approach using the bootstrap. Evolution, 1985:783-791.
[127]Gao T, Yao H, Song J, et al. Evaluating the feasibility of using candidate DNA barcodes in discriminating species of the large Asteraceae family. BMC Evolutionary Biology,2010. 10(1):324.
[128]Chen S, Yao H. Han J. et al. Validation of the 1TS2 region as a novel DNA barcode for identifying medicinal plant species. PloS one,2010,5(1):e8613.
[129]Newmaster SG, Fazekas AJ. Steeves RAD. et al. Testing candidate plant barcode regions in the Myristicaceae. Molecular Ecology Resources,2008,8(3):480-490.
[130]Sass C, Little DP, Stevenson DW. et al. DNA barcoding in the cycadales:testing the potential of proposed barcoding markers for species identification of cycads. PloS one,2007, 2(11):e1154.
[131]Kondo K, Shiba M. Yamaji H, et al. Species identification of licorice using nrDNA and cpDNA genetic markers. Biological and Pharmaceutical Bulletin,2007,30(8):1497-1502.
[132]Ren B Q. Xiang XG. Chen Z D. Species identification of Alnus (Betulaceae) using nrDNA and cpDNA genetic markers. Molecular Ecology Resources,2010,10(4):594-605.
[133]Liu Z. Zeng X, Yang D, et al. Applying DNA barcodes for identification of plant species in the family Araliaceae. Gene,2012,49:76-80.
[134]Shaw J, Lickey EB, Beck JT, et al. The tortoise and the hare 11:relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. American Journal of Botany,2005,92(1):142-166.
[135]Mast AR, Givnish TJ. Historical biogeography and the origin of stomatal distributions in Banksia and Dryandra (Proteaceae) based on their cpDNA phylogeny. American Journal of Botany,2002,89(8):1311-1323.
[136]Miller JT, Grimes JW, Murphy DJ, et al. A phylogenetic analysis of the Acacieae and Ingeae (Mimosoideae:Fabaceae) based on trnK, matK, psbA-trnH, and trnL/trnF sequence data. Systematic Botany,2003:558-566.
[137]137. Winkworth RC, Donoghue MJ. Vibumum phylogeny based on combined molecular data:implications for taxonomy and biogeography. American Journal of Botany,2005,92(4): 653-666.
[138]Li M, Ling KH, Lam H, et al. Cardiocrinum seeds as a replacement for Aristolochia fruits in treating cough. Journal of ethnopharmacology,2010,130(2):429-432.
[139]Li M, Cao H, But PPH, et al. Identification of herbal medicinal materials using DNA barcodes. Journal of Systematics and Evolution,2011,49(3):271-283.
[140]Groot GA, During HJ, Maas JW, et al. Use of rbcL and trnL-F as a two-locus DNA barcode for identification of NW-European ferns:an ecological perspective. PIoS one,2011,6(1): e16371.
[141]Kojoma M. Kurihara K, Yarnada K, et al. Genetic identification of cinnamon(Cinnamomum spp.) based on the trnL-trnF chloroplast DNA. Planta medica,2002,68(1):94-96.
[142]Zhao HG, Zhou JJ, Cao SS, et al. Analysis of interspecific relationship among Stellaria media and its related species based on ITS and trnL-F sequence differences. Journal of Plant Resources and Environment,2009,18(1):1-5.
[143]China Plant BOL Group. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plant. Proceedings of the National Academy of Sciences,2011,108(49):19641-19646.
[144]Yang ZJ, Zhang L, Zhao HX, et al. Chromosome numbers of some species of Salvia (Lamiaceae) from the Sichuan Province, China. Nordic Journal of Botany,2009,27(4): 287-291.
[145]Wang M. Zhang L, Ding CB, et al. Meiotic observations of eight taxa in the genus Salvia. Caryologia,2009,62(4):334-340.
[146]Sharma SK, Dkhar J, Kumaria S, et al. Assessment of phylogenetic inter-relationships in the genus Cymbidium (Orchidaceae) based on internal transcribed spacer region of Rdna. Gene, 2012.,495,:10-15.
[147]Fazekas AJ. Burgess KS, Kesanakurti PR. et al. Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PloS one,2008,3(7):e2802.
[148]曹珍,谢晓亮.丹参的不同鉴别方法.时珍国医国药,2007,18(8)：1861-1863.
[149]Fazekas AJ., Kesanakurti PR., Burgess KS, et al. Are plant species inherently harder to discriminate than animal species using DNA barcoding markers?. Molecular Ecology Resources,2009,9(s1):130-139.
[150]Pettengill JB, Neel MC. An evaluation of candidate plant DNA barcodes and assignment methods in diagnosing 29 species in the genus Agalinis (Orobanchaceae). American Journal of Botany,2010,97(8):1391-1406.
[2]董玉琛.我国作物种质资源研究的现状与展望.中国农业科技导报,1999,2：36-40.
[3]王国山,顾恒琴,侯忠.对作物品种资源工作的认识与思考.国外农学-杂粮作物,1997,3：34-36.
[4]张华峰,季彪俊,植物种质资源研究概论.宜春学院学报,2005,27(2)：92-95.
[5]梁艳荣.大葱种质资源鉴定与其生理特性研究.博士学位论文.内蒙古农业大学,2008.
[6]Tautz D, Arctander P, Minelli A, et al. DNA points the way ahead in taxonomy. Nature,2002, 418(6897):479.
[7]Hebert PDN, Cywinska A, Ball SL. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B:Biological Sciences,2003, 270(1512):313-321.
[8]Hebert PDN, Penton EH., Burns JM, et al. Ten species in one:DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United States of America,2004,101(41):14812-14817.
[9]Hebert PDN, Stoeckle MY, Zemlak TS, et al. Identification of birds through DNA barcodes. PLoS biology,2004,2(10):e312.
[10]Ward RD, Zemlak TS, Innes BH, et al. DNA barcoding Australia's fish species. Philosophical Transactions of the Royal Society B:Biological Sciences,2005,360(1462): 1847-1857.
[11]Hajibabaei M, Janzen DH, Burns JM, et al. DNA barcodes distinguish species of tropical Lepidoptera. Proceedings of the National Academy of Sciences of the United States of America,2006,103(4):968-971.
[12]Kerr KCR, Stoeckle MY, Dove CJ, et al. Comprehensive DNA barcode coverage of North American birds. Molecular Ecology Notes,2007,7(4):535-543.
[13]Chase MW, Cowan RS, Hollingsworth PM, et al. A proposal for a standardised protocol to barcode all land plants. Taxon,2007,56(2):295-299.
[14]Kress WJ, Erickson DL. A two-locus global DNA barcode for land plants:the coding rbcL gene complements the non-coding trnH-psbA spacer region. PloS one,2007,2(6):e508.
[15]Kim KJ, Lee HL. Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants. DNA research,2004,11(4):247-261.
[16]Pennisi E. A barcode for plants. Science,2007,318:190-191.
[17]Hollingsworth ML, Clark AA, Forrest LL, et al. Selecting barcoding loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants. Molecular Ecology Resources,2009,9(2):439-457.
[18]刘宇婧,刘越,黄耀江,等.植物DNA条形码技术的发展及应用.植物资源与环境学报,2011,20(1)：74-82.
[19]闫化学,于杰.DNA条形码技术在植物中的研究现状.植物学报,2010,45(1)：102-108.
[20]CBOL Plant Working Group. A DNA barcode for land plants. Proceedings of the National Academy of Sciences,2009,106(31):12794-12797.
[21]陈士林,宋经元,姚辉,等.药用植物DNA条形码鉴定策略及关键技术分析.中国天然药物,2009,7(5)：322-327.
[22]Meier R, Zhang G, Ali F. The use of mean instead of smallest interspecific distances exaggerates the size of the "barcoding gap" and leads to misidentification. Systematic Biology,2008,57(5):809-813.
[23]宁淑萍,颜海飞,郝刚,等.植物DNA条形码研究进展.生物多样性,2008,16(5)：417-425.
[24]Wolfe KH, Li WH., Sharp PM. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proceedings of the National Academy of Sciences,1987,84(24):9054-9058.
[25]Chase MW, Salamin N, Wilkinson M, et al. Land plants and DNA barcodes:short-term and long-term goals. Philosophical Transactions of the Royal Society B:Biological Sciences, 2005,360(1462):1889-1895.
[26]Newmaster SG, Fazekas AJ, Ragupathy S. DNA barcoding in land plants:evaluation of rbcL in a multigene tiered approach. Botany,2006,84(3):335-341.
[27]Kress WJ, Wurdack KJ, Zimmer EA, et al. Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences of the United States of America,2005, 102(23):8369-8374.
[28]Presting GG. Identification of conserved regions in the plastid genome:implications for DNA barcoding and biological function. Botany,2006,84(9):1434-1443.
[29]Taberlet P, Coissac E, Pompanon F, et al. Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding. Nucleic Acids Research,2007,35(3):e14-e14.
[30]唐建阳,周先治.植物DNA条形码研究现状及应用前景.中国农学通报,2009,25(24)：35-43.
[31]张凤春,张文国.“生物多样性”释义(上)：L生物多样性的概念机现状.环境保护,2010.1(9)：45-48.
[32]32.王洪新,胡志昂.植物繁育系统、遗传结构和遗传多样性的保护.生物多样性,1996,4(2)：92-96.
[33]33.钱迎倩,马克平.生物多样性研究的原理与方法.北京：中国科学技术出版社.1994：13-36
[34]沈浩,刘登义.遗传多样性概述.生物学杂志,2001,18(3)：5-8.
[35]陈灵芝,中国的生物多样性-现状及其保护对策.北京：科学出版社.1993：99-113.
[36]冯夏莲,何承忠,张志毅,等.植物遗传多样性研究方法概述.西南林学院学报,2006,26(1)：69-74.
[37]国家药典委员会.中华人民共和国药典(第二部).北京：化学工业出版社.2005：52-53.
[38]李家实.中药鉴定学(第四版).上海：科学技术出版社.1995：169.
[39]李玉梅,陈立忠,李影.近年来丹参的药理研究进展.近黑龙江医药,1996,9(4)：234-235.
[40]柴瑞震.丹参的药理研究近况.中国中医药科技,2003,10(6)：390-392.
[41]郭宝林.丹参种质资源研究进展.中国中药,2002,27(7)：492-495.
[42]中国药材公司.中国中药资源.北京：科学出版社.1995：31-33.
[43]张力文.川丹参新品系区域试验及近缘种植物光合特性研究.硕士学位论文.四川农业大学,2011.
[44]中国科学院中国植物志编委会.中国植物志(第六十六卷).北京：科学出版社.1977：79-194.
[45]钟国成.四川省中江县丹参品系综合评价.硕士学位论文.四川农业大学,2010.
[46]杨在君,缪金城,张利,等.四川和重庆鼠尾草属植物的种类及药用资源.时珍国医国药,2008,19(4)：904-908.
[47]肖小河,方清茂,夏文娟,等.药用鼠尾草属数值分类与丹参药材道地性.植物资源与环境,1997,6(2)：17-21.
[48]倪梁红,赵志礼.华东地区鼠尾草属药用植物资源.时珍国医国药,2010,21(10)：2653-2655.
[49]Farkas A, Papp N, Horvath G, et al. Anatomical and genetic differences between Salvia taxa. European Journal of Pharmaceutical Sciences,2007,32(1):S20.
[50]Kandemir N. The morphological, anatomical and karyological properties of endemic Salvia hypargeia Fich & Mey. (Lamiaceae) in Turkey. Pakistan Journal of Botanly,2003,35(2): 219-236.
[51]Baran P, OZDEMlR C. The morphological and anatomical characters of Salvia napifolia Jacq. in Turkey. Bangladesh Journal of Botany,2006,35(1):77-84.
[52]Kaya A, Goger F, Baser KHC. Morphological, anatomical and palynological characteristics of Salvia halophila endemic toTurkey. Nordic Journal of Botany,2007,25(5):351-358.
[53]马志刚,杨永建,谢錾,等.甘肃鼠尾草属五种植物根的生药鉴定.兰州医学院学报,1993,19(4)：212.
[54]李素芬,李金芩,李小娇,等.中国民族民间医药.戟叶鼠尾草的生药学研究,2008,17(7)：18-20.
[55]唐丽萍,梁晓源,韦群辉,等.滇丹参的生药学研究.云南中医学院学报,2003,26(1)：11-14.
[56]张利,杨在君,黄霞,等.丹参及四川鼠尾草属植物叶表皮微形态研究.四川大学学报(自然科学版),2008,45(3)：674-680.
[57]Corsi G, Bottega S. Glandular Hairs of Salvia officinalis:New Data on Morphology, Localization and Histochemistry in Relation to Function. Annals of Botany,1999,84(5): 657-664.
[58]郑宝江,于丽杰,邢怡,等.兰花鼠尾草(Salvia farinacea Benth.)两类腺毛发育过程中超微结构的研究.植物研究,2002,22(1)：23-25.
[59]Werker E. Function of essential oil-secreting glandular hairs in aromatic plants of the Lamiaceae--a view. Flavour and Fragrance Journal,1993,8:249-255.
[60]杨德奎,孙京田,王丙全.山东鼠尾草属花粉形态的研究及其在分类上的意义.山东科学,2003,16(1)：14-16.
[61]Stewart WS. Chromosome numbers of Californian Salvias. American Journal of Botany, 1939,26(9):730-732.
[62]Gill LS. Chromosome studies in Salvia (Labiatae):West-himalayan species. Cellular and Molecular Life Sciences,1971,27(5):596-598.
[63]Alberto C M, Sanso A, Xifresa CC. Chromosomal studies in species of Salvia (Lamiaceae) from Argentina. Botanical Journal of the Linnean Society,2003,141(4):483-490.
[64]Ozkan M. Karyotype analysis on two endemic Salvia L.(Lamiaceae) species in Turkey. International Journal of botany,2006,2(3):333-335.
[65]Ozdemir C, Senel G The Morphological Anatomical and Karyological Properties of S. sclerea. Turkish Journal of Botany,1999,23(1):7-18.
[66]Palomino G, Mercado P, Ramamoorthy TP. Chromosomes of Salvia subgenus Calosphace (Lamiaceae), a preliminary report. Cytologia,1986,51(2):381-386.
[67]蔡朝晖,高山林,郭蓓.丹参组织培养材料的染色体显微鉴定技术.中国医科大学学报,1993,24(1)：49-52.
[68]张兴国,王义明,罗国安,等.丹参品种资源特性的研究.中草药,2002,33(8)：742-747.
[69]Yang ZY, Gong X, Pan Y. Cytological study of six Salvia species (lamiaceae) from the Hengduanshan Mountains region of China. Caryologia,2004,57(4):360-366.
[70]赵红霞,张利,凡星,等.丹参、黄花鼠尾和雪山鼠尾染色体数目的研究.中国中药杂志,2006,31(22)：1847-1849.
[71]Yang ZJ, Zhang L, Zhao HX, et al. Chromosome numbers of some species of Salvia (Lamiaceae) from the Sichuan Province, China. Nordic Journal of Botany,2009,27(4): 287-291.
[72]徐红,王燕燕,魏丹红,等.不同产地丹参药材的ISSR分析与鉴别.中药新药与临床药理,2007,18(6)：454-457.
[73]李廷春,樊洪泓,高正良,等.丹参遗传多样性的SRAP标记分析.核农学报,2008,22(5)：576-580.
[74]郝岗平,孙立彦,史仁玖,等.山东产丹参遗传多样性的扩增片段长度多态性指纹分析.时珍国医国药,2007,18(7)：51-55.
[75]汪红,王强.丹参及鼠尾草属植物的rDNA ITS序列分析.中草药,2005,36(9)：1381-1385.
[76]王迎,李大辉,张英涛.鼠尾草属药用植物及其近缘种的ITS序列分析.药学学报,2007,42(12)：1309-1314.
[77]Skoula M, Hilali I E, Makris A M. Evaluation of the genetic diversity of Salvia fruticosa Mill. clones using RAPD markers and comparison with the essential oil profiles. Biochemical Systematics and Ecology,1999.27(6):559-568.
[78]藤艳芳,王峥涛,余国奠.丹参的药用资源研究进展.中国野生植物资源,2003,20(2)：1-3.
[79]P史密斯, R.索卡尔.数值分类学—数值分类的原理与应用.赵铁桥译.北京：科学出版社.1984：97-98.
[80]四川植物志编委会.四川植物志(第十卷).成都：民族出版社.1992：.330-377.
[81]徐克学,生物数学.北京：科学出版社.1999：72-73.
[82]孔德政,管志涛.基于主成分和聚类分析的荷花品种生物学性状的比较研究.中国园林,2008,8：86-89.
[83]罗应婷,杨钰娟.SPSS统计分析从基础到实践(第2版).北京：电子工业出版社.2010：248-263.
[84]陈珊珊,周明芹.浅析遗传多样性的研究方法.长江大学学报(自然科学版),2010.7(3)：54-57.
[85]Lewin R. Limits to DNA fingerprinting. Science,1989.243:2549-2551.
[86]Nakipoglu M. Turkiye'nin Salvia turleri uzerinde karyolojik Arastirmalar I. S.frutiosa Mill., S. tomentosa Mill., S. officinalis L., S. smyrnaea Boiss. (Lamiaceae). Doga Turkish Journal of Botany,1993a.17:21-25.
[87]Nakipoglu M. Turkiye'nin Bazi Salvia L. turleri uzerinde karyolojik arastirmalar Ⅱ. S. viridis L., S. glutinosa L., S. virgata Jacq., S. verbenaca L., S. argentea L (Lamiaceae). Doga Turkish Journal of Botany,1993b.17:157-161.
[88]Mani T, Thoppil JE. Influence of B-chromosome on essential oil content and composition in Salvia coccinea Buc'hoz ex Etl. (Lamiaceae). Caryologia,2005,58:246-248.
[89]舒志明,梁宗锁,孙群,等.丹参不育系Sh-B的植物学特征.西北农林科技大学学报(自然科学版),2007,35(7)：175-179.
[90]Sikdar B, Bhattacharya M, Mukherjee A, et al. Genetic diversity in important members of Cucurbitaceae using isozyme, RAPD and ISSR markers. Biologia Plantarum,2005,54(1): 135-140.
[91]Ye YM, Zhang JW, Ning GG, et al. A comparative analysis of the genetic diversity between inbred lines of Zinnia elegans using morphological traits and RAPD and ISSR markers. Scientia Horticulturae,2008,118(1):1-7.
[92]Viana AJC, Souza MM, Araujo IS, et al. Genetic diversity in Passiflora species determined by morphological and molecular characteristics. Biologia Plantarum,2010,54(3):535-538.
[93]Zheng D, Liang Y, Liu G, et al. RAPD analysis of germplasm resources of Kudingcha species in Oleaceae. Agricultural Sciences in China,2009,8(7):784-792.
[94]郭宝林,林生,冯毓秀,等.丹参主要居群的遗传关系及药材道地性的初步研究.中草药,2002,33(12)：1113-1116.
[95]徐红,王燕燕,王峥涛,等.不同产地丹参遗传关系的DNA标记分析.时珍国医国药,2008,19(12)：2970-2972.
[96]Khalil A, Hassawi DS, Kharma A. Genetic relationship among Salvia species and antimicrobial activity of their crude extract against pathogenic bacteria. Asian Journal of Plant Sciences,2005,4(5):544-549.
[97]田晔林,刘克锋,石爱平,等.一串红品种(系)遗传多样性RADP分析.中国农学通报,2006,22(5)：76-78.
[98]侯艳霞,汤浩茹,董晓莉,等.鼠尾草属植物基因组DNA提取及RAPD反应条件优化. 中国农学通报,2008,24(3)：72-77.
[99]Doyle JJ. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull,1987,19:11-15.
[100]Curley J, Jung G. RAPD-Based Genetic Relationships in Kentucky Bluegrass. Crop science, 2004,44(4):1299-1306.
[101]Rohlf FJ. NTSYS-pc. Numerical taxonomy and multivariate analysis system. Version 1.80. Exeter software, Setauket, New York.1993.
[102]Nei M, Li WH. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences,1979,76(10):5269-5273.
[103]Kaundun SS, Zhyvoloup A, Park YG. Evaluation of the genetic diversity among elite tea (Camellia sinensis var. sinensis) accessions using RAPD markers. Euphytica,2000,115(1): 7-16.
[104]赵飞,樊军锋,杨培华,等.十二个油松种群遗传多样性的RAPD分析.北方园艺,2011,11：112-116.
[105]周延清.DNA分子标记技术在植物研究中的应用.北京：化学工业出版社.2005：56-57.
[106]Kim C, Na HR, Choi HK. Genetic diversity and population structure of endangered Isoetes coreana in South Korea based on RAPD analysis. Aquatic Botany,2008,89(1):43-49.
[107]Bhutta WM, Akhtar J, Ibrahim M, et al. Genetic variation between Pakistani wheat (Triticum aestivum L.) genotypes as revealed by Random Amplified Polymorphic DNA (RAPD) markers. South African Journal of Botany,2006,72(2):280-283.
[108]Uma S, Sudha S, Saraswathi MS, et al. Analysis of genetic diversity and phylogenetic relationships among indigenous and exotic Silk (AAB) group of bananas using RAPD markers. Journal of horticultural science & biotechnology,2004,79(4):523-527.
[109]Zamani Z, Sarkhosh A, Fatahi R, et al. Genetic relationships among pomegranate genotypes studied by fruit characteristics and RAPD markers. Journal of Horticultural Science and Biotechnology,2007,82(1):11-18.
[110]Ebrahimi R, Zamani Z, Kashi A. Genetic diversity evaluation of wild Persian shallot (Allium hirtifolium Boiss.) using morphological and RAPD markers. Scientia Horticulturae, 2009,119(4):345-351.
[111]陈云鹏,曹家树,缪颖,等.芸薹类蔬菜基因组DNA遗传多样性的RAPD分析.浙江大学学报：农业与生命科学版,2000,26(2)：131-136.
[112]Matkowski A, Zielinska S, Oszmianski J, et al. Antioxidant activity of extracts from leaves and roots of Salvia miltiorrhiza Bunge, S. przewalskii Maxim., and S. verticillata L. Bioresource technology,2008,99(16):7892-7896.
[113]刘美子,宋经元,罗焜,等.DNA条形码序列对9种蒿属药用植物的鉴定.中草药,2012,43(7)：1393-1397.
[114]韩建萍,宋经元,姚辉,等.中药材DNA条形码鉴定的基因序列比较.中国中药杂志,2012,37(8)：1056-1060.
[115]庞晓慧,徐海滨,韩建萍,等.中药材薄荷的DNA条形码鉴定研究.中国中药杂志,2012,37(8)：1114-1116.
[116]刘震,陈科力,罗煜,等.忍冬科药用植物DNA条形码通用序列的筛选.中国中药杂志,2010,35(19)：2527-2532.
[117]Fay MF, Bayer C, Alverson WS, et al. Plastid rbcL sequence data indicate a close affinity between Diegodendron and Bixa. Taxon,1998,47:43-50.
[118]Cuenoud P, Savolainen V, Chatrou LW, et al. Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB and matK DNA sequences. American Journal Botany,2002,89:132-144.
[119]Zhang C, Fan X, Yu HQ, et al. Different maternal genome donor to Kengyilia species inferred from chloroplast trnL-F sequences. Biologia Plantarum,2009,53(4):759-763.
[120]Newmaster SG, Ragupathy S, Testing plant barcoding in a sister species complex of pantropical Acacia (Mimosoideae, Fabaceae). Molecular Ecology Resources,2009,9(s1): 172-180.
[121]Zhang L, Zhao H X, Fan X, et al. Genetic diversity among Salvia miltiorrhiza Bunge and related species inferred from nrDNA ITS sequences. Turkish. Journal of Biology,2012, 36(3):319-326.
[122]Tamura K, Dudley J, Nei M, et al. MEGA4:molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular biology and evolution,2007,24(8):1596-1599.
[123]Lahaye R, van der Bank M, Bogarin D, et al. DNA barcoding the floras of biodiversity hotspots. Proceedings of the National Academy of Sciences,2008,105(8):2923-2928.
[124]Meyer CP, Paulay G DNA barcoding:error rates based on comprehensive sampling,2005, 3(12):. PLoS biology,2005,3(12):e422.
[125]Armstrong KF, Ball SL. DNA barcodes for biosecurity:invasive species identification. Philosophical Transactions of the Royal Society B:Biological Sciences,2005,360(1462): 1813-1823.
[126]Felsenstein J. Confidence limits on phylogenies:an approach using the bootstrap. Evolution, 1985:783-791.
[127]Gao T, Yao H, Song J, et al. Evaluating the feasibility of using candidate DNA barcodes in discriminating species of the large Asteraceae family. BMC Evolutionary Biology,2010. 10(1):324.
[128]Chen S, Yao H. Han J. et al. Validation of the 1TS2 region as a novel DNA barcode for identifying medicinal plant species. PloS one,2010,5(1):e8613.
[129]Newmaster SG, Fazekas AJ. Steeves RAD. et al. Testing candidate plant barcode regions in the Myristicaceae. Molecular Ecology Resources,2008,8(3):480-490.
[130]Sass C, Little DP, Stevenson DW. et al. DNA barcoding in the cycadales:testing the potential of proposed barcoding markers for species identification of cycads. PloS one,2007, 2(11):e1154.
[131]Kondo K, Shiba M. Yamaji H, et al. Species identification of licorice using nrDNA and cpDNA genetic markers. Biological and Pharmaceutical Bulletin,2007,30(8):1497-1502.
[132]Ren B Q. Xiang XG. Chen Z D. Species identification of Alnus (Betulaceae) using nrDNA and cpDNA genetic markers. Molecular Ecology Resources,2010,10(4):594-605.
[133]Liu Z. Zeng X, Yang D, et al. Applying DNA barcodes for identification of plant species in the family Araliaceae. Gene,2012,49:76-80.
[134]Shaw J, Lickey EB, Beck JT, et al. The tortoise and the hare 11:relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. American Journal of Botany,2005,92(1):142-166.
[135]Mast AR, Givnish TJ. Historical biogeography and the origin of stomatal distributions in Banksia and Dryandra (Proteaceae) based on their cpDNA phylogeny. American Journal of Botany,2002,89(8):1311-1323.
[136]Miller JT, Grimes JW, Murphy DJ, et al. A phylogenetic analysis of the Acacieae and Ingeae (Mimosoideae:Fabaceae) based on trnK, matK, psbA-trnH, and trnL/trnF sequence data. Systematic Botany,2003:558-566.
[137]137. Winkworth RC, Donoghue MJ. Vibumum phylogeny based on combined molecular data:implications for taxonomy and biogeography. American Journal of Botany,2005,92(4): 653-666.
[138]Li M, Ling KH, Lam H, et al. Cardiocrinum seeds as a replacement for Aristolochia fruits in treating cough. Journal of ethnopharmacology,2010,130(2):429-432.
[139]Li M, Cao H, But PPH, et al. Identification of herbal medicinal materials using DNA barcodes. Journal of Systematics and Evolution,2011,49(3):271-283.
[140]Groot GA, During HJ, Maas JW, et al. Use of rbcL and trnL-F as a two-locus DNA barcode for identification of NW-European ferns:an ecological perspective. PIoS one,2011,6(1): e16371.
[141]Kojoma M. Kurihara K, Yarnada K, et al. Genetic identification of cinnamon(Cinnamomum spp.) based on the trnL-trnF chloroplast DNA. Planta medica,2002,68(1):94-96.
[142]Zhao HG, Zhou JJ, Cao SS, et al. Analysis of interspecific relationship among Stellaria media and its related species based on ITS and trnL-F sequence differences. Journal of Plant Resources and Environment,2009,18(1):1-5.
[143]China Plant BOL Group. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plant. Proceedings of the National Academy of Sciences,2011,108(49):19641-19646.
[144]Yang ZJ, Zhang L, Zhao HX, et al. Chromosome numbers of some species of Salvia (Lamiaceae) from the Sichuan Province, China. Nordic Journal of Botany,2009,27(4): 287-291.
[145]Wang M. Zhang L, Ding CB, et al. Meiotic observations of eight taxa in the genus Salvia. Caryologia,2009,62(4):334-340.
[146]Sharma SK, Dkhar J, Kumaria S, et al. Assessment of phylogenetic inter-relationships in the genus Cymbidium (Orchidaceae) based on internal transcribed spacer region of Rdna. Gene, 2012.,495,:10-15.
[147]Fazekas AJ. Burgess KS, Kesanakurti PR. et al. Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PloS one,2008,3(7):e2802.
[148]曹珍,谢晓亮.丹参的不同鉴别方法.时珍国医国药,2007,18(8)：1861-1863.
[149]Fazekas AJ., Kesanakurti PR., Burgess KS, et al. Are plant species inherently harder to discriminate than animal species using DNA barcoding markers?. Molecular Ecology Resources,2009,9(s1):130-139.
[150]Pettengill JB, Neel MC. An evaluation of candidate plant DNA barcodes and assignment methods in diagnosing 29 species in the genus Agalinis (Orobanchaceae). American Journal of Botany,2010,97(8):1391-1406.