【作者】 Frédéric Ngezahayo；

【导师】 刘宝；

【作者基本信息】 东北师范大学 ， 遗传学， 2009， 博士

【摘要】 人参是重要的药用植物,被称为“百草之王”。在野生人参与栽培人参之间巨大的价格差异的商业利益驱使下,人们对野生人参资源进行过度开采,以至于野生人参已经处于灭绝的边缘,现在已经不得不通过栽培来满足野生人参市场的需要。众所周知,对于植物物种的栽培驯化会导致其遗传多样性的大量丢失。为了了解野生和栽培人参类型之间遗传多样性的差异,我们开展了本文研究工作。然而,关于人参野生型和栽培型的遗传和表观遗传差异依然处于未知阶段,为解决此问题,我们首先选择中国人参(Panax ginseng)野生和栽培两个类群中的17个单株,西洋参的4个单株和一个五加科外群种(短梗五加Acanthopanax sessiliflorus)的1个单株。利用筛选的24对引物,进行了扩增片段长度多态性(AFLP)分析,共产生了1490个位点(平均每对引物62个位点),其中有1342个位点在22个单株中呈现多态性,多态性水平为90.06%。UPGMA聚类分析显示,在所有研究的人参单株中,只有西洋参形成一个单独的分组,野生型和栽培型没有形成明确的分组。在随后的分析中也证明了这一点:PCOORDA分析显示了相同的分组情况。我们发现西洋参与人参的几个亚组之间存在着很高水平的的遗传差异,范围在0.3699 (AMG vs. WLG)到0.4715 (AMG vs. CHB)之间。在栽培和野生型(WLG and WDM)人参中只发现了较低水平的遗传差异,除了“长脖”和“石柱”之外其他的都几乎具有相同的差异趋势。另外,这两个组(野生和栽培人参)之间遗传差异的相关系数很低(0.2057),说明只有25.57%的遗传差异属于两个组之间的,79.43%的遗传差异都属于这两个组之中单株之间的差异。AMOVA分析显示遗传多样性的大部分(91.64%; P <0.001)存在于组内(野生/栽培),而只有8.36% (P >0.001)的遗传多样性存在于两个组之间。我们进一步进行了甲基化敏感多态性分析(MSAP),通过筛选的28对引物进行扩增,总共生成1821个清晰可重复的位点,其中有379个(20.81%)是甲基化的位点,79.19%是非甲基化位点。甲基化不敏感多态性(MISP)分析的结果与AFLP分析结果相一致。结合AFLP和MIP结果分析显示,人参野生和栽培群体之间遗传距离非常近。与遗传多态性相反的是,甲基化敏感多态性(MSP)结果显示出不同的单株形成明显的分组,除了天然野生型(WLG)和栽培野生型(WDM)之外,每个亚组都能够单独聚类,前者仍然聚类成一组。PCOORDA分析显示出相同的分组结果确证了前述结论。同时在CG和CNG位点甲基化水平上野生型和一些栽培型中发现了明显的差异,“长脖”和“石柱”的甲基化水平最低,只有野生型组群(WLG或WDM)的三分之一左右。进一步AMOVA分析显示组间DNA甲基化多样性要比AFLP和MIP方法高(分别为8.36%和9.17%)。两组之间相关性分析,例如野生和栽培人参,显示了表观遗传差异在平均水平上高于遗传差异。这表明人参的驯化过程在全基因组水平上诱导产生了大量甲基化多态性。最后,通过亚硫酸盐测序对甲基化敏感多态性结果进行验证,结果显示栽培型(特别是“长脖”和“石柱”)中的甲基化水平较低,野生型人参的甲基化水平较高。基于Jaccard相似性相关系数的Mantel分析显示,在所研究的单株中,遗传和表观遗传多态性没有显著相关性,这进一步说明在人参驯化过程中,DNA甲基化水平和模式受到了较大的影响。将两个亚组之间呈现遗传和甲基化差异的条带回收测序并进行同源性分析,结果显示,有部分条带序列与已知功能或推测蛋白编码基因同源,然而很少甚至没有与重复序列如转座子和反转座子同源的序列。这暗示,这些野生和栽培人参中发生的表观遗传变异的序列可能影响了基因的差异表达。更多还原

【Abstract】 Orient Ginseng (Panax ginseng C. A. Meyer) is one of the most important global plants used in traditional medicine. The demand of this slow growing plant has exceeded its resources in the wild; this has necessitated a commercial cultivation although the wild type is still ten thousands costlier than the cultivated type in the markets. Hence, this valuable wild plant remains endangered due to the commercial differences between the wild and the domesticated types. Although studies in other plants have shown that the domestication of a plant has a tendency of narrowing genetic diversity, the genetic diversity and epigenetic variation between the wild plants and the cultivated landraces of the Orient ginseng remains unexplored.To address these issues, amplified fragment length polymorphism (AFLP) analysis was performed with 24 selected primers on seventeen Orient ginseng plants to represent: three natural wild plants (WLG), three cultivated wild plants (WDM), and eleven domesticated landraces; (Damaya (DMY), Yuanbangyuanlu (YUAN), Changbo (CHB), and Shizhu (SHIZHU), together with four cultivated American ginseng plants (AMG) and one Acanthopanax sessiliflorus as an outgroup. The selected AFLP primers generated a total of 1490 loci (average 62 loci per primer) of which 1342 loci were polymorphic among 22 plants with a high level of polymorphism (90.06%). UPGMA Cluster analysis showed that, apart from the American ginseng that formed a distinct cluster, no clear grouping for all studied plants of ginseng was evident either for the wild or the domesticated Orient ginseng landraces. This was corroborated by principal coordinate analysis (PCOORDA) that showed the same grouping. High levels of genetic distance were observed between American ginseng and each sub-group of the Orient ginseng, ranging from 0.3699 (AMG vs WLG) to 0.4715 (AMG vs CHB). Lower levels of genetic distance were observed between WLG and WDM and the cultivated plant groups (DMY, YUAN, CHB, and SHIZHU) and were almost in the same trend though Changbo and Shizhu tended to be distant from the others. In addition, the coefficient of genetic differentiation between the two groups (Wild and Cultivated ginsengs) was low (Gst = 0.2057), showing that only 20.57% of genetic differentiation resided between the two groups, while 79.43% resided in different plants of the two groups. Analysis of molecular variance (AMOVA) showed that a significant proportion of genetic variation (91.64%; P <0.001) resided within groups (wild/cultivated) while only 8.36% (P >0.001) of the total genetic variation resided between the two groups.Secondly methylation sensitive amplification polymorphism (MSAP) with 28 selected primers was performed on the same plants. This generated a total of 1821 clear and reproducible sites, of which 379 (20.81%) were methylated while 79.19% were un-methylated. Similar results to AFLP markers were observed by using methylation insensitive polymorphisms (MIP). The combined AFLP and MIP results showed that wild and cultivated groups of P. ginseng were not genetically distinct. On the contrary, methylation sensitive polymorphisms (MSP) showed a distinct grouping of different plants, each sub-group forming a cluster although the natural wild (WLG) and the cultivated wild (WDM) remained clustered together. This was corroborated by principal coordinate analysis (PCOORDA) that showed the same grouping. Clear differences in methylation levels at both CG and CHG sites between wild and some cultivated plants were also observed, the lowest levels of methylation being observed in Yuanbangyuanlu, Changbo and Shizhu being almost a third of the methylation level in wild groups (WLG or WDM). Moreover, AMOVA showed that the inter-group epigenetic variation was higher than AFLP and MIP methods (8.36 and 9.17% respectively). Coefficients of epigenetic differentiation showed a proportion higher than genetic (Gst = 34.59%) resided between the two groups, i.e., wild and cultivated ginseng plants. This indicates that domestication of ginseng engendered clear global methylation polymorphisms between wild and cultivated plants of P. ginseng.Finally, bisulfite sequencing analysis validated the MSP results showing lower levels of methylation in the cultivated type (especially in Changbo and Shizhu) while higher levels were shown in the wild groups of P. ginseng. Mantel test based on Jaccard coefficients of similarity showed absence of correlation between genetic and epigenetic polymorphisms between the studied plants, suggesting that only the cytosine DNA methylation was affected by domestication of ginseng. Sequence analysis of a subset of genetic and epigenetic variable bands between different sub-groups of ginseng showed homology to known-functional and putative protein-coding genes while repetitive sequences such as retrotransposons or transposons were less-represented.更多还原