RAPD Analysis Of Population Genetic Diversity And Genetic Structure Of Cinnamomum Camphora (L.) Presl

The genetic diversity and genetic structure of Cinnamomun Camphora (L) Presl population were studied by random amplified polymorphic DNA (RAPD). Thesis was composed of 4 parts:1. Based on the conventional CTAB method, an efficient procedure was developed. The major improvement included elimination a lot of secondary metabolites such as polyphenols etc, and collection of nuclei before free DNA. Then 3 X CTAB buffer which consisted of 100 m mol/L Tris (PH8.0), 25 m mol/L EDTA, 1.5 mol/L NaCl, 3.0% CTAB was added to extract total DNA. The results from ratios of A260 and A280, electrophoresis, digestion with restriction endonu-cleases and PCR amplification suggested that isolated DNA was suitable for RAPD analysis.2. A preservative medium which consisted of saturated NaCl-CTAB, 2% (w:v) PVP, 2% (v:v) (3 -sulfhydryl-ethanol, 50 m mol/L EDTA(pH8.0), was applied to field preservation of leaves tissue for DNA preparation of C. Camphora. It was indicated that the isolated DNA from the leaves tissue preserved in the medium for 7d 14d was similar to that from fresh leaves tissue.3. Effects of the content of DNA template, primer, dNTP, Mg2+ and Taq DNA polymerase on experimental result were tested by uniform test design, and the optimal reaction system of RAPD for C. Camphora was determined as follows: 8 n g/ u L DNA template, 0.2 n mol/L primer, 0.2mmol/L dNTP, 1.5 m mol/L MgCl2 and 1U Taq DNA polymerase in total 20 u L reaction volume. Effects of the denaturation time and annealing temperature on experimental result were tested as well. The optimal amplification procedure conditions were: 180s at 94 C. 30s at 94 C, 90s at 35C, 120s at 72C, cycling 42 times. 300s at 72C, then stored at4C.4. The genetic diversity and genetic structure of C. Camphora population were studied by RAPD. The analysis data of 168 RAPD loci of 21 random primers indicated that level of genetic variability in the 68 samples from 7 populations in Fujian was not high. The number of polymorphic bands was 75, The percentage of polymorphic bands (PPB) was 44.64%. The genetic diversity of Shaowu population (SW) was the highest, which PPB was 33.33%. And Fujian Academy of Forestry population (YS) was the lowest, which PPB was 22.62%. As analyzed by population genetic analysis (POPGENE) 1.32, Shannon's Information index, Nei's (1973) gene diversity and Gst among populations were 0.2496,0.1688 and 0.3498, respectively. Analysis of molecular variance (AMOVA) showed that there was 28.55% of variant component among populations, and 71.45% of variant component within populations. Usingunweighted pair group method arithmetic average (UPGMA) analysis, 7 populations of C. Camphora were mainly divided into two groups, there were 4 populations in one group including Fuzhou population (FZ) Shaowu population (SW), Shunchang population (SC) and Yong'an population (YA), and 3 populations in another group including Jian'ou population (JO) Pucheng population (PC) and Fujian Academy of Forestry population (YS).