【作者】 宋振巧；

【导师】 王洪刚； 王建华；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 丹参(Salvia miltiorrhiza Bge)是我国常用大宗药材,具有广泛的药理作用,药材使用量逐年增加。随着丹参野生资源的数量逐年减少,栽培面积逐年增大,但是栽培丹参存在严重混杂现象。目前丹参的种植主要集中于四川的中江、陕西、山东、河南、河北等地。对各产地丹参种源进行遗传多样性和群体遗传结构分析,明确各产地丹参资源的遗传基础和亲缘关系,可为丹参资源的收集保存、分类鉴定、合理开发利用奠定基础,对解决当前丹参种质资源混杂和指导丹参品种选育具有重要的意义。本研究以来自山东省内外的9个丹参居群为材料,从形态学、细胞学、分子生物学等方面对其形态性状、开花生物学及繁殖特点进行了鉴定和观测,对供试丹参资源各居群的遗传多样性及亲缘关系进行了分析,对11个丹参分离类型进行了产量和品质性状的评价,从中筛选出适于泰安种植的优良栽培类型。获得的主要研究结果如下:1.通过对9个居群的丹参种质资源地上部性状分析证明,42个性状中叶柄颜色、叶柄长、叶长、叶宽、叶形指数等叶片性状、花色、花萼颜色、株高、冠幅、分枝数等在丹参种质资源中的多样性较大;其遗传多样性存在于居群内和居群间且两者变异程度相近。通过各居群的遗传多样性比较发现,泰安地区的丹参居群多样性程度最高,菏泽和四川中江的丹参居群较低,其它居群的多样性指数比较接近;省间材料聚类分析结果表明,河南内乡与河南卢氏丹参居群的亲缘关系最近,山东泰安与其它居群的亲缘关系最远;山东省内潍坊与临沂居群的亲缘关系最近,菏泽与其它居群的距离最远。42个性状聚类可分成6组,综合为7个主成分,其累积贡献率达70.17%;根据主成分分析的结果与性状间的相关性,选出影响力较大的16个性状,包括花部因子、茎部因子、叶部因子等地上部性状,可较好的代替42个性状用于种质聚类分析。利用欧式遗传距离进行UPGMA聚类可将90个材料分为6个类群;根据主成分分析结果和各类群特点,提出了丹参的分类标准,该标准对今后丹参种质的评价具有一定的指导意义。2..利用根尖细胞对9个居群丹参材料的染色体数目进行了鉴定,并对其核型进行了分析。结果表明,9个丹参居群的根尖细胞染色体数目均为2n=16,没有观察到多倍体细胞和B染色体;9个材料除四川中江丹参的核型公式是2n = 16 = 6m + 10sm,其它8个材料的核型公式均为2n = 16 = 8m + 8sm;在其中6个材料中观察到2条具有随体的染色体;利用染色体相对长度和臂比进行聚类,可将9个居群的材料分为三类。花粉母细胞减数分裂观察结果表明,不同材料的16条染色体在减数分裂中期I均能正常配对形成8个二价体体,丹参的减数分裂属于同时型。在丹参的减数分裂中期I观察到频率较高的细胞融合现象。3.以山东泰安、临沂、莱芜、菏泽和潍坊5个居群的56个丹参株系为材料,利用ISSR和SRAP引物进行群体遗传结构的分析。结果表明: 8个ISSR引物和5个SRAP引物在5个居群中分别检测出219和82个位点,在种级水平上多态性位点百分比为97.72%和91.46%,省内5个丹参居群在种级水平上的遗传多样性丰富;在居群内每个引物平均分别检测到16.6和9.36个多态性位点,多态性位点比例为60.63%和57.07%。根据ISSR和SRAP检测的遗传分化系数Gst表明丹参资源的遗传多样性主要存在于居群内,居群间的遗传变异分别占总变异的13.05%、20.19%;两种标记检测的5个居群遗传多样性表明,泰安丹参居群的遗传多样性最高,其次临沂和莱芜较高,菏泽丹参居群较低,潍坊居群最低。由两种标记分析的各居群间的遗传关系表明,临沂和潍坊居群遗传距离较近首先聚类,其次与菏泽遗传距离也较近,三者可聚为一类,泰安与其它居群的亲缘关系最远聚在另一类,莱芜居群使用标记不同而聚类结果不一致;山东丹参各居群间的遗传距离与地理距离间具有一定的相关性。利用现有的ISSR和SRAP标记不能将白花丹参与紫花丹参区分开。4.利用ISSR、SRAP标记对国内5个居群的丹参种质进行了遗传多样性和群体遗传结构分析。5个ISSR引物和6个SRAP引物分别检测出120、122个位点,在种级水平上多态性位点百分比分别为100%和90.10%,表明丹参在种级水平上的遗传多样性丰富;每个ISSR、SRAP引物在居群内分别平均扩增出14.48、11.27条多态性条带,多态性比率分别为48.27%、55.10%。根据遗传分化系数Gst表明丹参资源的遗传多样性主要存在于居群内,居群间的遗传变异不足20%。利用两种标记检测的不同丹参居群的遗传多样性分析表明,河南卢氏居群的遗传多样性最高,泰安居群次之,四川中江居群最低,根据nei遗传距离和UPGMA聚类分析结果,四川中江与陕西商洛居群的遗传距离较小可聚在一类,河南卢氏与内乡居群的亲缘关系较近,泰安与其它4个居群的亲缘关系最远,但可与河南两居群聚于另一类。基于Nei氏无偏估计的遗传一致度的聚类分析表明,多数居群的遗传距离与地理距离间有一定的相关性。5.对丹参11种栽培类型分别进行了地上部性状调查、单株根鲜重等根部性状测量,以及有效成分含量测定,结果表明紫花单叶圆形类型田间农艺性状较好、单株产量和有效成分含量处于较高水平,较适合于山东泰安等丹参道地产区种植。6.对丹参的开花生物学初步研究结果表明,丹参单花花期约5～7 d,从花前2 d到开花6 h内花粉活力和柱头可授性都较大,两者有效可遇期约1.5 d,丹参自交结实和自然结实主要是通过同株异花传粉获得。结合花粉/胚珠比、杂交指数以及繁育处理的结果表明,丹参的繁育类型为兼性异交,自交亲和,需要传粉者,传粉媒介主要是蜂类。初步推断丹参为常异花授粉植物,并对丹参育种中适宜采用的杂交方法及育种途径进行了探讨。更多还原

【Abstract】 Salvia miltiorrhiza Bge is a traditional Chinese herb plant, its root has been used for centuries to treat diseases such as angina pectoris, cerebral vascular diseases, degenerative diseases, acute or chronic liver injuries, and has extensive and important curative effects in practice. While quantity need for this medicinal material increased and wild resource dropped off year after year, the planting area was added, but there existed severity farrago in cultivate regions. Now the plant region mainly distributed in Sichuan, Shanxi, Shangdong, Henan and Hebei province in China. Research about genetic diversity and genetic structure of different S. miltiorrhiza geographical populations in China would benefit for understanding of the genentic base and genetic relations among geographical populations, and also benefit to the germplasm collection, sonservation, utilization, and improvement of S. miltiorrhiza.In this study, S. miltiorrhiza germplasms from nine geographical populations in China were analysised by morphological, cytology, molecule biology method. Morphological variation, the floral biology and breeding system of S. miltiorrhiza was preliminary studied, the genetic diversity and relation between populations was investigated Moreover the suitable variety for cultivation in the Shandong region was selected based on root character and the content of active ingredients. The results are as follows:.1. There were extensive variations in morphology characters indifferent geographical populations of S. miltiorrhiza. Among forty-two morphologic traits, the most significant variance lay in petiole color and length, leaf width and length, leaf shape index, flower color, plant highth and plant breadth, ramification number. Inter-/intro-population variation occurred significantly. Genetic differentiation between and within five eco-geographical populations of S. miltiorrhiza was estimated by Shannon-Wiber diversity index, which showed that genetic variance existed within and among populations with same frequency. Comparing with Shannon-wiber diversity index for nine populaitions in China, the genentic diversity of Tai’an was the highest, Heze and Zhongjiang populution were less. Fourty-two morphological traits could be divided into seven groups Principal component analysis(PCA) showed that the forty-two characters can be consolidated for the seven principal components and their accumulative contribution ratio amounted to 70.17%. according to the relativity between characters and principal component analysis , sixteen major discriminating charactics were selected, synthesizing flowr factors, blanches and leaves factors. These characters would better used to analysis and cluster for S. miltiorrhiza replacing 42 characters. Based on UPGMA analysis of Euclidean distance among individual cultivars of S .miltiorrhiza, germplasms were clustered into 6 groups. Moreover based on the characteristics of each group and the resultof PCA the classification of S. miltiorrhiza was taked out which would benenfit for assession of S.miltiorrhiza germplasm in future.2. Mitosis study showed that the chromosome numbers of nine geographical populations of S. miltiorrhiza were also 2n=16, no polyploidy cells and B chromosome were observed. The karyotype of Zhongjiang population was 2n = 16 = 6m + 10sm, and the karyotype of other population were 2n = 16 = 8m + 8sm. Among six populations, two chromosomes with satellite was observed. Nine population could be cluster into three type based on the relative chromosome length and arm ratio. Meiosis study showed that all the sixteen could be paired into eight bivalents at metaphase I, and high frequency of cytomixis were observed in prophase I.3. Fifty-six S. miltiorrhiza lines from five different geographical populations located at Tai’an, Linyi, Laiwu, Heze and Weifang in Shandong province of China were studied by ISSR and SRAP markers. The results indicated that percentage of polymorphic loci (P) on species level was 97.72%, 91.46% respectively, it indicated that there were abundant genetic diversity on species level. Average 16.6 polymorphic bands with in every ISSR primer in each population were detected and the percentage of polymorphic loci (P) was 60.63%. Average 9.36 polymorphic bands in every SRAP primer in each population were detected, the percentage of polymorphic loci (P) was 57.07%. Genetic differentiation between and within five eco-geographical populations of S. miltiorrhiza was estimated by genetic differentiation coefficient (Gst=0.1352, 0.1892) which showed that 13.05%, 20.19%genetic variance was among populations revealed by ISSR and SRAP markers. More than 80% genetic variance existed within populations. The genentic diversity Comparing with percentage of polymorphic loci, Nei’s gene diversity index and Shannon’s information index, the genentic diversity from Tai’an was the highest, Linyi and Laiwu was higher and secondly Heze with the last. UPGMA cluster analysis indicated that Laiwu population and Linyi population were higher in genetic identity which was nearer in genetic relationships, the genetic distance between heze and above two populations was also near therefore the three populations could be cluster into one group, however the genetic distance between Tai’an population and other four populations were farther therefore into another group. Furthmore the genetic relationships beween Laiwu population and other populations was different revealed by ISSR and SRAP markers.. Based on Nei’s unbiased measures of genetic identity, UPGMA cluster analysis measures of five eco-geographical populations. It indicated that there was a correlation between genetic relationships and geographical distance among the populations. Based on the study of population genetic structure, owing to the highest genetic diversity, Tai’an population should be given a high priority consideration in Salvia miltiorrhiza populations in situ germplasm conservation strategies.Moreover4. Sixty-two S. miltiorrhiza from five different geographical populations located different province of China were studied by ISSR and SRAP markers. The results indicated that a total of 120 and 122 loci were respectively amplified from S. miltiorrhiza with five ISSR primers and six SRAP primers, the percentage of polymorphic loci (P) on species level was 100%, 90.10% respectively by two different markers which revealed that there were abundant genetic diversity on species level. Average 14.48 and 11.27 polymorphic bands with in every ISSR and every SRAP primer in each population were detected with the average percentage of polymorphic loci (P) was 60.63%, 57.07% respectively. Genetic differentiation between and within five eco-geographical populations of S. miltiorrhiza was estimated based on genetic differentiation coefficient (Gst), which showed that 80% genetic variance existed within populations, and less than 20% genetic variance was among populations revealed by ISSR and SRAP markers. Comparing with percentage of polymorphic loci, Nei’s gene diversity index and Shannon’s information index, the genentic diversity of Lushi population was the highest, secondly Tai’an population, Shangruo population from Shanxi province and Neixiang population from Henan province were high too with the last.Zhongjiang population from Sichuan province. UPGMA cluster analysis indicated that Zhongjiang population and Shangruo population also higher in genetic identity were clustered into one group, the genetic distance between two populations of Henan province was nearer , Tai’an population were relative independent populations, but Tai’an population and two populations of Henan provinc relative near therefore the three populations could be cluster into another group. Based on Nei’s unbiased measures of genetic identity, UPGMA cluster analysis measures of five eco-geographical populations. indicated that there was a correlation between genetic relationships and geographical distance among the populations.5. Different lines of Salvia miltiorrhiza were separated in field based on the biological characteristics, the overground biological characters and root character and contents of active ingredients including TanshinoneⅡA, Cryptanshinone and Salvianolieaeid B of them were evaluated. The results showed that unifoliate round leaf typr of S. miltiorrhiza have better agronomic characters , weighter root and higher active ingredients, it was more suitable for cultivation in the Shandong region.6. Floral biology of Salvia miltiorrhiza showed that the flowers of S. miltiorrhiza were bisexual and never-ending, the blossom of a single flower could last approximately 5?7 days. Pollination peak last from 2 days before blossom to 6 hours after blossom, and the higher stigma receptive stage was the same time. Self-pollination was possible by temporal and spatial superposition of male and female organs for 2 day within the same flower. Seed set percentage of autogamous selfing in isolation without emasculation condition and geitonogamous selfing mainly pollinated through different flower in one plant. The results indicated that no apomixis occurred, the breeding system was outcrossing with self-compatibility and pollinators were needed based on the pollen-ovule ratio(p/o), out-crossing index and the results of emasculation, bagging and artificial pollination studies. The most frequent floral visitors were bees. It was inferred elementary that S. miltiorrhiza belong to often cross-pollinated plant. The crossing method and breeding strategy were discussed.更多还原