【作者】 卢毅军；

【导师】 傅承新；

【作者基本信息】 浙江大学 ， 生态学， 2013， 博士

【摘要】 蜡梅(Chimonanthus pracox)是蜡梅科蜡梅属植物,为我国特产,是著名的冬季园林观花植物。在中国大部分省市广泛栽培,栽培历史悠久。本研究通过运用单亲遗传的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)探讨了蜡梅的栽培起源。基于ITS、cpDNA序列和ITS+cpDNA联合序列构建了蜡梅属的系统发育树,结合形态学分类数据,探讨了蜡梅属内种间关系,并在此基础上对蜡梅属内物种提出分类处理建议。主要研究内容与结果如下：1)蜡梅属的系统发育与物种界定运用ITS和cpDNA的三个片段,以美国蜡梅(Calycanthus floridus)为外类群,对蜡梅属进行了分子系统学分析,ITS分析揭示了蜡梅属6个种分成明显的2个分支：西南蜡梅(Ch. campanulatus)与蜡梅(Ch.praecox)有明显的亲缘关系,支持率很高；山蜡梅(Ch.nitens)、浙江蜡梅(Ch. zhejiangensis)、柳叶蜡梅(Ch.salicifolius)和突托蜡梅(Ch. grammatus)形成另一支。在第2支的四个种中,表现出柳叶蜡梅与突托蜡梅关系更近,浙江蜡梅与山蜡梅近缘；cpDNA序列分析的结果也支持西南蜡梅和蜡梅之间的亲缘关系,但对其他四个种分辩率不高。ITS和cpDNA联合数据分子树确立了西南蜡梅与蜡梅的姐妹群关系,推测两者来自一个共同祖先,从花期、花色、叶揉碎后无气味等相似特征上更说明他们之间的近缘关系；同时表明山蜡梅与浙江蜡梅近缘,柳叶蜡梅和突托蜡梅近缘。对蜡梅种内不同群体的关系进行的分析表明,野生蜡梅中湖北保康(WBK)、神龙架(WSLJ)和宜昌(WYCH)的群体形成一个亚支,揭示地理位置较近的群体来自共同祖先的可能性很大,基因交流比其他群体之间更容易：湖北武汉(CWH)栽培群体和重庆巫溪(WWX)野生群体形成另一个亚支,揭示了武汉栽培群体中部分个体可能来自于重庆巫溪野生群体；其他各群体均表现为平行枝,说明蜡梅的种下变异很少,无论栽培的群体还是野生的群体都一样,需要进一步群体遗传学的研究。本研究的外部形态性状数据的PCoA分析也表明浙江蜡梅和山蜡梅相互重叠,难以区分。在分布区上,山蜡梅在湖南、福建、广西、贵州等地均有分布,但浙江未见分布,而浙江蜡梅仅分布于浙江龙泉,因此,结合形态、分子及地理分布特征,我们认为浙江蜡梅是山蜡梅分布区的最东边界的一个群体,可能只是山蜡梅的一种生态型(ecotype),而不是独立的分类单位。因此,赞成将浙江蜡梅合并入山蜡梅,作为山蜡梅的异名处理。浙江蜡梅、突托蜡梅和山蜡梅曾统称为山蜡梅复合体,在形态上的微小变异可能是环境因素的影响造成的。本文ITS和形态学数据研究结果也表明突托蜡梅在分子水平以及叶片大小、形态和花被片等形态方面与浙江蜡梅和山蜡梅存在一定区别,不支持并入山蜡梅,需要进一步增加基因片段进行深入的研究。2)基于cpDNA和AFLP的蜡梅栽培起源基于cpDNA的trnH-psbA、trnS1-G1和trnS2-G2片段和全基因组AFLP分子标记3个引物组合EcoRI-CAA(FAM)/MseI-TC,EcoRI-TTT(M)/MseI-AGG和EcoRI-CAA (FAM)/MseI-TA,对蜡梅13个栽培群体、8个野生群体进行了全面的群体遗传和亲缘地理研究。结果表明在蜡梅21个群体282个个体中,三个叶绿体片段共检测到9个多态位点,共有9种单倍型。单倍型网络图呈现以在群体中出现频率最高的A单倍型为中心的星状拓扑结构,以1-3步衍生出B、C、D、E、F、G、H、I单倍型。野生群体共有7种单倍型(占单倍型总数的77.8%),但仅2个野生群体(12.5%)具有单倍型多态性,而栽培群体有6种单倍型,其中有8个栽培群体(61.5%)具有单倍型多态性。野生群体与栽培群体有4种共有单倍型(A、B、C、D),后裔单倍型中有5个单倍型为群体特有,4个共有单倍型是在不同的野生群体内发现。结果表明野生群体具有较高的单倍型多样性和核苷酸多样性：hT=0.819,πT=0.0011；AMOVA分析表明栽培群体和野生群体的中基因流Nm分别为0.52和0.14,表明都已经发生遗传分化,栽培群体内的遗传变异与群体间的遗传变异分别为50.81%和49.19%,而野生群体的遗传变异主要存在群体之间(78.12%)。cpDNA序列单倍型揭示了在蜡梅野生群体中,遗传变异大部分存在于各个群体间,栽培群体中,群体间的遗传变异与群体内的遗传变异没有明显差异。从单倍型分析显示蜡梅的栽培起源为多地多次起源。AFLP结果表明309个条带(98.41%)具有多态性。核基因水平表明遗传多样性程度最高的群体是重庆静观(CCQ)栽培群体(h=0.1518),遗传多样性最低的是四川峨眉山(CEMS)栽培群体(h=0.0724)。野生群体遗传多样性水平略高于栽培群体(野生群体的h=0.2834；栽培群体的h=0.2383)。AMOVA分析同样揭示在蜡梅野生群体中,遗传变异大部分存在于各个群体间,栽培群体中,群体间的遗传变异与群体内的遗传变异没有明显差异。21个蜡梅群体间的分化指数分别为0.7478(cpDNA)和0.6187(AFLP)。PCoA分析、Neighbor-Joining分析、UPGMA分析和Structure计算结果揭示所有腊梅群体由两个不同基因池组成,形成了两个种内谱系。13个栽培群体中有8个与野生的重庆巫溪(WWX)和浙江临安(WLA)共有相同基因池,而广西阳朔(CYS)、江西抚州(CJXF).湖南湘潭(CXT)、福建福州(CFZ)和安徽合肥(CHF)5个栽培群体与其余6个野生群体具有相同基因池。综合cpDNA和AFLP,可以推测蜡梅可能是多地、多次的起源。浙江临安所在的中国东部为现代栽培蜡梅的一个可能起源地,重庆巫溪、四川万源、贵州贵阳所在的中国西南地区为现代栽培蜡梅的另一个可能起源地。这一结果与蜡梅现在的栽培中心结果相吻合。江西抚州(CJXF).湖南湘潭(CXT)和安徽合肥(CHF)的栽培群体的地理位置位于西部重庆和东部浙江之间的我国中部地区,具有多个单倍型,包括特有单倍型,该地区是否存在另外的野生群体,值得深入研究。上述研究也揭示蜡梅在栽培过程中受到的奠基者效应、人工选育和克隆繁殖等因素影响不明显,未经历明显的遗传瓶颈,加上商业、文化上的广泛交流,使栽培群体保持较高的遗传多样性。基于上述分析,本研究对中国特有的观赏植物蜡梅的就地保护和迁地保护提出要重视重点群体的就地保护,并对重点栽培群体的所有成员进行遗传普查,建立基因档案,以便对一些特殊个体进行重点保存和利用。并提出不同的群体中拥有各自的特异单倍型,是栽培蜡梅选育优质基因和优良品种的重要资源库,可以为蜡梅的栽培育种提供基础。更多还原

【Abstract】 Wintersweet (Calycanthaceae, Chimonanthus praecox), widely cultivated in many provinces in china, is a Chinese endemic winter-flowering plant which has a long history of cultivation. In this study, cpDNA sequences and AFLP (Amplified Fragment Length Polymorphism) markers were applied to study the domestication of Ch.praecox firstly. Then phylogenetic trees of Chimonanthus were constructed based on ITS and cpDNA sequences. With the combination of morphology data, we finally discussed the intragenic relationships in Chimonanthus and some taxonomic recommendations were given. There are2main conclusions of our research:1. Phylogeny and species defined of ChimonanthusSamples including species of Chimonanthus and Calycanthus floridus were all surveyed by ITS and cpDNA fragments. The molecular phylogenetic tree of ITS revealed that Chimonanthus were divided into two lineages:Ch.praecox and Ch.campanulatus had obvious relationship and the support rate is very high; Ch.zhejiangensis, Ch.salicifolius, Ch.nitens and Ch.grammatus were clustered together. The latter was also divided into two subgroups:Ch.salicifolius and Ch.grammatus had close relationship, Ch.zhejiangensis and Ch.nitens had close relationship. cpDNA sequence supports the relationship of Ch.praecox and Ch.campanulatus. The united data of ITS and cpDNA established Ch.praecox and Ch.campanulatus are sister group and suggest both species come from a common ancestor, the similar characteristics of flowering, flower color, leaf crumple odorless also showed the close relationship in both species.The analysis of relationships among the populations of Ch. praecox shows WBK, WSLJ and WYCH were formed a sub branch; it revealed those geographically close populations maybe from the same ancestor and gene communication more easily than other groups. CWH and WWX formed another sub branch showed some individuals of CWH were introduced from WWX. The other groups showed parallel branches. Regardless of the populations of the cultivated or wild, it need for further study of population genetics.PCoA analysis of the external morphological characters the show that Ch.zhejiangensis and Ch.nitens overlap and difficult to distinguish. Ch.nitens distribute in Hunan, Fujian, Guangxi, Guizhou and other provinces but Zhejiang province hasn’t distributed, Ch.zhejiangensis only distributed in Longquan, zhejiang province. Combined with the features of morphological, molecular and geographical distribute, we induced Ch.zhejiangensis is the most eastern boundary group and only is the kind of ecological type (ecotype) of Ch.nitens rather than independent taxa. Therefore, we supports Ch.zhejiangensis merge into Ch.nitens, as the synonym of Ch.nitens. Ch.zhejiangensis, Ch.nitens and Ch.grammatus had called by a joint name as the Ch.nitens complex; the small variations may be caused by environmental factors. ITS and morphological data of this paper also showed Ch.grammatus had some differences at the molecular level, as well as leaf size, shape and tepals with Ch.zhejiangensis and Ch.nitens and not support merge into Ch.nitens. It needs to be further study by increasing the gene fragment.2. Domestication of Ch. praecox based on cpDNA sequences and AFLP.cpDNA sequence (psbA-trnH, trnS1-G1and rrnS2-G2) and AFLP(EcoRI-CAA (FAM)/MseI-TC,oRI-TTT (FAM)/Mse-AGG andoRI-CAA (FAM)/MceI-TA) were used in13cultivated populations and8wild populations of Ch. praecox.9polymorphic sites classified into9haplotypes were detected in these three fragments for282individuals of21populations. The whole haplotype network is a star-style topology with haplotype A (highest frequency) as a radiating center, deriving from it are the other eight haplotypes (B, C, D, E, F, G, H and I). There were7haplotypes (77.8%of total) in wild populations and only2(12.5%) harbored haplotype diversity, while6haplotypes were in cultivated populations and8(61.5%) harbored haplotype diversity. Results of haplotype network showed that4haplotypes (A, B, C and D) in cultivated populations shared with wild populations,5derived haplotypes is peculiar to populations,4shared haplotypes found in different wild populations. The wild populations has a high haplotype diversity and nucleotide diversity:/ht=0.819, πT=0.0011; AMOVA analysis showed that the gene flow (Nm) was0.52and there was obvious genetic differentiation in the cultivated populations. The genetic variation of cultivated populations was49.19%among populations and50.81%within the populations. In the wild populations, the genetic variation among populations was78.12%and gene flow (Nm) was only0.14. The cpDNA sequence suggested that most genetic variance occurred among populations in wild populations and in cultivated populations the two statistical numbers were similar. Results of haplotype network showed that Ch. praecox might experience multiple origin events.315bands were amplified in282individuals by3pairs of AFLP markers and309bands were polymorphic (98.41%). The highest genetic diversity occurred in CCQ cultivated population (h=0.1518) and the lowest in EMS cultivated population (h=0.0724).Genetic diversity in wild populations were a little higher than cultivated populations (h in cultivated populations were0.2383; in wild populations were0.2834). AMOVA analysis results keep consistent to cpDNA sequence. In all of populations of Ch. praecox, strong differentiation among21populations was found suggested that genetic variance occurred among populations (cpDNA:0.7478and AFLP:0.6187)。Results of PCoA analysis, Neighbor-joining and UPGMA analysis and Structure calculation revealed that all populations by two different genes pools and formed two intraspecific lineages.8of13cultivated populations with WWX and WLA shared the same gene pool,5cultivated populations of CYS,CJXF,CXT,CFZ and CHF the remaining six wild populations shared the same gene pool. cpDNA and AFLP data suggests Ch.praecox may be multiple origins. One origination center of modern cultivated C. praecox is hypothesized to eastern China where Zhejiang LA is; while southwest China is another origination center including Chongqing Wuxi, Sichuan Wanyuan and Guizhou Guiyang. It consistent with the modern cultivation center of Ch. praecox. Cultivated populations of CJXF, CXT and CHF with some endemic haplotypes located in central China. It worthy of further study to know whether there were wild population existences.The study also reveals Ch. praecox is not experienced obvious genetic bottleneck for founder effect, artificial breeding and clonal propagation in the cultivation process insignificant, and the commercial, cultural intercourse make cultivated populations of Ch. praecox maintain higher genetic diversity.Based on the above analysis, this study suggests the important populations of Ch. praecox should pay attention to in situ conservation, the major cultivated populations should carry out the genetic screening to all members and establish a genetic profile in order to conservation and use some special individuals. The specific haplotype in different populations is an important repository to breeding fine varieties and quality gene and provide the basis for the cultivation and breeding of Ch. praecox.更多还原