RAPD Analysis Of Population Genetic Diversity And Genetic Structure Of Alsophila Spinulosa

A preservative medium which consisted of 1.5% SDS (sodium dodecyl sulfate ), 100 mmol/LTris (pH8.3), SOmmol/LEDTA (pH8.0), 500 m mol/L NaCl and 500 m mol/L P -sulfhydryl-ethanol, was applied to field preservation of leaves tissue for DNA preparation of Alsophila spinulosa. It was indicated that the isolated DNA from the leaves tissue preserved in the medium for 7d^ 15d, 30d> 90d> 180d was 48kb, and DNA yield was 600 ~ 1200 v g /g'FW. This results was highly similar to that from fresh leaves tissue. The isolated DNA from the preserved leaves tissue were used for RAPD analyzing and it was showed that the preserved leaves tissue from same individual was obtained a same result as from fresh leaves. High-quality DNA of 91 individuals from 8 populations of Alsophila spinulosa was prepared by this method. This isolated DNA has been use to experiments base on PCR technology.The influencing factors of RAPD of Alsophila spinulosa population genetic diversity research were studied and the experimental parameters were optimized. The optimal RAPD conditions are: 10 u 1 reaction volume , containing 2ng/u 1 template DNA, 2.0mmol/L MgCl2, 0.3 u mol/L primer, 300 u mol/L dNTP, 0.5U Taq DNApolymerase. Amplification program is : 4min at 94℃; 30s at 94℃, 40s at 36℃, 90s at 72℃, 40 cycles; 7min at 72 ℃ then stored at 4℃.The genetic diversity and genetic structure of Alsophila spinulosa population were studied using random amplified polymorphic DNA (RAPD ) analysis. The analysis data of 183 RAPD loci of 20 primes indicated low level of genetic variability in the 65 individuals from 8 populations of three Alsophila spinulosa distribution areas of Fujian, Sichuan and Guizhou. The number of polymorphic bands was 48, the percentage of polymorphic bands (PPB) was 26.2%, and the Nei's gene diversity and Shannon diversity index were 0.0746 and 0.1140 respectively. The population of Jinsha had the highest genetic diversity of the 8 examinated populations, which PPB was 11.56%, at the same time, population of Guaxi had the lowest diversity level, which PPB was 1.25%, these data indicated that genetic diversity of population was extremely low. Genetic distance between populations was correlative to geographic distance. There existed prominent population differentiation among populations with Gst value as high as 0.7015. Analysis of molecular variance (AMOVA) also showed that there were 64.13% of genetic variance existed among populations. Alsophila spinulosa possiblily experiented severe bottleneck effect during its evolution. To make effective protect policy for alsophila spinulosa, we should pay attention to protect as many different populations as possible .