【作者】 郑红梅；

【导师】 呼天明；

【作者基本信息】 西北农林科技大学 ， 水土保持与荒漠化防治， 2008， 博士

【摘要】 嵩草属植物是多年生草本植物,它们具有草质柔软、营养丰富等特点,为各类家畜所喜食,嵩草是青藏高原的主要建群种,它们的良好生长对维持青藏高原的生态平衡起着重要作用。牧草种质资源研究是我国草地生物多样性正常发展和持续利用的必要保证。然而由于环境及人为的影响,嵩草植物面临的问题比较严峻,如草地退化、载畜能力下降等,而且嵩草属植物大多是无性繁殖,致使其对环境变化及不良的环境适应性不强。由于青藏高原极端的自然环境,对嵩草属植物的系统发育关系及其适应性研究较少,因此探索其种内遗传多样性、遗传结构、系统发育关系及种子特性,为进一步研究高寒草甸生态系统的结构,功能及其演化提供理论依据。本文首先研究了嵩草种群维持和更新的另一重要过程-通过有性生殖产生的种子的萌发特性研究。在实验室条件下,分别对7种嵩草的千粒重、种子吸水曲线及不同溶液处理发芽率进行了比较,结合种子微形态结构、变温层积及生理指标测定等,探讨了嵩草种子的萌发特性及解除休眠的技术。结果表明嵩草都具有坚硬而厚的种皮,40%氢氧化钠处理3h,种皮的通透性有所改善,大多数嵩草的发芽率明显高于对照(P<0.05),且在萌发初期显著地提高了大花嵩草、高山嵩草、矮生嵩草的过氧化物酶、超氧化物歧化酶和脱氢酶的活性。通过电镜扫描发现嵩草的种皮都坚硬而厚,保护组织的厚度占果实横切面厚度的比例之大是少见的;嵩草属植物小坚果电镜扫描微形态特征虽然种皮基本纹饰相似,但从种子形态来看,还是有一定的区别,此外受材料数目限制,要通过电镜扫描种皮微形态性状还是难于确定嵩草属种间的关系,在本试验中只能作为一个系统发育关系划分的参考依据。从分子水平研究嵩草品种资源之间的遗传多样性,对扩增长度多态性(AFLP)和随机扩增多态性DNA(RAPD)几个关键参数进行试验优化,旨在建立适合嵩草的AFLP和RAPD技术体系。结果表明AFLP体系用于酶切的嵩草基因组DNA模板600 ng为宜,连接最适反应时间为10h过夜,预扩增产物最适稀释倍数为5倍。采用优化好的体系,以嵩草为材料进行AFLP标记,E-ACC+M-CAG、E-ACA+M-CAG、E-ACC+M-CAA和E-ACA+M-CTG四个引物均获得多态性强且质量好的AFLP指纹图谱。用筛选出的4对E+3和M+3引物及优化好的嵩草AFLP体系对11份嵩草基因组DNA进行AFLP扩增,结果共得到164条清晰可辨条带,多态性条带154条,多态性位点百分率为93.96%,嵩草平均Nei’s基因多样性指数为0.2430,Shannon’s多样性指数为0.4012,表明嵩草种质间存在丰富的遗传多样性。通过UPGMA聚类分析,将11个嵩草居群划分为4类。对11个嵩草群体的RAPD遗传多样性进行分析表明,10个随机引物共扩增出170个清晰可辨的条带,扩增带数最多的引物为22条,多态率达98.16%;带数最少的引物为10条,多态性检出率最低为45.45%。当遗传相似系数为0.66时,11个嵩草居群被分为4个类群。我们的研究表明嵩草居群基于RAPD (H = 0.2221和I = 0.3592)数据的遗传多样性相对低于AFLP (H = 0.2430和I = 0.4012)。基于RAPD和AFLP遗传相似系数数据的Mantel检验表明两者有着显著的相关性(r = 0.6553, p = 0.002),说明RAPD和AFLP方法在嵩草遗传多样性的研究中都是有效的。根据AFLP和RAPD两种分子标记方法,嵩草居群的遗传相似系数与海拔之间没有相关性,嵩草居群的生境的异质性影响遗传分化。序列分析为嵩草属的系统分类、探讨其种间系统关系提供了重要的科学依据。系统发育树不仅表明了各种间的亲缘关系,而且对探讨嵩草的起源与演化具有重要的意义。虽然RAPD和AFLP分子标记是遗传多样性研究的有效方法,然而其不能得到足够的信息与前人的研究相比较。我们用测基因nrITS、cptrnT-L-F、cpndhF及trnK-RPS16序列的方法进行进一步研究。研究结果表明大花嵩草(L)居群和其它的嵩草居群有着较远的亲缘关系。具有相似的生境或具有相似适应性的嵩草居群倾向聚在一起。大多数的聚类分析表明在11个嵩草居群中存在着基因变异,可能是由于基因突变或由于嵩草居群间生境的异质性引起的。更多还原

【Abstract】 The genus Kobresia is the dominant perennial forage in the Tibetan plateau, which has good nutritional quality preferred by livestock, important ecological functions. However, Kobresia pastures are seriously subject to environmental degradation and decreasing carrying capacity due to natural and anthropogenic impacts, and Kobresia species of the Tibetan plateau have not been studied about plant phylogenetic relationship and adaptationin in detail, in part because of the difficulty in accessing this vast cold, harsh, and remote region. Vegetative growth is the dominant means of reproduction for the Kobresia. Studies suggest that substantial vegetative growth can reduce genetic diversity and renders populations less able to buffer changing extreme conditions. It is necessary to protect the genetic diversity of Kobresia to understand how they are able to thrive in harsh environment. In this study the genetic diversity, genetic structure and seed characters of Kobresia species were studied to provide theory and techinical reference of understanding the mechanisms for adaptation and evolutionary history on Kobresia plants collected from the Tibetan plateau.Study on another process of maintaining and renewing-germination mechanism of seed. Seed germination of Kobresia and the techniques of relieving dormancy were studied through examining thousand-grain weight, seed water absorbing and germination rate, with different treatments together other experiments such as stratifying seeds under different temperatures, morph-dissecting, physiological items and so on. The results is that seed coat of Kobresia is hard and thick, seeds treated by 40% NaOH 3h, permeability of seed coat improved, germination rate were significantly higher than those of non-treated seeds (P < 0. 05), and increased significantly activity POD, SOD and dehyolrogenase of K. macrantha Boeck(L), K. humilis C.A.Mey(N), K. pygmaea C.B(N) during early seed germination. Some enzymes activity relating to germinating capacity are low, which lead to low germination rate on seed Physiological aspects. Seed coat of Kobresia is hard and thick, the thickness of protect tissuses is seldom found. The seed coat ornament is similar by scanning electron microscope, but there are some differents in morphology of seed, it is difficult to identify phylogenetic relationship of Kobresia owing to limiting number of materials.This study analyzed the genetic diversity of Kobresia accessions, in the molecular level. The study presented was intended to establish the AFLP and RAPD reaction system of Kobresia. Several main factors that influenced the results of the AFLP and RAPD methods were optimized. The usage of DNA template is 600ng, the optimal time is 10 hours for ligation, 5 times of dilution for the products of pre-amplification is suggested for selective amplification. Genomic DNA of Kobresia was amplified with four E + 3 and M + 3 primer pairs with AFLP (E-ACC+M-CAG, E-ACA + M-CAG, E-ACC + M-CAA and E-ACA + M-CTG). AFLP analysis produced 164 scorable bands, among them 154 (93.96%) were polymorphic. The mean Nei’s gene diversity index (H) was 0.2430, the Shannon’s information index (I) was 0.4012, indicating that genetic diversity of Kobresia is abundant.The number of 11 Kobresia accessions from Tibetan plateau can be classified into four groups after cluster analysis based on the UPGMA method.Ten primers were identified after screening 100 primers. RAPD markers which amplified 170 scorable bands showed high level of polymorphism by the PIC value, ranging from 45.45% to 95.45% with an average of 67.33% across the germplasm assayed. 11 Kobresia accessions from Tibetan plateau can be classified into four groups after cluster analysis based on the RAPD data, when similarity is 0.66.Our study revealed that the genetic diversity of Kobresia acessions level RAPD (H = 0.2221 and I = 0.3592) was relatively lower than AFLP (H = 0.2430 and I = 0.4012). A Mantel test showed a significant correlation between the RAPD and AFLP-based Genetic similarity (r = 0.6553, p = 0.002). In general, there was abundant genetic diversity among Kobresia accessions resources, and the genetic coefficient was unrelated to their geographic latitude. Maybe natural habitat influenced genetic diversity of Kobresia. RAPD marker, AFLP marker, and sequence alignment of nrDNA ITS, cpDNA trnT-L-F spacer sequences, cpDNA ndhF DNA sequences and TrnK-RPS16 sequences were used to determine genetic diversity and phylogenetic relationship among these Kobresia accessions.In the dendrograms, K.macrentha (L) species forming a particular clade, species’inhabiting similar habitats or having similar adaptations tended to be together. These trees showed that the difference of genetic variation and adaptation might be taking place in these Kobresia accessions, possibly because of different natural habitat or sexual recombination in the Tibetan plateau. All same species accessions were not identical but still considerably more similar than accessions from different species, evidenced by their clustering together in the dendrogram.更多还原