Molecular Markering And Genetic Diversity Of Endangered Plant Bretschneidera Sinensis Hemsl.

The community and the genetic diversity of endangered plant Bretschneidera sinensis in Fujian province were investigated and sampled to analyze the community characteristics, genetic structure and genetic diversity. The survival condition and endangered mechanism of Bretschneidera sinensis population were also researched in the paper in order to provide basis to protect and make use of Bretschneidera sinensis resources. The results were as follows:(1) The community structure and species composition of concomitant community of Bretschneidera sinensis were investigated in Minjiangyuan Nature Reserve, Jianning county, Fujian province. Results showed that the species composition was complex. There were 39 species belonging to 26 families, 34 genera in the community. Shannon-Wienner index was 1.89, Pielou index was 0.33 and Simpson index was 0.52. Bretschneidera sinensis was the dominant species in the community. The concomitant species were abundant, but the relationships among Bretschneidera sinensis and its concomitant species were unstable.(2)The single-factor method was applied to optimize ISSR system and the reliable ISSR analysis system was established. The optimized reaction system of ISSR-PCR was: 20μl reaction volume containing 2 mmol/L Mg²⁺(10 mmol/L), 0.5 mmol/L primer(10μmol/L), 0.6 mmol/L dNTP(10 mmol/L), 16 ng DNA template(20 ng/μl) , 1.5 U Taq polymerase(5 U/μl). The optimized amplification was under the following conditions: 5 minutes at 94℃for predenaturing, followed by 45 seconds at 94℃for denaturing, 1 minute at 52～56℃(different primers with different annealing temperature) for annealing, 90 seconds at 72℃for extending, for 38 cycles, and 10 minutes at 72℃for a final extension, finally stored at 4℃. After primary screening and final screening, 10 ISSR primers with high resolution and multiple polymorphic bands were screened to analyze the genetic structure and genetic differentiation of geographic populations.(3) ISSR molecular marker was used for study the offspring-parent relation for the first time. Results showed that there were some differentiation between mother tree and its offspring with the low level of genetic diversity. That the genetic identity between mother tree and offspring was above 0.8500, and the genetic distance was below 0.1500 might be adopted to comfirm the offspring-parent relation.(4) ISSR was adopted to examine the genetic differentiation of Bretschneidera sinensis populations in different scales, samples from 3 special subpopulations, 19 normal subpopulations in Minjiangyuan Nature Reserve and 7 geographic populations in Fujian province. Results showed that: 1)72 amplification sites were detected with 10 primers. The differences among different scales populations in levels of genetic diversity were obvious, with the highest level(PPB=69.44 %) among geographic populations, the lowest level(PPB=58.22 %) in A^C special subpopulations, the PPB of 22 subpopulations was 62.50 % in Minjiangyuan Nature Reserve. 2) There was a high genetic differentiation within A^C subpopulations and a low genetic diversity among A^C subpopulations, whereas the opposite conclusion in both the 22 subpopulations and 7 geographic populations. It meant that Bretschneidera sinensis populations had a low genetic diversity in a small scale while a high one in a big scale.(5) The Mantel test showed that the genetic distance among geographic populations was significantly positively correlated with geographical distance (r=0.69, P<0.05).The correlation analysis between genetic diversity and physical environmental factors, biological environmental factors showed that population genetic diversity was prominently positively correlated with longitude and latitude, and was significantly negatively correlated with aspect(r=0.99). There was no correlation between genetic diversity and important values, Pielou index(r=0.35⁰.64). The species numbers, Shannon-Wienner index and Simpson index had weak correlation with genetic diversity.The species composition of Bretschneidera sinensis community was complex. The relations between Bretschneidera sinensis and its concomitant species were unstable. The ISSR analysis system was optimized and established. The judge indexes were established based on ISSR technology to confirm the offspring-parent relation. The correlation between genetic diversity and geographic distance, physical environmental factors and biological environmental factors were analysed respectively. The survival condition and endangered mechanism of Bretschneidera sinensis population were also researched in this paper. All the results above might laid a foundation for setting conservation strategy and might provide scientific evidence to avoid extinction of Bretschneidera sinensis and guarantee the resource sustainable.