半夏遗传多样性分子标记及质量评价研究
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摘要
半夏[Pinellia ternata(Thunb．)Breit．]为天南星科半夏属植物，以其干燥块茎入药，药材名半夏(Rhizoma Pinelliae)。半夏为东亚特有种，分布于中国、朝鲜和日本。在我国大部分省区有分布。半夏是我国传统的大宗药材，因其疗效确切、临床应用范围广泛，近年来国内外需求逐渐增加，5年内市场价格增长近一倍，出现了供不应求局面。由于人们长期过度采挖、耕作方式改变和自然环境恶化等原因，半夏野生资源急剧减少。20世纪70年代半夏开始家种后，出现了许多栽培类型，但由于连年栽培，品种严重退化，极大地影响了半夏生产，致使半夏供求缺口日益增大。另外，栽培类型混乱也严重影响着半夏用药稳定性。因此半夏种质资源亟待整理和评价，以便为半夏核心种质资源库的建立提供有力保障。
本研究收集了我国半夏分布区大部分种质，在分子水平上分析了半夏遗传多样性，了解其群体遗传结构和多态性水平，并进行了类群的划分，为制定和实施半夏资源保护利用策略提供有力理论依据。其次，采用3种方法分离获得了半夏的微卫星序列，并分析了其特点，为SSR分子标记在半夏中的应用奠定了基础。最后，本文进行了半夏种质资源药用品质、安全性和农艺性状质量评价。具体研究内容和结果如下：
1．利用分子标记技术分析我国半夏种质资源遗传多样性
以取自全国半夏主要分布区的16个省的24份种质的240个样本进行遗传多样性分析。利用RAPD标记技术分析，结果表明在物种水平上，多态位点百分率(PPL)为68.77％，Nei’S基因多样性指数(He)为0.2062．居群水平上的多态位点百分率在9.88％-58.02％之间，平均为27.21％，最高为沅陵居群，其次为南宁和贵阳居群，最低为南昌居群。群体间遗传分化系数(G_(ST))为0.5282，居群间基因流(Nm)为0.4466。数据显示居群间分化较大，居群内存在一定程度变异。聚类分析显示，当相似系数为0.896时，可将24份材料分为5个类群，其中南宁、昭阳、芜湖和运城四个居群与其它大部分半夏居群相似性较低。
利用ISSR分子标记技术分析半夏遗传多样性，结果表明，在物种水平上，PPL为97.92％，He为0.308。居群水平上的PPL在14.0％～72.9％之间，平均为41.1％，最高为菏泽居群(72.92％)，其次为磐安(68.75％)，最低为泰州居群(14.58％)．G_(ST)为0.4765，Nm为0.5494。结果也表明半夏居群间存在遗传分化程度较高，居群内也存在一定的遗传变异。聚类分析表明，当相似系数为0.820时，可将供试的24个半夏居群分为5个类群。
SRAP标记是一种新型技术，本文分析SRAP标记在半夏遗传多样性研究适用性。结果表明在物种水平上，PPL为97.86％，He为0.330。居群水平上PPL在35.04％～68.80％之间，平均为48.25％，最高南宁居群(68.80％)，其次为运城(68.38％)，最低为彝良居群(35.04％)。G_(ST)为0.4006，Nm为0.7482。聚类分析显示，当相似系数为0.862时，可将24个居群分为7个类群。将SRAP标记结果同RAPD和ISSR结果分析比较，认为该新型标记可以用于半夏遗传多样性研究。
通过测定半夏核糖体ITS碱基序列，分析其特点，了解种内变异类型。结果得到我国半夏主要的15个居群18个样本rDNA中的ITS和5.8SrDNA完全序列。ITS1、5.8s和ITS2序列长度分别为276bp、162bp和246bp。ITS2碱基频率差异显著，ITS1较为保守。根据两者序列以邻接法建立分子系统发生树，表明半夏种内至少存在7个类型。结合RAPD、ISSR和SRAP三种分子标记的研究结果，可将供试材料分为四个等级加以保护和利用。
2．半夏微卫星的开发
SSR标记具有共显性特点，开发半夏微卫星对于遗传多样性研究具有重要意义。通过分析评价几种有代表性的SSR标记开发方法，为微卫星开发方法的选择提供参考信息。在现有方法基础上提出两种半夏微卫星序列开发的策略。
其一，根据链亲和素磁珠和生物素特异结合的特性，将微卫星探针5’端生物素化后与链亲和素磁珠特异结合，用磁珠和探针的结合物与两端连接已知序列人工接头的半夏基因组DNA酶切片段杂交，洗脱未杂交DNA片断后，建立微卫星文库。以此为模板用人工接头序列为引物进行PCR扩增，产物直接克隆，经菌液PCR筛选后测序分析．
其二，将特异接头连接到限制性内切酶消化的基因组DNA上，以此为模板用兼并引物抑制性PCR获得SSR文库，克隆后经过两次菌液PCR筛选进行测序分析。结果表明，这两种方法可以快速、高效地开发出微卫星序列。
最后，搜索了数据库中半夏微卫星序列，结合前面从基因组DNA中已开发的微卫星序列，分析了半夏SSR特点。结果发现二核苷酸重复类型在半夏SSR占绝对优势，出现频率近80％，二核苷酸重复中以(AC)n重复基元为主，达67.57％。此外，对SSR位点总共设计了62对引物。
3．半夏种质资源的质量评价
利用RAPD技术鉴别了半夏的混淆植物水半夏、滴水珠和虎掌半夏．结果筛选到12条随机引物可用于半夏分子鉴别，并将引物P26转化为SCAR标记。
半夏块茎中含有多种化学成分，但《中国药典》(2005年版)没有规定半夏质量评价的指标成分．本研究将不同地区半夏种质栽培于同一生境，以鸟苷和总生物碱为指标成分，结合农艺性状单株产量，比较分析其差异。测得半夏鸟苷和生物碱含量分别为0.0136％～0.0264％和0.0155％～0.0652％，单株产量为0.5536～2.9740g。居群间单株产量、鸟苷和生物碱含量存在显著差异。聚类分析将半夏居群分为3类，认为应加强第Ⅱ类型类群的选育工作。
另外，测定了6个半夏居群药材(生半夏)及其土壤中的Mg、Ca、Fe、Zn、Ni、Se6种人体必需元素(前5种为植株必需元素)。通过比较含量差异，了解半夏对元素吸收特性，分布规律，为半夏的品质评价提供参考依据。结果显示，各元素在皮中含量最高。不同半夏居群各元素含量存在着显著性差异，磐安居群含量最高，西和居群含量最低。药材中元素间Zn和Se、Fe和Ni、Fe和Ca相关性显著。药材和土壤元素含量相关性不显著，说明半夏主动吸收矿质元素。
为评价药材安全性，测定不同居群半夏药材及其土壤Cu、Pb、Cd、As、Hg、Cr6种重金属和有机氯农药的残留。结果表明，药材中昭阳、菏泽居群As元素含量超标，磐安居群Cd元素超标，昭阳、菏泽、芜湖和磐安居群Pb元素超标，昭阳和菏泽居群半夏重金属总量超标。各居群均未检测到Cr元素含量。土壤中，西和居群Hg元素超标，昭阳、泰州、磐安和西和居群Cd元素超标，昭阳居群Cu元素超标。各居群半夏药材及其土壤中没有检测到六六六、DDT两种有机氯农药的残留。同时分析了半夏对重金属的吸收特性，半夏对重金属没有显著的富集作用，为低积累植物。结合各居群重金属含量的水平认为《中国药典》(2005年版)对西洋参等6种药材重金属限量标准可以适用于半夏的质量控制。
半夏对环境胁迫的适应性是一个重要的农艺性状，本文研究了不同半夏居群生理指标对高温胁迫响应。高温处理(35℃/25℃)结果表明，各居群半夏SOD活性先增高后下降，与脯氨酸含量趋势相同，MDA含量持续升高，根系活力则显著下降。居群间对高温胁迫响应差异显著。
Pinellia ternata(Thunb.) Breit. is from Genus Pinellia of Araceae Family, with dry tubers into medicine, named Rhizoma Pinelliae. P. ternata is endemic to East Asia, distributed in China, Korea and Japan. Most provinces and autonomous regions in China have distribution. P. ternata is Chinese traditional staple ingredient, because of the exact effect and a broad range of clinical applications. In recent years, domestic and foreign demand is growing, at five years the market price growth nearly doubled. Due to over exploitation, fanning changes and the reasons for the deterioration of the natural environment, P. ternata wild resources drop dramatically. After success of P. ternata cultivation in the 1970s, many"local variety" appeared. However, variety degraded seriously for consecutive cultivation, which greatly influenced the P. ternata production and resulted in P. ternata growing supply shortfall. In addition, chaos of variety is serious impact on the stability of Rhizoma Pinelliae. Therefore, germplasm resources of P. ternata need collation and evaluation to protect and exploit resources effectively
Germplasm were collected in most P. ternata distribution of the provinces of china. By analysis of P. ternata genetic diversity at the molecular level, we understand its genetic structure and level of polymorphism. Moreover, the delineation of the group provided the strong theoretical basis for the development and implementation of source protection-use strategy. Secondly, we successfully isolated microsatellite sequence of P. ternata by three methods, and analyze its characteristics for SSR markers application in P. ternata. Finally, we evaluated quality of germplasm resources of P. ternata in this paper on chemical composition, safety, and agronomic traits. Specific studies and results as follow:
1. Analysis of the genetic diversity of P. ternata germplasm resources in China by Molecular marker technology
240 samples were utilized which from 24 germplasm resources in 16 provinces of P. ternata main distribution. Based on analysis of RAPD data, the results showed.that at species level polymorphic loci percentage(PPL) is 68.77%, Nei'S gene diversity index(He) 0.2062. At population level, PPL is 9.88%～58.02%, averaging at 27.21%, Yuncheng the highest population, followed by the Nanning and Guiyang populations, Nanchang, the lowest of populations. Groups of genetic differentiation factor(Gst) is 0.5282, population gene flow(Nm=0.4466). Data show that among populations much differentiation. Cluster analysis revealed that when similar coefficient of 0.896, 24 can be divided into five material groups. Nanning, Zhaoyang, Wuhu and Yuncheng four populations was low similar to most of the other populations.
Analysis genetic diversity based on P. ternata ISSR data, the results showed that at species level PPL is of 97.92%, He 0.308. At population level PPL is in 14.0%～72.9%, averaging at 41.1%, Heze highest population(72.92%), followed by Pan'an(68.75%), Taizhou the lowest population(14.58%). G_(ST) is to 0.4765, and Nm of 0.5494. The results also show populations have much differentiation, and the population has a certain level of genetic differentiation. Cluster analysis showed that as the coefficient of 0.820, 24 populations of P. ternata were divided into five groups.
SRAP marker is a new technology. It was tested that SRAP marker applicability in analysis of the genetic diversity of P. ternata. The results show that at species level, PPL of 97.86%, He 0.330. While at population level PPL is in 35.04%～68.80%, averaging at 48.25%. Nanning, the highest population(68.80%), followed by Yuncheng(68.38%), Yiliang is the lowest population(35.04%). G_(ST) is to 0.4006, Nm of 0.7482. Cluster analysis revealed that as coefficient of 0.862, 24 populations were divided into 7 groups. By comparing SRAP, RAPD, ISSR data, the new technology can be used in P. ternata genetic structure study.
To understand types of variability within species and genetic diversity, ribosomal ITS characteristics Were analyzed based on sequence results. rDNA ITS complete sequences were obtained which from 18 samples of 15 main P. ternata populations. ITS1, 5.8s and ITS2 length was 276bp, 162bp, and 246bp respective. ITS1 was more conservative than ITS2. According to the sequences phylogenetic dendrogram establishment by Neighbor-adjacent method, it showed there are at least seven varaties in P. ternata. Combined with RAPD, ISSR, and SRAP molecular markers data, it was suggested that 24populations should be divided into four grades to be protected and utilized.
2. Micro-satellite development of P. ternata
The main strategies of micro-satellites development were reviewed in the paper. Analysis and evaluation of several representative methods are to select appropriate method. In addition, two micro-satellites development strategies for P. ternata were provided based on the existing methods.
First method: Because of the strong affinity of biotin for streptavidin, microsatellite probe of the 5'end biotin was combined with magnesphere paramagnetic particles, and then combinations hybridization with the digested P. ternata DNA fragments both ends of them connected with special adaptor. Elution of the other DNA fragments, the micro-satellite library was established. The adaptors as PCR primers clone the products directly, and screening the results by technology of bacteria PCR amplification in the end.
Second method: Connect the special adaptors to digested genomic DNA fragments, as a template. The micro-satellite library was established by inhibition PCR with mergers primer. Then analyze results of clone sequence after two screening by bacteria PCR. The results show that the two methods are fast, efficient to develop micro-satellite.
Finally, by searching NCBI database micro-satellites of P. ternata were obtained.With the developed micro-satellites, the characteristics of P. ternata SSR was analysized. The results showed dinucleotide repeat type in P. ternata SSR is absolute advantage, frequency nearly 80%. Dinucleotide repeat motif(AC)n is up to 67.57%. In addition, 62 pairs of SSR primers were designed.
3. Quality asseccment of P. ternata germplasm
RAPD technology was used to identify Pinellia ternata, Typhonium flagelliforrne, Pinellia cordata and Pinellia pedatisecta. The results showed that 12 random primers can identify P. ternata at molecular level. Primer P26 was converted into SCAR marker successfully.
P. ternata tubers contain a variety of chemical ingredients, but the Chinese Pharmacopoeia does not rule evaluation index components. P. ternata from different areas was planted in the same ecological environment in the study. Guanosine and total alkaloid of index components combined with yield of agronomic traits were measured to comparative analyze their differences in the study. The result showed that guanylin and total alkaloid content is in 0.0136%～0.0264% and 0.0155%～0.0652% respectively, the yield per plant in 0.5536～2.9740g.There are significant differences between populations. Cluster dendrogram reveal that 24 populations were divided into three categories, and category II should be strengthen in breeding work.
Beside, it were measured that Ca, Fe, Zn, Ni, Se six essential elements(the first five are plant indispensable elements)contents in the six P. ternata populations of P. ternata and soil samples. Compare the differences in content, in order to provide quality asseccment reference. Understanding absorption and distribution characteristics of the P. ternata, the results showed that elements content in the epidermal is the highest. In total element contents, there are significant differences between populations, Pan'an the highest and Xihe the lowest. In herb, elements Zn and Se, Fe and Ni, Fe and Ca were correlation with each other significantly. It is not significantly relation between herbs and soil content, which illustrate that P. ternata is initiative of absorption elements.
To evaluate the safety of herbs from different populations, Cu, Pb, Cd, As, Hg, Cr heavy metals and organic pesticide residues were detected in P. ternata herbs and soil. The results showed that As content of Zhaoyang, Heze populations exceeded the limit, Cd of Pan'an, Pb of Zhaoyang, Heze, Wuhu, Pan'an, total heavy metal of Zhaoyang and Heze also exceeding the limit. All the populations were not detected Cr. In soil, Hg contents of Xihe population exceeding the limit, Cd of Zhaoyang, Taizhou, Pan'an, Xihe, Cu of Zhaoyang exceeding the limit. All the herbs and soil samples from the different population were not detected 666, DDT organic pesticide residues. It is analyzed absorption properties of heavy metals. There is no significant role of P. ternatain the heavy metal enrichment. Combining the level of heavy metal content in P. ternata, it showed that the limit rule of heavy metals for American ginseng et al six materials in the 2005 version Pharmacopoeia can be applicable to the quality control of P. ternata.
Adaptability of P. ternata to the environment stress is an important agronomic trait. It studied response of different P. ternata populations to high temperature stress in this paper. By high temperature(35℃/25℃) treatment, it was measured SOD activity, MDA and the proline in leaves and root vigor. The results showed that SOD activity increased then dropped the same to proline content trend. MDA continues to rise, and root activity has dropped significantly. Different populations respond to heat stress significantly differently.
本研究收集了我国半夏分布区大部分种质，在分子水平上分析了半夏遗传多样性，了解其群体遗传结构和多态性水平，并进行了类群的划分，为制定和实施半夏资源保护利用策略提供有力理论依据。其次，采用3种方法分离获得了半夏的微卫星序列，并分析了其特点，为SSR分子标记在半夏中的应用奠定了基础。最后，本文进行了半夏种质资源药用品质、安全性和农艺性状质量评价。具体研究内容和结果如下：
1．利用分子标记技术分析我国半夏种质资源遗传多样性
以取自全国半夏主要分布区的16个省的24份种质的240个样本进行遗传多样性分析。利用RAPD标记技术分析，结果表明在物种水平上，多态位点百分率(PPL)为68.77％，Nei’S基因多样性指数(He)为0.2062．居群水平上的多态位点百分率在9.88％-58.02％之间，平均为27.21％，最高为沅陵居群，其次为南宁和贵阳居群，最低为南昌居群。群体间遗传分化系数(G_(ST))为0.5282，居群间基因流(Nm)为0.4466。数据显示居群间分化较大，居群内存在一定程度变异。聚类分析显示，当相似系数为0.896时，可将24份材料分为5个类群，其中南宁、昭阳、芜湖和运城四个居群与其它大部分半夏居群相似性较低。
利用ISSR分子标记技术分析半夏遗传多样性，结果表明，在物种水平上，PPL为97.92％，He为0.308。居群水平上的PPL在14.0％～72.9％之间，平均为41.1％，最高为菏泽居群(72.92％)，其次为磐安(68.75％)，最低为泰州居群(14.58％)．G_(ST)为0.4765，Nm为0.5494。结果也表明半夏居群间存在遗传分化程度较高，居群内也存在一定的遗传变异。聚类分析表明，当相似系数为0.820时，可将供试的24个半夏居群分为5个类群。
SRAP标记是一种新型技术，本文分析SRAP标记在半夏遗传多样性研究适用性。结果表明在物种水平上，PPL为97.86％，He为0.330。居群水平上PPL在35.04％～68.80％之间，平均为48.25％，最高南宁居群(68.80％)，其次为运城(68.38％)，最低为彝良居群(35.04％)。G_(ST)为0.4006，Nm为0.7482。聚类分析显示，当相似系数为0.862时，可将24个居群分为7个类群。将SRAP标记结果同RAPD和ISSR结果分析比较，认为该新型标记可以用于半夏遗传多样性研究。
通过测定半夏核糖体ITS碱基序列，分析其特点，了解种内变异类型。结果得到我国半夏主要的15个居群18个样本rDNA中的ITS和5.8SrDNA完全序列。ITS1、5.8s和ITS2序列长度分别为276bp、162bp和246bp。ITS2碱基频率差异显著，ITS1较为保守。根据两者序列以邻接法建立分子系统发生树，表明半夏种内至少存在7个类型。结合RAPD、ISSR和SRAP三种分子标记的研究结果，可将供试材料分为四个等级加以保护和利用。
2．半夏微卫星的开发
SSR标记具有共显性特点，开发半夏微卫星对于遗传多样性研究具有重要意义。通过分析评价几种有代表性的SSR标记开发方法，为微卫星开发方法的选择提供参考信息。在现有方法基础上提出两种半夏微卫星序列开发的策略。
其一，根据链亲和素磁珠和生物素特异结合的特性，将微卫星探针5’端生物素化后与链亲和素磁珠特异结合，用磁珠和探针的结合物与两端连接已知序列人工接头的半夏基因组DNA酶切片段杂交，洗脱未杂交DNA片断后，建立微卫星文库。以此为模板用人工接头序列为引物进行PCR扩增，产物直接克隆，经菌液PCR筛选后测序分析．
其二，将特异接头连接到限制性内切酶消化的基因组DNA上，以此为模板用兼并引物抑制性PCR获得SSR文库，克隆后经过两次菌液PCR筛选进行测序分析。结果表明，这两种方法可以快速、高效地开发出微卫星序列。
最后，搜索了数据库中半夏微卫星序列，结合前面从基因组DNA中已开发的微卫星序列，分析了半夏SSR特点。结果发现二核苷酸重复类型在半夏SSR占绝对优势，出现频率近80％，二核苷酸重复中以(AC)n重复基元为主，达67.57％。此外，对SSR位点总共设计了62对引物。
3．半夏种质资源的质量评价
利用RAPD技术鉴别了半夏的混淆植物水半夏、滴水珠和虎掌半夏．结果筛选到12条随机引物可用于半夏分子鉴别，并将引物P26转化为SCAR标记。
半夏块茎中含有多种化学成分，但《中国药典》(2005年版)没有规定半夏质量评价的指标成分．本研究将不同地区半夏种质栽培于同一生境，以鸟苷和总生物碱为指标成分，结合农艺性状单株产量，比较分析其差异。测得半夏鸟苷和生物碱含量分别为0.0136％～0.0264％和0.0155％～0.0652％，单株产量为0.5536～2.9740g。居群间单株产量、鸟苷和生物碱含量存在显著差异。聚类分析将半夏居群分为3类，认为应加强第Ⅱ类型类群的选育工作。
另外，测定了6个半夏居群药材(生半夏)及其土壤中的Mg、Ca、Fe、Zn、Ni、Se6种人体必需元素(前5种为植株必需元素)。通过比较含量差异，了解半夏对元素吸收特性，分布规律，为半夏的品质评价提供参考依据。结果显示，各元素在皮中含量最高。不同半夏居群各元素含量存在着显著性差异，磐安居群含量最高，西和居群含量最低。药材中元素间Zn和Se、Fe和Ni、Fe和Ca相关性显著。药材和土壤元素含量相关性不显著，说明半夏主动吸收矿质元素。
为评价药材安全性，测定不同居群半夏药材及其土壤Cu、Pb、Cd、As、Hg、Cr6种重金属和有机氯农药的残留。结果表明，药材中昭阳、菏泽居群As元素含量超标，磐安居群Cd元素超标，昭阳、菏泽、芜湖和磐安居群Pb元素超标，昭阳和菏泽居群半夏重金属总量超标。各居群均未检测到Cr元素含量。土壤中，西和居群Hg元素超标，昭阳、泰州、磐安和西和居群Cd元素超标，昭阳居群Cu元素超标。各居群半夏药材及其土壤中没有检测到六六六、DDT两种有机氯农药的残留。同时分析了半夏对重金属的吸收特性，半夏对重金属没有显著的富集作用，为低积累植物。结合各居群重金属含量的水平认为《中国药典》(2005年版)对西洋参等6种药材重金属限量标准可以适用于半夏的质量控制。
半夏对环境胁迫的适应性是一个重要的农艺性状，本文研究了不同半夏居群生理指标对高温胁迫响应。高温处理(35℃/25℃)结果表明，各居群半夏SOD活性先增高后下降，与脯氨酸含量趋势相同，MDA含量持续升高，根系活力则显著下降。居群间对高温胁迫响应差异显著。
Pinellia ternata(Thunb.) Breit. is from Genus Pinellia of Araceae Family, with dry tubers into medicine, named Rhizoma Pinelliae. P. ternata is endemic to East Asia, distributed in China, Korea and Japan. Most provinces and autonomous regions in China have distribution. P. ternata is Chinese traditional staple ingredient, because of the exact effect and a broad range of clinical applications. In recent years, domestic and foreign demand is growing, at five years the market price growth nearly doubled. Due to over exploitation, fanning changes and the reasons for the deterioration of the natural environment, P. ternata wild resources drop dramatically. After success of P. ternata cultivation in the 1970s, many"local variety" appeared. However, variety degraded seriously for consecutive cultivation, which greatly influenced the P. ternata production and resulted in P. ternata growing supply shortfall. In addition, chaos of variety is serious impact on the stability of Rhizoma Pinelliae. Therefore, germplasm resources of P. ternata need collation and evaluation to protect and exploit resources effectively
Germplasm were collected in most P. ternata distribution of the provinces of china. By analysis of P. ternata genetic diversity at the molecular level, we understand its genetic structure and level of polymorphism. Moreover, the delineation of the group provided the strong theoretical basis for the development and implementation of source protection-use strategy. Secondly, we successfully isolated microsatellite sequence of P. ternata by three methods, and analyze its characteristics for SSR markers application in P. ternata. Finally, we evaluated quality of germplasm resources of P. ternata in this paper on chemical composition, safety, and agronomic traits. Specific studies and results as follow:
1. Analysis of the genetic diversity of P. ternata germplasm resources in China by Molecular marker technology
240 samples were utilized which from 24 germplasm resources in 16 provinces of P. ternata main distribution. Based on analysis of RAPD data, the results showed.that at species level polymorphic loci percentage(PPL) is 68.77%, Nei'S gene diversity index(He) 0.2062. At population level, PPL is 9.88%～58.02%, averaging at 27.21%, Yuncheng the highest population, followed by the Nanning and Guiyang populations, Nanchang, the lowest of populations. Groups of genetic differentiation factor(Gst) is 0.5282, population gene flow(Nm=0.4466). Data show that among populations much differentiation. Cluster analysis revealed that when similar coefficient of 0.896, 24 can be divided into five material groups. Nanning, Zhaoyang, Wuhu and Yuncheng four populations was low similar to most of the other populations.
Analysis genetic diversity based on P. ternata ISSR data, the results showed that at species level PPL is of 97.92%, He 0.308. At population level PPL is in 14.0%～72.9%, averaging at 41.1%, Heze highest population(72.92%), followed by Pan'an(68.75%), Taizhou the lowest population(14.58%). G_(ST) is to 0.4765, and Nm of 0.5494. The results also show populations have much differentiation, and the population has a certain level of genetic differentiation. Cluster analysis showed that as the coefficient of 0.820, 24 populations of P. ternata were divided into five groups.
SRAP marker is a new technology. It was tested that SRAP marker applicability in analysis of the genetic diversity of P. ternata. The results show that at species level, PPL of 97.86%, He 0.330. While at population level PPL is in 35.04%～68.80%, averaging at 48.25%. Nanning, the highest population(68.80%), followed by Yuncheng(68.38%), Yiliang is the lowest population(35.04%). G_(ST) is to 0.4006, Nm of 0.7482. Cluster analysis revealed that as coefficient of 0.862, 24 populations were divided into 7 groups. By comparing SRAP, RAPD, ISSR data, the new technology can be used in P. ternata genetic structure study.
To understand types of variability within species and genetic diversity, ribosomal ITS characteristics Were analyzed based on sequence results. rDNA ITS complete sequences were obtained which from 18 samples of 15 main P. ternata populations. ITS1, 5.8s and ITS2 length was 276bp, 162bp, and 246bp respective. ITS1 was more conservative than ITS2. According to the sequences phylogenetic dendrogram establishment by Neighbor-adjacent method, it showed there are at least seven varaties in P. ternata. Combined with RAPD, ISSR, and SRAP molecular markers data, it was suggested that 24populations should be divided into four grades to be protected and utilized.
2. Micro-satellite development of P. ternata
The main strategies of micro-satellites development were reviewed in the paper. Analysis and evaluation of several representative methods are to select appropriate method. In addition, two micro-satellites development strategies for P. ternata were provided based on the existing methods.
First method: Because of the strong affinity of biotin for streptavidin, microsatellite probe of the 5'end biotin was combined with magnesphere paramagnetic particles, and then combinations hybridization with the digested P. ternata DNA fragments both ends of them connected with special adaptor. Elution of the other DNA fragments, the micro-satellite library was established. The adaptors as PCR primers clone the products directly, and screening the results by technology of bacteria PCR amplification in the end.
Second method: Connect the special adaptors to digested genomic DNA fragments, as a template. The micro-satellite library was established by inhibition PCR with mergers primer. Then analyze results of clone sequence after two screening by bacteria PCR. The results show that the two methods are fast, efficient to develop micro-satellite.
Finally, by searching NCBI database micro-satellites of P. ternata were obtained.With the developed micro-satellites, the characteristics of P. ternata SSR was analysized. The results showed dinucleotide repeat type in P. ternata SSR is absolute advantage, frequency nearly 80%. Dinucleotide repeat motif(AC)n is up to 67.57%. In addition, 62 pairs of SSR primers were designed.
3. Quality asseccment of P. ternata germplasm
RAPD technology was used to identify Pinellia ternata, Typhonium flagelliforrne, Pinellia cordata and Pinellia pedatisecta. The results showed that 12 random primers can identify P. ternata at molecular level. Primer P26 was converted into SCAR marker successfully.
P. ternata tubers contain a variety of chemical ingredients, but the Chinese Pharmacopoeia does not rule evaluation index components. P. ternata from different areas was planted in the same ecological environment in the study. Guanosine and total alkaloid of index components combined with yield of agronomic traits were measured to comparative analyze their differences in the study. The result showed that guanylin and total alkaloid content is in 0.0136%～0.0264% and 0.0155%～0.0652% respectively, the yield per plant in 0.5536～2.9740g.There are significant differences between populations. Cluster dendrogram reveal that 24 populations were divided into three categories, and category II should be strengthen in breeding work.
Beside, it were measured that Ca, Fe, Zn, Ni, Se six essential elements(the first five are plant indispensable elements)contents in the six P. ternata populations of P. ternata and soil samples. Compare the differences in content, in order to provide quality asseccment reference. Understanding absorption and distribution characteristics of the P. ternata, the results showed that elements content in the epidermal is the highest. In total element contents, there are significant differences between populations, Pan'an the highest and Xihe the lowest. In herb, elements Zn and Se, Fe and Ni, Fe and Ca were correlation with each other significantly. It is not significantly relation between herbs and soil content, which illustrate that P. ternata is initiative of absorption elements.
To evaluate the safety of herbs from different populations, Cu, Pb, Cd, As, Hg, Cr heavy metals and organic pesticide residues were detected in P. ternata herbs and soil. The results showed that As content of Zhaoyang, Heze populations exceeded the limit, Cd of Pan'an, Pb of Zhaoyang, Heze, Wuhu, Pan'an, total heavy metal of Zhaoyang and Heze also exceeding the limit. All the populations were not detected Cr. In soil, Hg contents of Xihe population exceeding the limit, Cd of Zhaoyang, Taizhou, Pan'an, Xihe, Cu of Zhaoyang exceeding the limit. All the herbs and soil samples from the different population were not detected 666, DDT organic pesticide residues. It is analyzed absorption properties of heavy metals. There is no significant role of P. ternatain the heavy metal enrichment. Combining the level of heavy metal content in P. ternata, it showed that the limit rule of heavy metals for American ginseng et al six materials in the 2005 version Pharmacopoeia can be applicable to the quality control of P. ternata.
Adaptability of P. ternata to the environment stress is an important agronomic trait. It studied response of different P. ternata populations to high temperature stress in this paper. By high temperature(35℃/25℃) treatment, it was measured SOD activity, MDA and the proline in leaves and root vigor. The results showed that SOD activity increased then dropped the same to proline content trend. MDA continues to rise, and root activity has dropped significantly. Different populations respond to heat stress significantly differently.
引文
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