【作者】 薛建华；

【导师】 卓丽环； 周世良；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2006， 博士

【摘要】 莲(Nelumbo nucifera Gaertn)是一个古老的孑遗物种,在系统与进化研究中有重要意义。又是食用、药用和观赏资源,还蕴藏着丰富的文化内涵。 野生莲是栽培莲的祖先,是培育优良莲品种的天然基因库。黑龙江流域是我国确实存在野生莲的少数地区之一。考古学者曾在黑龙江省依兰县第三纪的地层中发现莲叶化石。虽然有些学者于10年前对黑龙江省的野生莲做过资源调查和形态方面的观测,但对整个黑龙江流域野生莲的资源现状、遗传多样性的水平及分布格局等方面的研究仍为空白。 本文作者于2004年8-9月和2005年7-8月两次对黑龙江流域野生莲资源进行了实地考查和取样工作。两次踏查共发现56处野生莲的分布点,涉足45处莲的自然分布点,14处移栽点。在俄罗斯境内找到8个分布点。这些莲群体有些生长在人口密集的都市中,有些则在荒无人烟的沼泽中繁衍生息。作者将这些分布点用GPS进行了准确定位,同时与多数分布点建立了通讯联系,便于今后的跟踪监测。作者将野生莲群体的生存状况与前人调查的结果对比,随着降水量的减少和人们对湿地资源的不合理开发,黑龙江流域的野生莲面临着生境片断化、种群的面积减小甚至正在消失的严重威胁。随着风景点的建设引入栽培品种,当地的野生莲面临栽培品种基因污染的形势越来越严峻。 作者分别用RAPD、ISSR和SSR三种分子标记方法,在野生莲个体和群体水平上分析了遗传变异的大小及其遗传结构。 作者用RAPD和ISSR分子标记方法对来自黑龙江省47份野生莲、俄罗斯兴安斯克保护区的2份野生莲和中国其他省份的27份栽培莲进行了遗传多样性研究。20条RAPD引物共扩增出113个位点,多态位点占71.68%,期望杂合度0.1583。其中,野生莲的多态位点占50.44%,期望杂合度0.1241;栽培莲的多态位点占53.98%,期望杂合度0.1651。16条ISSR引物扩增出90个位点,多态位点占41.11%,期望杂合度0.0851。野生莲的多态位点占28.89%,期望杂合度0.0661;栽培莲的多态位点占32.22%,期望杂合度0.0963。说明野生莲的遗传多样性较低,且低于栽培莲。野生莲中,乌苏里江流域、松花江流域和黑龙江中下游流域之间的遗传分化很小,流域之间的遗传方差仅占21.68%(RAPD,Gst=0.1312)和15.11%(ISSR,Gst=0.1352)。在所有的遗传变异中,野生型和栽培型之内的遗传方差占73.25%(RAPD)和81.11%(ISSR),野生型和栽培型之间的遗传方差占19.17%(RAPD)和13.17%(ISSR),而3个野生流域群和1个栽培群之间的遗传方差仅占7.58%(RAPD)和5.72%(ISSR)。NJ分析表明,黑龙江流域的野生莲与其他地方的栽培莲有明显的遗传分化。在野生莲中,松花江中游地区的野生莲可能是黑龙江野生莲的残遗中心,由松花江中游向下游地区和黑龙江中下游与乌苏里江流域扩散。更多还原

【Abstract】 As an ancient relict species, lotus (Nelumbo nucifera Gaertn) plays an important role in system and development evolution studies. The lotus is food, medicine, and the plant widely used for gardening and landscaping. It has rich cultural values.Wild lotus is the ancestor of cultivar lotus, and serve as a natural gene bank for breeding of fine lotus varieties. The Heilong River Valley is one of the few places where are the natural habitats for the wild lotus. The archaeological scholar once had found the lotus leaf fossils in tertiary period stratum in Yilan County of Heilongjiang Province (middle reach of Songhua River). Although some studies did on field surveys and morphology observations on the wild lotus in Heilongjiang Province ten years ago, we only know little on its status, genetic diversity level and distribution pattern.The large scale of field surveys and samplings in Heilong River Valley from August to September in 2004 and from July to August in 2005. We identified 57 distributed sites for the wild lotus. There were 8 sites were from Russia. 44 the natural distribution sites and 14 transplanted places were staded. Some of these lotus communities grew in the densely populated cities, and others in the isolate and uninhabited marsh. We located and recorded the distributed sites by GPS. We also established a communication network with most of the places in order to follow up in the future. Comparing our results with previous studies, we found that the habitats of wild lotus in the Heilong River Valley has been facing with serious threats, such as fragmentation of the habitat, habitat loss and population declining due to climate change and illogical exploitation of wetland resource. Native wild lotus is facing more and more genetic diversity invasion with the construction of the scenic spots, cultivars were introduced.By using RAPD, ISSR and SSR three molecular marks, we revealed the variation sizes and the genetic structures at individual or colony levels.We study the genetic variations by using RAPD and ISSR markers to track 47 samples of wild lotus from Ussuri River, Songhua River and 2 samples from Heilong River Valleys in Russia and 27 samples of cultivated lotus from other provinces in China. Twenty RAPD primers generated 113 loci, of which 71.68% were polymorphic across all samples. The expected heterozygosity was 0.1583. The percentage of polymorphic loci and expected heterozygosity in the wild lotus were 50.44% and 0.1241, respectively. The parameters of the cultivated lotus were slightly higher than that of wild, 53.98% and 0.1651, respectively. Sixteen ISSR primers produced 90 loci. The percentages of polymorphic loci and expected heterozygosity were 41.11 and 0.0851. Polymorphic loci and expected heterozygosity were28.89% and 0.0661 for the wild lotus, and 32.22% and 0.0963 for the cultivated lotus. ANOVA analysis of the wild lotus showed that small variances existed among the 3 river valleys (21.68% for RAPD with Gst=0.1312 and 15.11% for ISSR with Gst=0.1352). The genetic variances of within the samples of the wild and cultivated lotuses were 73.25% for RAPD and 81.11% for ISSR. However the variances between the wild and the cultivated lotus were 19.17% for RAPD and 13.17% for ISSR, and variations among the valleys and the culta were least important (7.585 for RAPD and 5.725 for ISSR). Neighbor-joining analysis illustrated that there were significant differences between the wild and the cultivated lotus. The wild lotus at middle reaches of the Songhua River Valley might be the centre of remnants, from which it expanded to the Ussuri River Valley and the lower and middle reach of Heilong River Valley.4 pairs of SSR molecular markers were used to analyze 493 samples of 5 populations of wild lotus.and the preliminary results indicated each living colony of wild lotus was polyclonal, and the clone numbers were 19 to 41. The total clone numbers from 5 populations were 74. Each colony had a certain number of basic clone plants. The longest colony plant was close to the maximum sampling distance, which proved the lotus’ strong ability of colonial reproduction. The average allelic gene number of from 5 populations were 4.0000, The effective allelic number of 5 populations were 2.2311, and the average expected heterozygousity Nei,s was 0.3756. All of these suggest that at average colony level of the genetic diversity was still relatively low, but genetic similarity was very high (0.9314-0.9954) , divergences were small Fit (0.3490) . The most genetic variations occured within the colonies. Gene flow (Nm) among colonies was relatively high (6.8073). Allelic gene frequency was relative high and that could be caused by their seeds dispersal depending on water flows. Base on low contribution to genotype (25.68%-55.41%) , the valid group of the wild lotus was littlesmall, so the sampling tactics should aim at multiple colonies.In addition, comparing to cultivar lotus, wild lotus in Heilong River Valley possesses many differences in morphological characteristics, such as petal number, flower color, height of plants, and shapes of their roots and seeds. Within the wild lotus, the differences were not significant.The genetic diversity of a species was formed in the long evolution process. It was also influenced by colony origin, colony history, reproduction pattern and environmental variation, etc. The very limited genetic diversity of the wild lotus provided that the species had experienced severe bottle neck, founder and rebirth effects.Considering its long evolutiony, poor genetic diversity, small and isolated populations, fragmented of its habitat, and importance in wetland ecosystems, we suggest to take strongmeasures to protect the wild lotus in Heilong River, especially the populations in middle reaches of the Songhua River Valley, that could be the origin of the species.The research results on genetic diversity of wild lotus in the Heilong River Valley has filled the research gap in this respect, and which provided the solid theoretical foundation for preserving and utilization of its genetic diversity, and its origin and evolution process.更多还原