【作者】 陈川；

【导师】 傅承新；

【作者基本信息】 浙江大学 ， 植物学， 2011， 博士

【摘要】 药用植物玄参(Scrophularia ningpoensis Hemsley)是玄参科玄参属多年生草本植物,为我国特产,是著名中药材“浙八味”之一。根药用,有滋阴降火,消肿解毒的功效,在浙江、四川、湖北等地广有栽培。目前,随着中药标准化的推行,中药的种质鉴定和栽培研究也受到了越来越多的关注。本研究通过ISSR (Inter-Simple Sequence Repeats)分子标记研究了栽培玄参的遗传多样性和群体遗传结构,并在此基础上开发了CAR(Sequence Characterized Amplified Region)分子标记,用于鉴定浙江种源的玄参。运用单亲遗传的cpDNA序列变异和双亲遗传的nrDNA的扩增长度多态性(Amplified Fragment Length Polymorphism, AFLP)探讨了玄参的栽培起源。基于ITS和cpDNA序列构建了东亚玄参的系统发育树,阐明了东亚玄参的属内种间关系,并在此基础上对确立的玄参复合种进行了亲缘地理研究。主要研究内容与结果如下：1.栽培玄参遗传多样性、群体遗传结构及SCAR分子标记的研制12条ISSR引物对玄参8个栽培群体及2个野生群体的分析结果表明两个野生群体天目山群体TM1W和磐安天网群体TWW的多态位点百分比较高,分别为48.39%和35.48%。栽培群体的多态位点百分比普遍较低,为9.68%-20.97%,其中磐安窈川乡YC,而磐安尚湖PA群体最低。AMOVA分析揭示了遗传多样性所占比率为：种群间76.67%,种群内23.33%,说明各个群体间分化明显。在根据Nei’s遗传距离利用UPGMA法所构建的10个玄参群体的遗传关系聚类图中,大部分栽培群体聚为一大支,其中磐安种源的栽培群体存在较近的亲缘关系,揭示了其道地性的遗传基础。在ISSR实验的基础上,发现引物UBC874扩增结果中1500bp左右出现的条带是浙江群体所特有的,其他地区的玄参均不存在此条带。通过克隆测序,发现此片段长度为1306bp,针对此片段设计的特异性引物CC874u/CC874d在对所有地区的玄参进行检测后发现,只有浙江的玄参能扩增出单一、清晰的条带。因此这对特异性引物可以作为SCAR分子标记用于浙江种源玄参的鉴定。2.栽培玄参的起源进一步运用cpDNA psbA-trbH和trnL-F片段,结合AFLP分子标记对玄参14个栽培群体、14个野生群体进行了研究。结果表明在玄参28个群体379个个体中,两个叶绿体片段共检测到27个多态位点,共得到22种单倍型。其中,栽培群体仅有4种单倍型,每个栽培群体只拥有一种单倍型,没有多态性,单倍型多样性为0.486,核苷酸多样性为0.002；野生群体共有21种单倍型：单倍型多样性为0.919,核苷酸多样性为0.003。揭示仅有限的野生个体参与了玄参的栽培起源,多年的克隆繁殖使栽培玄参的遗传多样性水平已相当低,栽培群体基因流仅0.161,远低于野生群体(0.649)。单倍型MP严格一致树和网状支系图揭示栽培玄参的四个单倍型(E、A、B、P)与多个不同的野生群体共享,表明栽培玄参的多次多地起源,并发现江西冷水野生群体LSW是浙江玄参的最可能起源地。AFLP中6个引物组合在315个个体中共扩增出289个稳定、清晰、可判读的条带,其中261个条带(90.31%)具有多态性。野生群体的遗传多样性水平普遍高于栽培群体(栽培群体的h=0.0076-0.0875；野生群体的h=0.0791-0.1614)。从扩增的多态条带数目来看,多态性条带的百分比在野生群体中为22.15%-50.87%,在栽培群体中为3.11%-27.68%。并且栽培玄参群体间分化大于野生玄参的：在栽培玄参中76.97%的遗传变异来自于群体间,23.03%的遗传变异来自于群体内(FST=0.7697)；在野生玄参中仅39.60%的遗传变异来自于群体间,60.40%的遗传变异来自于群体内(FST=0.3961)。AFLP标记的PCoA分析、Neighbor-Joining分析和UPGMA分析结果一致表明栽培玄参遗传一致度较高,并在聚类图中聚为一支,其中与栽培玄参关系最近的是湖南平江HNW和江西冷水LSW两个野生群体,确定了这两个野生群体参与了玄参的栽培起源,而并非来自浙江的野生群体。最终揭示浙江产区的栽培玄参(PAC和XJC)以及引种自浙江的福建光泽栽培群体GZC已与其他地区的栽培玄参之间出现了明显的遗传分化,形成了浙玄参的道地性的基础。研究揭示野生玄参虽然分布较广,但资源同趋减少,认为部分群体拥有丰富的遗传多样性,是选育优质基因和优良品种的重要资源库,可以为玄参的栽培育种提供基础。3.东亚玄参的系统进化为进一步搞清药用玄参在东亚玄参属的地位,本研究运用ITS和cpDNA的trnQ-rps16、psbA-trnH和trnL-F三个片段对东亚分布的玄参属(包括中国21个种、同本5种、韩国3种),并以欧美玄参22种为对照(含15种北美玄参的序列来自Genebank)进行了分子系统学分析,结果表明东亚分布的玄参形成了四个谱系：广布种玄参(S. nignpoensis)、丹东玄参(S. kakudensis)、北玄参(Sbuergeriana)和双锯齿玄参(S. yoshimurae)形成的一个非常稳定的复合群；倍体的砾玄参组；分布中国华中-西北-西南的其它玄参类群；以及分布于韩国和日本的特有种(S. takesimensis、S. grayana、S. duplicata-serrata、S. musashiensis)与北美玄参和欧洲的S. nodosa构成的稳定单系。玄参、北玄参、丹东玄参和双锯齿玄参具有相似的化学成分,在分子系统树上表现为一个具很高支持率的单系类群,种与种之间界限不明确,缺乏分辨率,结合地理和形态特征认为东亚的这四个种很明显是一个多倍化过程中的类群,提出将这四个种定为典型的玄参复合种(S. ningpoensis species complex)。此外,首次根据分子系统树推测了东亚玄参的2个来源：一是可能来自北美,即日本-韩国-远东的特有种类,另一支来自欧洲-中亚(以S. canina-S. incisa为基础)。揭示玄参属在东亚形成了2支：包括一支华中-西南至西北分布的玄参类群和另一支华东-华北-东北-韩国-日本分布的药用玄参复合种。结合松散分子钟模型推断东亚玄参的四个谱系形成在52.12mya：华中-西北-西南分布的玄参的早期分化发生在41.25mya的第三纪始新世,喜玛拉雅山脉隆起之前；中亚-西北-华北-东北分布的砾玄参组几乎在同时分化形成(38.81mya)；到中新世(26mya左右),东亚-东北亚的玄参复合种开始出现,而韩国和日本的非复合种玄参应该是在10.7-7.25mya的上新世从北美迁移而来。4.玄参复合种的亲缘地理研究对确立的玄参复合种,运用cpDNA psbA-trbH和trnL-F片段,结合AFLP分子标记对其42个群体(玄参28个群体,北玄参7个群体,丹东玄参6个群体,双锯齿玄参1个群体)进行了亲缘地理研究。cpDNA结果表明538个个体中共有34个多态位点,27种单倍型。其中玄参与北玄参共享单倍型2种；北玄参与丹东玄参共享单倍型1种；双锯齿玄参只拥有一种单倍型,与玄参共享。玄参与北玄参(分布于中国辽宁凌源的群体)共享的单倍型位于网络图的中心位置,根据溯祖理论推测其为祖先单倍型,结合染色体数目的统计(北玄参2n=30,玄参2n=90,丹东玄参2n=36)推测辽宁凌源的的北玄参可能是该复合种现存的祖先类型。江西冷水(LSW)和浙江天目山(TM2W)分别拥有5个和6个cpDNA单倍型,以及特有单倍型S10和S6,推测这2个群体很可能是玄参复合种在末次冰期的避难所。AFLP分析揭示了玄参复合种的Nei’s遗传多样性(h)：玄参、北玄参、丹东玄参和双锯齿玄参依次为：0.2202,0.1543,0.1512,0.0685。将所有群体按照玄参、北玄参、丹东玄参和双锯齿玄参分为四个组后进行分组分层AMOVA分析,仅检测到32.62%的遗传变异发生于组间,表明种间分化不显著。PCoA分析、Neighbor-Joining分析和STRUCTURE分析揭示了这四个种的关系：认为从核基因水平,除了双锯齿玄参外,玄参、北玄参和丹东玄参之间的分化是存在的,虽然这种分化程度有限(仅32.62%),但还是可以进行物种的区分；而相对保守、单亲遗传的cpDNA序列反应的群体历史信息表明,这四个种可能来自于同一个祖先,并且分化时间不长。综合cpDNA单倍型,核DNA序列和AFLP分析,推测玄参复合种的分化和迁移路线：玄参复合种是玄参属随着东亚古气候从干旱生境(现存二倍体砾玄参组植物)向湿润生境转变的结果。二倍体的北玄参是该复合种的根基,现存河北辽宁交界的凌源野生北玄参LYW群体是它的典型代表。认为该复合种的祖先在中新世早期(20mya左右),当时气候开始变温暖潮湿,北玄参向东迁移分化形成丹东玄参,向南迁移形成玄参和双锯齿玄参。cpDNA单倍型和AFLP分析结果都表明台湾的双锯齿玄参与玄参的关系非常密切,来自同一个基因池,首次提出了对双锯齿玄参的分类地位进行修订,建议降为亚种置于玄参(S. ningpoensis Hemsley)种下。更多还原

【Abstract】 Scrophularia ningpoensis Hemsley, known as one of the famous Traditional Chinese Medicine (TCM)--"Zhebawei", used in the Chinese Materia Medica (CMM) belonging to the family Scrophulariaceae, has a long history of widespread use in China. Root of this medicinal herb is used to treat inflammation, laryngitis, tonsillitis, abscesses of carbuncles, constipation and this species is widely cultivated in Zhejiang, Sichuan, Hubei Provinces and so on. Nowadays, with the standardization of TCM, domestication of medicinal herbs and molecular authentication become more and more important. In this study, firstly, we used the ISSR (Inter-Simple Sequence Repeats) molecular markers to detect the genetic diversity and population genetic structure in cultivated S. ningpoensis. Further more, one pair of SCAR (Sequence Characterized Amplified Region) primers was developed to identify S. ningpoensis originated from Zhejiang Province. Secondly, cpDNA sequences and AFLP (Amplified Fragment Length Polymorphism) markers were applied to study the domestication of S. ningpoensis. Thirdly, phylogenetic trees of East-Asian Scrophularia were constructed based on ITS and cpDNA sequences. Finally, the Phylogeography of S. ningpoensis species complex was studied by cpDNA sequences and AFLP molecular markers. There are4main conclusions of our research:1. Genetic diversity and population genetic structure of cultivated S. ningpoensis and the development of SCAR markersTwelve ISSR universal primers were applied in8cultivated population and2wild populations of S. ningpoensis, which revealed that two wild populations (TM1W, TWW) harbored higher polymorphic percentage (48.39%,35.48%) than cultivated populations (9.68%-20.97%). In cultivated populations, the polymorphic percentage of population YC and PA were the lowest. AMOVA analysis suggested that genetic variance occurred among populations is76.67%and that occurred within population is23.33%, which resulted from differentiation in populations. UPGMA dendrogram for ten populations of S. ningpoensis based on Nei’s genetic distance revealed that cultivated populations grouped in one clade, within which populations from Pan’an County were closer than others.Based on ISSR analysis, primer UBC874provided an approximately1500bp band unique to populations originated from Zhejiang Province. After gel purified, cloned and sequenced, this DNA fragment turned out to be1306bp. A pair of22bp SCAR primers (CC874u and CC874d) was designed for the amplification of this DNA fragment. All samples from different regions were amplified by SCAR primers CC874u and CC874d and PCR products show that a single band about1000bp was only in accessions originated from Zhejiang Province, which proved that CC874u and CC874d are useful for identifying S. ningpoensis originated from Zhejiang Province.2. Domestication of S. ningpoensiscpDNA (psbA-trbH and trnL-F) and AFLP molecular markers were used in14cultivated populations and14wild populations of S. ningpoensis.27polymorphic sites classified into22haplotypes were detected by cpDNA. There were only4haplotypes in cultivated popoulations and every population only harbored single haplotype:the haplotype diversity is0.486and nucleotide diversity is0.002; whereas21haplotypes were in wild populations:haplotype diversity is0.919, nucleotide diversity is0.003. Results of haplotype network showed that four haplotypes in cultivated populations shared with many wild populations were located in different clades, which suggested that S. ningpoensis might experience multiple origin events and LSW wild population in Jiangxi Province probably was involved in the origin of cultivated S. ningpoensis in Zhejiang Province.Two hundreds eighty nine bands were amplified in315individuals by6pairs of AFLP markers and261bands were polymorphic (90.31%). Genetic diversity in wild populations were higher than cultivated populations (h in cultivated populations were from0.0076to0.0875; in wild populations were from0.0791to0.1614). As to the percentage of polymorphic bands, in wild populations were from22.15%to50.87%, in the cultivated were from3.11%to27.68%. Moreover, the differentiation in cultivated populations was larger than which in the wild:in cultivated populations, genetic variance occurred among populations were76.97%, genetic variance occurred in populations were23.03%; in the wild, those two statistical numbers were39.60%and60.40%respectively.Results of PCoA、Neighbor-Joining and UPGMA analysis were consistent, which showed that all cultivated populations grouped together with two wild populations: HNW and LSW, located in Hunan and Jiangxi Provinces respectively. This clarified that the origin of cultivated S. ningpoensis were Hunan and Jiangxi Province, precisely, the HNW and LSW wild populations. That point contrary to current belief that Zhejiang Province was involved in the origin of cultivated S. ningpoensis. Moreover, cultivated populations from Zhejiang (PAC, XJC, GZC—introduced from Zhejiang) occurred in same cluster represented genetic identity, which can be the genetic evidence for geo-authentic S. ningpoensis from Zhejiang. Due to habitat deterioration and over exploitation, the wild genetic resources of S. ningpoensis have suffered rapid declines. But some native populations (LSW, JHW etc.) have high genetic diversity may contain special genes that are very important for the plant’s growth and use.3. Phylogeny of East Asian ScrophulariaSamples including21species of Scrophularia in China mainland,5species in Japan,3species in South Korea and7species in America-Europe were all surveyed by ITS and cpDNA fragments (trnQ-rps16, psbA-trnH, trnL-F), combined with sequences of15North American species which were download from Genebank. The molecular phylogenetic tree revealed that Scrophularia in East Asian were divided into four lineages:S. ningpoensis species complex including S. ningpoensis, S. buergeriana, S. kakudensis and S. yoshimurae; diploid clade with Sect. Tomiphyllum; the rest Scrophularia species distributed in China; South Korea-Japan-North America clade. S. ningpoensis, S. buergeriana, S. kakudensis and S. yoshimurae form a monophyletic clade with high bootstrap support and shared with very similar morphological characters can be treated as S. ningpoensis species complex. Our study also threw light on the origin of East Asian Scrophularia:Species in South Korea and Japan might be derived from North America; Species in China might be from Europe-Central Asian and then evolved into two lineages. Based on relaxed clock model in BEAST analysis, time dating was estimated:four lineages in East Asian formed at52.12mya; S. ningpoensis species complex rose at26mya in the Miocene epoch; other Scrophularia species in China were evolved at41.25mya in the Eocene epoch, the same time as diploid clade; South Korea and Japan groups were migrated from North America at10.7-7.25mya in the Pliocene epoch. 4. Phylogeography of S. ningpoensis species complexSamples of S. ningpoensis species complex comprises28populations of S. ningpoensis,7populations of S. buergeriana,6populations of S. kakudensis and one population of S. yoshimurae. cpDNA fragments (psbA-trbH and trn"L-F) combined AFLP molecular markers were used for all the42populations.Thirty four polymorphic sites and27haplotypes were detected in538individuals by cpDNA sequences. Two haplotypes were share by S. ningpoensis and S. buergeriana; one haplotype was share by S. buergeriana and S. kakudensis; S. yoshimurae only contained one haplotype which was share with S. ningpoensis. The haplotype shared by S. ningpoensis and S. buergeriana (population in Lingyuan, Liaoning) located in the center of the haplotype network. By Coalescence Theory, the haplotype in the center of the network can be the candidate of ancestors. Considered with chromosome numbers in Scrophularia (S. buergeriana:2n=30; S. ningpoensis:2n=90; S. kakudensis:2n=36), we indicated that LYW population of S. buergeriana might be the ancestor of S. ningpoensis species complex. Two wild populations LSW (Jiangxi Province) and TM2W (Mt. Tianmu) harbored five and six cpDNA haplotypes respectively, and contain S10and S6rare haplotypes. So we indicated the glacial refugia for S. ningpoensis were Jiangxi and Mt. Tianmu.Results of AFLP analysis showed the Nei’s genetic diversity in S. ningpoensis species complex were:S. ningpoensis was0.2202, S. buergeriana was0.1543, S, kakudensis was0.1512and S. yoshimurae was0.0685. Hierarchical analysis of molecular variance revealed that genetic variance occurred among species were32.62%; genetic variances occurred among populations in species were38.10%and that within populations were29.28%. Results of PCoA, Neighbor-Joining and STRUCTURE analysis all clarified the differentiation among three species:S. ningpoensis, S. buergeriana and kakudensis. So we confirmed the taxonomic status of these three species. But results from cpDNA sequences suggested that these three species must from the same ancestor. S. ningpoensis species complex might rise in early Miocene epoch when Weather changed from dry to warm and humid. At that time,s. buergeriana (wild population in Liaoning, China--LYW) considered as diploid migrated towards east to form S. kakudensis, and migrated towards south to form S. ningpoensis and S. yoshimurae.S. yoshimurae and S. ningpoensis grouped together and came from the same gene pool. So these two species might be synonym and we suggested that S. yoshimurae can be treated as a subspecies of S. ningpoensis Hemsley.更多还原