【作者】 李莹莹；

【导师】 郑成淑；

【作者基本信息】 山东农业大学 ， 观赏园艺， 2013， 博士

【摘要】 牡丹（Paeonia suffruticosaAndr.），世界名花，不仅观赏价值极高，经济价值也较高。它起源于我国，野生种和品种众多，蕴藏着极为丰富的基因资源。利用DNA分子标记方法对牡丹野生种和栽培品种开展遗传多样性分析和品种鉴定研究，对牡丹种质资源的有效保护和种质创新具有重要的指导意义。CDDP是一种新型目标分子标记技术，偏向于产生候选功能标记，所得标记可能是目的基因的一部分或与目的基因紧密连锁，属于显性标记，多态性好、操作简单、费用低，探讨该技术在牡丹种质资源遗传多样性研究中的应用不仅能够为牡丹种质资源的研究提供新途径和新方法，也能为其他植物开展相关研究提供借鉴。本研究在探索适用于牡丹基因组CDDP-PCR反应体系的基础上，以9个野生种、8个国外品种和299个我国中原牡丹品种共316份材料为试材，利用18条CDDP标记引物对其进行了遗传多样性分析、品种特异性分析和一致性检验，筛选了区分牡丹种质资源的核心引物并探讨了分子身份证的构建方法。主要研究结果如下：1．通过正交试验设计和应用Mix试剂对比CDDP-PCR扩增效果后，确定了20L牡丹CDDP-PCR反应体系：2×Es Taq MasterMix（含染料）10L、10pmol/L引物1.0L、30ng/LDNA模板2L和ddH2O7L，并进一步对该体系进行稳定性和重复性试验，认为该体系可用于牡丹种质资源遗传多样性等的分析研究。2．从21条CDDP引物中筛选出18条多态性好、条带清晰的引物用于牡丹种质资源的遗传多样性分析，揭示了牡丹野生种、国外品种以及国内牡丹品种遗传多样性的差异。结果显示，22个国内外栽培品种的平均Nei s基因多样性H为0.1950，平均有效等位基因数Ne为1.3060，平均Shannon信息指数I是0.3128，不仅明显高于299个我国中原牡丹品种的相应指标（分别为0.1658、1.2569和0.2695），而且分别比野生种的平均值高出20.14%、5.42%和16.63%。这说明国外牡丹品种的遗传多样性最高，我国中原牡丹品种居其次，而野生种最低。聚类结果显示，牡丹栽培品种与野生种矮牡丹聚为一类，表明牡丹栽培品种可能起源于矮牡丹；来自不同国家的品种没有独自分别成类，日本品种与我国中原牡丹品种聚在了一起，而美国品种与法国品种聚在了一起。3．10大花色群体的遗传多样性分析表明：10个花色群体的平均Nei s基因多样性指数、平均有效等位基因数和平均Shannon信息指数依次为0.1451、1.2313和0.2306。综合分析各项指标得出：红色系和紫色系具有较高的遗传多样性，复色系和绿色系牡丹的遗传多样性较低。花色群体间的遗传距离较近，平均为0.0271；遗传一致度较高，平均为0.9735；遗传分化系数为0.1252，表明只有12.52%的遗传分化存在于群体间；群体间还具有较大基因流值（3.4939）。遗传多样性分析和UPGMA聚类结果在分子水平上验证了牡丹品种资源的花色演化趋势：以粉色系和红色系为中心，渐渐演化出紫红、紫、蓝和白色系，再进化出黄色系和黑色系，绿色系和复色系属于退化的色系。4．综合分析品种间遗传距离的平均值、最大值和最小值，最后确定遗传距离临界阈值T=0.20为牡丹品种特异性评判的标准，即品种间遗传距离大于0.20时，则表明2个品种具有特异性。按此标准，对分别由12个单株构成的8个品种群体进行了品种内遗传距离的计算和分析，结果表明，供试牡丹品种具有较好的遗传一致性。5．对18条引物的多态性指标进行综合分析，将Pr2、Pr5、Pr12、Pr19、Pr20和Pr21确定为进行品种区分和鉴定的核心引物，并分别统计核心引物中单引物、双引物及三引物对牡丹种质资源的区分情况，筛选出了Pr2+Pr19+Pr20三引物组合用于构建分子身份证，并运用十进制数字串条形码的形式构建了316份牡丹种质资源的18位分子身份证，可以为实现牡丹种质资源的数字化检索提供参考。更多还原

【Abstract】 Tree peony (Paeonia suffruticosa Andr.) is a famous flower in the world for its highornamental and economic value with origin in China. There are many wild species and lots ofcultivars containing abundant gene resources. Study on the genetic diversity and cultivaridentification of tree peony by DNA markers will has an important guiding significance to theeffective protection for tree peony germplasm resources and its germplasm innovation. CDDPis a kind of new method belonging to gene-targeted markers technique. This method tends togenerate candidate function markers which may be a part of or linked with the gene targeted.It is a kind of dominant marker with good polymorphism, easy operation and low cost.Researches on tree peony germplasm genetic diversity by CDDP technique can provide notonly a new way or method for tree peony researches but also an available reference for otherplants to carry out related researches.In this study, optimized CDDP-PCR reaction system for tree peony was established and316genome DNA templates composed of9wild species,8foreign cultivars and299Zhongyuan tree peony cultivars were amplified with18informative and reliable CDDPprimers. Then, the amplification results were used to analyze the genetic diversity, the cultivardistinctness and uniformity of tree peony. At last, core primers were selected to indentifydifferent tree peony germplasm resources and to set up their molecular ID codes. The mainresults were as following:1. After comparing the results of orthogonal experimental design and PCR-Mix reagentapplication, the optimized CDDP-PCR reaction system(20L) were2×Es Taq MasterMix(dyed)10L,10pmol/L primer1.0L,30ng/L DNA template2L and ddH2O7L.Researches on the stability and repeatability of the above reaction system resulted that it couldbe used for genetic diversity analysis and its follow-up studies of tree peony.2. Eighteen out of21CDDP primers with good polymorphism and clear bands wereselected to reveal the differences among genetic diversities of wild species, foreign cultivarsor Zhongyuan cultivars in China. Results showed that the average Nei s gene diversity (H),average Shannon information index (I) and average effective number of alleles of22individuals composed of abroad and Zhongyuan cultivars was0.1950,0.3128and1.3060,respectively. As for299Zhongyuan cultivars, the above indexes were0.1658,1.2569and 0.2695in turn, which were all lower than those of22cultivars. And the values of299Zhongyuan cultivars were higher than those of wild species by20.14%,5.42%and16.63%inturn. These results indicated that the genetic diversity of abroad cultivars was the mostabundant and that of Zhongyuan cultivars was more while that of the wild species was thelowest. In addition, clustering results showed that the22foreign and domestic cultivars wereall clustered with P. spontanea, indicating that tree peony cultivars should originate from P.spontanea, while cultivars from different countries didn t get into a class respectively. As forforeign cultivars, Japan cultivars were in together with Zhongyuan cultivars in China andUSA or France cultivars were clustered together.3. There were10populations with different color. Results of their genetic diversityanalysis showed that average Nei s gene diversity (H), average Shannon information index (I)and average effective number of alleles was0.1451,1.2313and0.2306, respectively.Comprehensive analysis results indicated that the genetic diversity of red or purple populationwas higher while that of multicolor or green population was lower. The genetic distanceamong10populations was close with an average of0.0271and the genetic identity was highwith an average of0.9735. The coefficient of gene differentiation among them was0.1252,showing that there were12.52%genetic variation among populations. The gene flow among10populations was high and its value was3.4939. Results obtained from genetic diversityanalysis and UPGMA dendrogram based on Nei s genetic identity proved the evolutionarytrend of tree peony s flower color at molecular level: pink and red cultivars might be at thecenter of Zhongyuan tree peony resources, then gradually fuchsia, purple, blue, white, yellowor black cultivars were evolved, while green or multicolor cultivars might be degraded.4. After comprehensively analyzing the average, the maximum and the minimum valuesof genetic distances between an arbitrary cultivar and the others cultivars, the criticalthreshold T=0.20was determined as the evaluation criteria of tree peony cultivar distinctness,meaning that when the genetic distance between2cultivars was higher than0.20, they wereregarded as2different individuals. Then the genetic distance among12individuals belongingto the same cultivar were calculated and8cultivars were involved. According to the abovecriteria, tree peony cultivars used in this study had good genetic uniformity.5. Several polymorphism indexes of18CDDP primers showed that Pr2, Pr5, Pr12, Pr19, Pr20or Pr21could be core primers to identify tree peony germplasm resources. Based on theirability to distinguish tree peony gemplasm alone, two or three different primers combination,three primers combination of Pr2+Pr19+Pr20was selected and applied to set up the molecularID of tree peony. In the end, the IDs with18decimal numbers of316tree peony germplasm resources were structured and provided a new method for the realization of digital searchingfor tree peony resources.更多还原