Study On The Biological Characteristics Of Cyclobalanopsis Glauca And The Establishment Of EST - SSR Optimization System

Swetia mileensis is an endemic and medical plant in China belong to the Swertia genus of Gentianaceae Family and which is endangered in extinction for the severe fragmentation due to the commercial over exploitation. However, for a long time, the research of S. mileensis mainly focus on the chemical constituents and pharmacological activities, and the study of biology is relatively scarce at home and abroad. This study intends to use the method of reproductive biology and molecular biology to study the genetic diversity research of endemic medicinal plant S. mileensis.The results of the study are summarized as below:1. Through field investigation, mastered the flowering phenology of S. mileensis, we follow the flowering dynamics process, observed in S. mileensis single flower opening time is 5~6 D, the group in florescence 9~10 month..2. Based on measuring the plant height, numbers of divaricating, the length of leaf, the length of pedicel and the length of ovary among five natural populations of S. mileensis in Mi-Le Yunnan were studied. The results as follows: There were extremely significant differences(P<0.01) among different populations in five morphological characteristics factors. The largest morphological variation was the numbers of divaricating, following that, in order, were: the length of pedicel, the length of leaf, the plant height and the last was the length of ovary. This article used the One-Way ANOVA and LSD multiple comparing analysis method to analyze morphological characteristics factors among the five populations which being investigated.3. The flower characteristics and breeding system of S. mileensis were explored by field location investigations using data of TTC method, benzidine hydrogen peroxide method, pollenovule ratio(P/O), out crossing index(OCI) and bagging experiment. The result indicated that the single flowering was 2-3 d and the flower span of the population was about 120 d; there are a series of mechanism to guarantee the cross pollination in S. mileensis; the maturity of stamen was about 24 h earlier than the maturity of pistil, the duration of pollen vigor was 36-48 h and the overlap between stigma receptivity and pollen vitality was about 10-12h; the pollen-ovule ratio(P/O) and out-crossing index was 1918.63 ± 1053.56 and 5, which indicated that the breeding system was out-crossing with partial self-compatibility, requiring pollinators. Different frombreeding system reflected by the floral syndrome, we found that the frequency of flower-visiting insects was low and there was self-compatibility in S. mileensis for the reason that the seed setting rate of bagging was 68.37% in artificial cross pollination while it was 42.13% in bagging without emasculation. Although the pollination efficiency evaluated by pollen vigor decreased after shedding 30 h, the stamen cling to pistil in positive and then turn around on the back of the pistil and deviate from the pistil 30-45 degrees, which could pollinate bottom-up in one plant by shake slightly. The geitonogamy of the S. mileensis belongs to delayed self-cross type. Self-pollination occurs between upper and lower single flowers in cyme from one plant, before this every single flower still remained the mechanism of cross-pollination. This latency mechanism of the self-pollination provides reproductive assurance when cross-pollination fails due to lack of pollinators in harsh environments. In addition, the seed setting rate of the cross-pollination in different populations is higher than the rate of natural pollination in the same population, which confirms that the sexual reproduction system of the fragment population of S. Mileensis is susceptible to limitation of pollen and the lack of pollinators.4.This study optimized the EST-SSR system and selected an certain amount of primer pairs by using the genomic DNA extracted from leaves of S. mileensis with the modified CTAB method,the current study was designed single factor selection and L16(45) orthogonal experiment to optimize the main factors of the EST-SSR PCR system. The results showed that the optimized system for 20 μL is 50 ng DNA template, 150 μmol/L d NTPs, 0.1 μmol/L primer, 1.5 U Taq DNA polymerase and 2.5 μmol/L Mg2+. At the same time, 10 primer pairs were selected from 60 primer pairs for the SSR analysis based on their reproducible and clear banding patterns, which could be used for further SSR research.