【作者】 程远辉；

【导师】 周昌华；

【作者基本信息】 西南大学 ， 作物栽培与耕作学， 2007， 硕士

【摘要】 目前，何首乌野生资源濒临灭绝，人工种植刚刚起步，而栽培用种混乱，尚处在原始的混杂群体状态，严重影响产量的提高和质量的稳定。所以急需加强野生资源的保护和引种驯化研究，加快对种质资源的调查整理，筛选出遗传性状独立的优良材料，符合国家GAP(中药材生产质量管理规范)要求来进行生产推广。而用作中药种质研究的方法有多种，包括最初的形态学研究；细胞学标记技术即核型分析和解剖学鉴定；随后产生的生化大分子标记包括同功酶和蛋白质指纹标记；也有通过化学成分进行种质鉴定和分类。但以上各种途径所获结果都要受环境条件影响或植物本身的限制，或者是操作烦琐，花费较多人力、物力，更为致命的是它们技术本身所存在的弊端导致所得结论并不是种质遗传性状的直接反映。而分子标记可以比较直接的反映作物的遗传特性，特别是DNA分子指纹图谱技术的快速发展和应用使中药种质从分子水平即真正意义上的鉴定成为可能。此类技术在中药资源整理、种质鉴别等工作中已得到广泛的应用。本研究通过遗传因子和生理生化指标两方面对重庆何首乌种质进行整理、评价，探求不同形态和地区何首乌种质资源的遗传关系和丰产性状差异，为下步选种、育种打下良好的理论基础。1遗传分析采用SRAP一种分子技术对重庆何首乌种质资源进行了聚类分析和遗传鉴定，该标记技术具有简便、稳定、中等产率等特点。结果显示了极大的优越性和可行性，尤其是扩增的条带多而且清晰，易于分辨。从104对引物扩增得到的数据，根据Nei和Li的公式计算出了取自重庆不同地区的16组何首乌材料间的遗传距离。其中采自重庆石柱的材料10与北碚的16的遗传距离最大，为0.77；1与2间的遗传距离最小，为0.089；16份何首乌的平均遗传距离为0.44，说明16组不同区域的何首乌在分子水平具有较大差异。其中材料9与所有材料的平均遗传距离为0.63，远高于平均水平，可视为特异材料。根据各实验材料之间的遗传距离，以0.50(Eu-clidean distances)为临界值，可将16份材料大致划分成两大类和一个变异材料9，同一地区的材料大体上聚到一起，地理位置较远的在聚类图上分的也比较开。聚类结果显示，何首乌资源遗传关系大体上符合地域差异，与王凌晖等结论相符，但也不能完全按照地理位置来判定它们的亲缘关系，应有少量的变异。作者从重庆石柱地区取的五个材料中10、11、13三个材料关系相邻，而材料9和12则都具有了各自较为独立的遗传特性，可能是由于环境影响和长期演化所导致的变异。另外何首乌是自花授粉植物，理论上变异和分化的几率较低，而作者在取材范围较为狭窄的情况下所得出的遗传数据差异，说明了何首乌也有很丰富的遗传多态性。2生理指标测定分析分别对17个何首乌材料进行了叶面积、叶绿素含量、叶片厚度、光合、叶片夹角、根系活力等生理指标的测定和观察，结果表明贵州材料17在理论上应为遗传差异最大的材料，其生理指标表现也较为突出。材料12从聚类图上来看也为较特殊的材料，其形态和生理指标表现也非常特殊。而在遗传分析中最为特殊的材料9在生理指标上却表现平平。3综合分析遗传距离近的材料生理指标也大致相关，如材料1和材料2，材料10和材料11，材料5和12等。但也有例外，如材料15和16在叶绿素含量、叶面积和根系活力上等都相差较大。最后确定了较为优异的材料，材料4、材料10和材料17为优良材料，可以考虑作为生产用种；材料9和材料12可以作为特异种质保留，做为进一步选育的资源材料。遗传性状特异的材料其生产指标表现并非较好，所以作者认为为生产来选择优良材料，仅从现有资源中，筛选出丰产性状好的材料即可，只是要尽量保证种质纯度的一致性。而深一步研究可采用遗传分析方法进行资源分类和鉴定，来保护何首乌资源的遗传多样性。更多还原

【Abstract】 At present,the wild species of the Polygonum multiflorumtm Thunb is on the edge of extinction,the cultivated one has just began,however,the use of cultivated seeds is on the original disorder bulk status,that will badly influence the improving of the yield and the stability of the quality. So it is crying for reinforcing the protection of wild sources and the research of introduce domestication,stepping the investigation and arrangement of germplasm on the juice and selecting the superior material which conforms GAP to promote the yield. There are several methods for the research of traditional Chinese drugs germplasm,the morphology marker,cytology marker that is konyotype analysis and anatomy identification came out originally,then followed by biochemical macromolecules marker that contains enzyme and protein fingerprint marker. To identify and category the germplasm,analysising the chemical constituent is also feasible. All the results of those methods are influenced by environment and limited by plants themselves.Their procedures are very complicated,cost too much labor and material,however, the more fatal shortcoming is the result is not the direct reflection of the character of the germplasm due to the shortcoming of their technology.The results of molecular marker can reflect the plant’s inheritable character especially with the development and application of the DNA molecular fingerprint technology,it is possible for traditional Chinese drugs germplasm on the molecular level that is also on the real meaning.It has been widely used for resource management and identification,and other research of traditional Chinese drugs. This study assessed and collated Chongqing Polygonum multiflorumtm Thunb germplasm and search for genetic relationship and high-yield character among different morphology and area of Polygonum multiflorumtm Thunb germplasm. That is to theoretical base for seeds selection and breeding.1 Genetics analysis A new marker system SRAP was used to detect the polymorphisms of Polygonum multiflorumtm Thunb in Chongqing. Collect different shape of Polygonum multiflorumtm Thunb from major production areas.The SRAP was used to asses divergence among 16 populations.The data were analyzed using unweighted pairgroup method, based on arithmetic averages(UPGMA) bootstrap analysis.Cluster analyses were performed by using DPSv3.01 software,the alkaloid was extracted from P.ternate with chlorolform.104 combinations generated 250 polymorphic bands,the cluster analysis indicated 16 materials could be distinguished into two main groups and one special type,Nei&Li similarity coefficient ranged from0.23～0.99,and the average distance is 0.44.The most close two materials are numberl&2,on the contrary,the most further two materials are number 10 and 16 separatly from Shizhu and Beibei.The cluster analysis shows that affinity relationship conform their distribution areas, coincidenting with the study of WangLing-hui.But we can’t differ them by their areas absoultly,there exist differentiation yet.In the stuff of Shizhu 10,11 and 13 are very close,but 9 and 12 both have theirs independence,that maybe result from the effect of circumstance and the long time evolution.In addition,polymorphisms of Polygonum multiflorumtm Thunb is self-pollinated plant,there is lower differetntintion in theory,but the author choose a very narrow area in this study,in spite of that, the results of the study showed a potential application of SRAP fingerprinting for the identification of Polygonum multiflorumtm Thunb.2 Physiologic index analysis The author mensurated the leaf area,chlorophyll content,thickness, morphologic observation,included angle and the root system activity of 17 Polygonum multiflorumtm Thunb materials respectively,the result indicated that the 17 materials’ physiologic index performance is most prominent,the material 12 is a special stuff shown from the dendrogram,and it’ morphology & physiologic index performance looks exceptional. The most distinctive material in genetic analysis is number 9,but it’ s physiologic index is very common.3 Comprehensive study The adjacent materials of genetic distance shows very correlative in physiologic index,such as number l&2,number 10&11,number 5&12,and so on.But there is also exception,such as numberl5&16 differs largely in the leaf area, chlorophyll content, and the root system activity.Screened out some excellent materials,numberl,10 and 17,we can take them as releasing variety;number 9 and 12 could be conserced as germplasm resources.The special material in Genetics analysis are not always exclent in production,so in my opinion ,when we choose the good breed for out production,we just search them from the existing resource on hand,but we should try our best to maintain the variety puritify.If we want to study it in-depth,we had better sort the resources and identify them firstly by this way,and then we can protect diversity natural resources.更多还原