Studies On Genetic Diversity Of An Endanger Species,Trillium Tschonoskii

Trillium tschonoskii Maxim, locally named "a pearl on head" , a perennial herb of Liliaceae, mainly distributes in east Asia and north America. In China, T. tschonoskii is mainly distributed in Sichuan Province at elevations ranging from 1500 to 3000m and can be found in northwest of Yunnan, northwest of Zhejiang, north of Fujian, and southeast of Ganshu Province. The rhizome and leaves of T. tschonoskii has been found to be a source of dioscin and flavonoids, the effective agents in the treatment of dizziness, hypertension and neurasthenic disease. Furthermore, with the typical characteristics of plants in arctic pole tertiary geographic plant region, T. tschonoskii holds an important status in plant systematics and evolution. Recently, the number of wild T. tschonoskii decreases rapidly because of habitat fragmentation, overcollecting, long dormancy duration, and low percentage of seed germination and so on. The species is classified as endangered in the Chinese Plant Red Book. It is essential to excute studies on conservation genetics of T. tschonoskii for discussing its extinct mechanism. To estimate the level and distribution of geneticvariation in endangered species is a primary objective of conservation genetics.Regarding the genetic diversity studies of T. tschonoskii, previous studies were mostly focused on cytology. In the DNA level, genetic diversity and genetic structure of T. tschonoskii populations are studied by ISSR and AFLP markers in the study. The aims are to make the genetics conditions of T. tschonoskii clear and analyze the extinct mechanism, which afford science theory evidence for the conservation measure. In order to rescue the sources of T. tschonoskii, we study the plantlets of T. tschonoskii regeneration, we also excute regression analysis between diosgenin content and soil factor of different area. The results are as follows:1. The inter-simple sequence repeat (ISSR) analysis was conducted on 7 natural populations of T. tschonoskii in order to investigate genetic diversity and genetic structure of the populations. Out of 100 ISSR primers, 12 generated highly reproducible and stable DNA fragments. Using these primers, 135 discernible DNA fragments were produced. Of these, 46 (PPB= 34. 07%) were polymorphic loci, which indicated that low levels of genetic variation existed in the populations. The result of POPGENE analysis indicated that the level of genetic variation of T. Tschonoskii (He=0. 0759, Ho=0.1200) was lower than other endangered plants. The Wolong population possessed the highest level of genetic diversity (PPB= 18. 52% , He= 0.0417, Ho=0. 0684), while the Dabazi population exhibited the lowest levels of genetic diversity (PPB=8. 89%, He=0.0220, Ho=0. 0348). A high level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis(55. 54%) and analysis of molecular variance (52.53%). Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. Based on ecological and genetic information available for T. Tschonoskii,We propose an appropriate strategy for conserving the genetic resources of T. tschonoskii in China.2. Genetic diversity and genetic structure of T. tschonoskii were investigated by using amplified fragment length polymorphism (AFLP) marker. Eight primer combinations were carried out on 105 different individuals sampled from seven populations. Six hundred and nineteen discernible DNA fragments were generated with 169 (27. 30%) being polymorphic. The percentage of polymorphic bands (PPB) within populations ranged from 4. 52% to 10. 50%. The Wolong population possessed the highest level of genetic diversity (PPB= 10. 50%, He=0.0379, Ho =0.0562), while the Dabazi population exhibited the lowest levels of genetic diversity (PPB=4. 52%, He = 0. 0130 , Ho = 0. 0204). Genetic differentiation among populations was detected based on Nei's genetic diversity analysis (53.03%) and AMOVA analysis (52.53%). Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. The study results indicate ISSR and AFLP markers are good for assessing of the genetic diversity and genetic structure in T. tschonoskii.3. In order to rescue the sources of T. tschonoskii, We study plant regeneration by propagation in vitro. The results indicate that the optimum explant is tender bud, the second are ovary and rhizome, and the last are leaf and root. According to combination of hormone in medium, we deduce the combination BA and 2, 4-D or BA and NAA are helpful to the induction of callus. Granular callus grows slow and is't easy to differentiate. T. tschonoskii explants grow better with the lower temperature and weak light. The system of propagation in vitro of T. tschonoskii is preliminary established.3. HPLC is used to mensurate the active components (diosgenin) in T. tschonoskii. The results indicate that the Dabazi place possessed the highest diosgenin content (2.65%) while Mamade is second (1.81%), andWolong is the lowest (0. 13%). We analyze soil part nutrient element of 7 places in order to explain the cause of diosgenin content. By linear stepwise regression analysis between diosgenin content and soil factors, we find that the main influence factor of diosgenin content. That are youjizhi and water content.