| Camellia fascicularis is an extremely small population plant of Yunnan.The emergency plan for the rescue and protection of the extremely small populations species in Yunnan Province during 2010 to 2015 has included C.fascicularis.According to the IUCN assessment criteria,this species is classified as a "critically endangered species"(CR A1c).In this study,SSR molecular markers were used to analyze the genetic diversity of different populations of C.fascicularis.The genetic variation of different species of C.fascicularis was elucidate in the molecular level.Then,the tender stems with axillary bud used as explants to established tissue culture system of C.fascicularis.Accoring to comparing the effects of disinfection,proliferation culture and rooting culture in different disinfection durations,media types and hormone concentrations effect,respectively,An complete and stable in vitro rapid propagation system of C.fascicularis was established.The main results were as follows:1.In this study,a total of 95,979 SSR-containing ESTs were obtained based on transcriptional sequencing date,which enriched the C.fascicularis EST database,and 14 pairs of EST-SSR primers with high polymorphism were screened.The number of alleles of the 14 polymorphic loci were 2~8,and 68 alleles were amplified.The average number of alleles was 4.8571,and the average number of effective alleles was 2.7130.The average Shannon's diversity index(I)was 1.0925,showing a high polymorphism.2.The percentage of polymorphic loci(PPB)in 8 populations of C.fascicularis ranged from 42.86% to 100%.There were significant differences in genetic parameters among different populations,but their polymorphisms were high.The gene flow(Nm)values ranged from 0.0503 to 0.9820 with a mean value of 0.5440 <1,indicating that the gene exchange among C.fascicularis populations was low.Based on the analysis of genetic structure,eight populations were roughly divided into three groups.Genetic variation was 49.95% of the total variation,and mainly occurred in the population.3.A high level of genetic diversity was found in C.fascicularis,and its rich genetic diversity might be determined by the diversity of habitats and the long evolutionary history of the species.There was no gene exchange among the natural populations of C.fascicularis,and the population differentiation level was low.4.The tender stems of C.fascicularis used as explants were disinfected with 75% alcohol for 10 s,sterilized with 0.1% mercury for 10 min,and then inoculated in medium MS+6-BA 2.0 mg/L + IAA 0.5 mg/L with the contamination rate of 15 axillary buds germination rate of 90%,and the adventitious buds grew well;WPM medium as a basic culture medium for C.fascicularis was suitable.The best proliferation medium was WPM+3.0 mg/L 6-BA+0.3 mg/L IAA,the proliferation coefficient was 6.83,the average seedling height was 3.5cm,the seedlings were thick and grow well;1/2 MS +IBA 2.0 mg/L+ NAA 0.3 mg/L+0.3 g/L activated charcoal was the best rooting medium with average number of rooting strips of 4,and rooting rate of 100%;The plantlets transplanted in matrix(laterite: Humus=2:1),the survival rate reached 95% after 50 days.The seedling growth was good. |
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