Embryology And Genetic Diversity Of The Endangered Species Psathyrostachys Huashanica Keng.

Psathyrostachys huashanica Keng is a perennial grass and belongs to genus Psathyrostachys in Triticeae. Psathyrostachys distributes in center of middle Asia and Caucasus Mt., while P. huashanica, a species endemic to China, is only located in Mountain Huashan in Shaanxi Province. At present, the populations of this species are decreasing, and reaching the edge of extinction. Due to the distributing limitation and importance as breeding material for germplasm storage, it has been considered as the first class of national protected rare plants. For this reason, the present study is significant in probing plant flora, origin and evolution of Triticeae and crop breeding. The results showed as follows: 1. The embryology of P. huashanicaIn this paper, the microsporogenesis, the megasporogenesis, the male gametophyte development, the female gametophyte development and the development of embryo and endosperm of P. huashanica were observed by means of paraffin. The results are as follows: (1) Microsporal mother cells divide according to successive type, tetrads range symmetrically, pollens get mature at 3-celled stage, these characteristics are in line with that of Triticum. (2) The megasporogenesis in P. huashanica begins with archesporial cell. Successive cytokinesis in the megasporocyte follows meiosis and the megaspore tetrads are linear. Functional megaspore is in the chalaza and the monosporic embryo sac is chenopodiad type. Mature embryo sac has 8 nuclei. Egg cell has obvious polarity. One synergid degenerates before fertilization, and the other does after fertilization. Two polar nuclei array in three ways. The antipodal cells degenerate lately. (3) The formation and development of the embryo and endosperm is in accordance with the typical graminaceous plants. The embryogeny conforms to the Asterad type. The top cell and the base cell take part in the structure of embryo. There are 5 stages during thedevelopment of embryo, including the stage of 2-celled proembryo, multicellular proembryo, globular proembryo, pyriform proembryo, differential embryo and mature embryo. The mature embryo is composed of radicle, coleorhiza, plumule, coleoptile, scutellum and epiblast. That is the typical structure of the mature embryo of grass. The endosperm development follows the Nuclear type and includes three successive stages, which are free endosperm nucleus stage, cellularization stage and nurture stage. The cell wall is formed after the free endosperm nucleus is formed. The outmost layer of endosperm cell differentiates into the aleurone layer, while the other cells are filled with starch grains. It has three features as follows: 1) pollen abortion near two-celled stage is observed; 2) the double archesporial cells, the double embryo sacs and the double spherical original embryo are discovered; 3) the antipodal cell disintegrates earlier; 4) the number of nucleolus is numerous at the stage of free endosperm nucleus and cellularization.2. Genetic structure and differentiation of P. huashanica populations detected with RAPD markersRandomly amplified polymorphic DNA (RAPD) markers were used to analyze genetic structure and differentiation of P. huashanica populations sampled in three valleys (Huangpu, Xian and Huashan valleys) in Mt. Huashan. One hundred and twenty-two RAPD fragments were obtained in all 266 individuals with 20 primers with mean of 6.1 (2~ 10) fragments per primer. The percentage of polymorphic loci (PPB) is 60.66% in Huangpu Valley, 90.98% in Xian Valley, 95.08% in Huashan Valley, and the total PPB is 95.08%, which indicated a highly genetic variability of P. huashanica. The Shannon's Information index and Gst are 0.3306 and 0.3263, respectively, indicating that there were more genetic variations within the subpopulations than those among the subpopulations. The gene flow among the subpopulations of P. huashanica (Mw=l .0322) is much less than that of the common anemophytes (Nm=5.24). Mean genetic distance is 0.1571 (range: 0.0022 ~ 0.2901). The highest value of genetic distance was found between the subpopulations (hpl) of Huangpu Valley and the highest altitude subpopulations (h8) of Huashan Valley. Correlation analysis detected significant correlation between genetic distance and vertical distance of altitude. Clustering analysis and principal coordinate analysis revealed the genetic differentiation among thepopulations of P. huashanica. Differentiation mainly occurred between the higher altitude subpopulations and the lower altitude subpopulations, suggesting that altitude might be the major factor that restricted the gene flow between different altitude subpopulations and resulted in differentiation of subpopulations. 3. Genetic diversity analysis of the endangered and endemic species P. huashanica natural populations using simple sequence repeats (SSRs) markersThe microsatellites of barley were used to analyze genetic diversity of P. huashanica populations sampled from three valleys (Huangpu, Xian and Huashan valleys) in Mt. Huashan. A total of 33 alleles of 11 loci were detected from 266 individuals. The observed average number of alleles (A) is 2.75; the effective number of alleles (Ae) is 1.67. The percentage of polymorphic loci (PPB) is 58.33% in Huangpu Valley, 75% in Xian Valley, 83.33% in Huashan Valley, and the total PPB is 83.33%. About 77.6% of (Fst=0.324 ) genetic diversity was observed within the subpopulations. Genetic differentiation within the subpopulations was higher than that among the subpopulations. Mean genetic distance is 0.17 (range: 0.010~0.401). Furthermore, this study has found a high level of transferability of SSR markers among related genera in the Gramineae plants.