| Bretschneidera sinensis Hemsl. (Bretschneideraceae, Bretschneidera) is a monotypic endangered plant with a sparse distribution in the provinces south of the Yangtze River in China, and has already been listed in the First Grade of the List of Wild Plants Under State Protection. As an ancient relict species, B. sinensis has a very important scientific value in the research of the phylogenesis, the flora, the paleogeography and the paleoclimate of angiosperm. In this study, SSR markers were used to assess the genetic diversity and genetic structure of 15 natural populations of B. sinensis collected from six provinces in order to provide population genetics background for the development of its genetic resources conservation strategies. The main conclusions are as follows:(1)The optimization of SSR-PCR system for B. sinensisThe main factors affecting SSR-PCR system for B. sinensis were optimized through single factor experiments and orthogonal design, and an optimal SSR-PCR system for B. sinensis was established, containing 1.25mmol/L Mg2+,0.2mmol/L dNTP,0.5U Taq DNA polymerase,0.3μmol/L primers,90ng DNA template in 20μl reaction volumes. The PCR program was predenaturing at 95 ℃ for 5min; 35 cycles of denaturing at 95 ℃ for 30s, annealing at the optimized annealing temperature for 45s and extending at 72℃ for 90s; extending finally at 72 ℃ for 10min and then stored at 4℃.(2)The genetic diversity of B. sinensisThe optimized SSR-PCR system and 4 pairs of polymorphic primers screened were used to estimate the genetic diversity of 287 samples from 15 natural B. sinensis populations and 17 alleles were detected. At the species level, the observed number of alleles(A), the effective number of alleles(Ae), the Shannon’s Information index(I), the observed heterozygosity(Ho), the expected heterozygosity(He), the Nei’s expected heterozygosity(H) were 3-5,1.1183-3.0770,0.2325-1.1664,0.1115-0.5122,0.1060-0.6762, 0.1058-0.6750 with the mean of 4.25,1.9829,0.7683,0.3371,0.4061,0.4054, respectively. At the population level, the observed number of alleles(A), the effective number of alleles(Ae), the Shannon’s Information index(I), the observed heterozygosity(Ho), the expected heterozygosity(He), the Nei’s expected heterozygosity(H) were 2-3.25, 1.3441-1.9617,0.3400-0.7445,0.1833-0.5577,0.1886-0.4253,0.1853-0.4168 with the mean of 2.5,1.6188,0.5413,0.3521,0.3314,0.3217, respectively. All results showed that the genetic diversity of B. sinensis were both relatively high at the species and population level. The combined ranks of genetic diversity for the populations based on all the genetic indexes showed that the genetic diversity of HS population was the highest and that of JGS population was the lowest.(3)The genetic differentiation of B. sinensis populationsThe genetic differentiation coefficient(FST) of each locus ranged from 0.0536 to 0.3045 with a mean of 0.2143. The gene flow (Nm) among populations was 0.5710-4.4127 with a mean of 0.9163. And the analysis of molecular variance suggested that the genetic variations within populations was 79.80%, that among populations was 20.20%(P<0.001), and the genetic differentiation coefficient (GST) was 0.2020. The results showed that the gene flow among B. sinensis populations was less and the genetic differentiation was significant.(4) The structure of B. sinensisThe unbiased genetic distance among 15 populations of B. sinensis was between 0.0085 and 0.5284. It showed that the unbiased genetic distance between TX population and MS population was the nearest (GD= 0.0085), however, that between MS population and SHS population was farthest (GD= 0.5284). According to the unweighted pair-group method with arithmetic mean (UPGMA) dendrogram based on Nei’s unbiased genetic distance,15 natural populations of B. sinensis could be divided into 2 categories:SHS, HS and TBS populations as Group I, and the other populations as Group II. Meanwhile, the outcomes of STRUCTURE analysis also showed 287 samples of B. sinensis involved in this study should be divided into 2 groups, however, it was slightly different from the result of UPGMA. The STRUCTURE also indicated that the ancestry of several samples was complex, which showed there was gene flow among most populations.(5)The endangered reasons and conservation strategies for B. sinensisBased on all the results above, B. sinensis maintains a relatively high level of genetic diversity with big evolutionary potential and it is not the genetic diversity that caused B. sinensis endangered. It may relate to its own reasons (e.g. low rate of seed, difficulty in seed germination, etc) and excessive human interference (e.g. the habitat destruction, excessive utilization, etc). It is proposed that in-situ and ex-situ conservation strategies should be taken and the populations with high genetic diversity (e.g. HS, LP and RY population) should have the priority to be protected. At the same time, the government should strengthen publicity to the public to improve their awareness of protecting the endangered plants. |
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