Study On Seed Germination Characteristics And Molecular Phylogeny On Kobresia In Tibetan Plateau

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.