RAPD Analysis On The Germplasm Resources Of Camellia Cultivars

In this paper,a set of methods suitable for total DNA extraction of Camellia plants were put forward. Appropriate primers were screened and the fingerprints of 23 camellia cultivars from home and abroad were generated using RAPD method. In addition, the relationship between Camellia cultivars and their genetic diversity were studies with the RAPD technique. The feasibility of using RAPD method to analysis on the germplasm resources of Camellia Cultivars was discussed.The main results were as follows:1. The total DNA from the fresh young leaves of camellia cultivars was successfully extracted by using the developed CTAB method .In light of the influence of secondary compounds, such as polyphenols in camellia cultivars on the quality of extracted total DNA , some measures were taken to eliminate them including a proper amount of PVP was added during grinding to prevent oxidation of phenolic compounds,1%β-mercaptoe-thanol added to the extracte solution to separate the phenolic compounds, using CTAB/NaCl solution to separate polysaccharides. The results of ratio of A₂₆₀ and A₂₈₀ ,electrophoresis and amplification suggested that the developed CTAB method could obtain high quality DNA with better purity and integrity compared with other methods.2. A steady system of PCR reaction for camellia cultivars was built with orthogonal design. The optimal PCR system for RAPD analysis was as follows: 80ng DNA template, 0.3μmol/ L random primer,0.2 mmol/L dNTPs, 2mmol/L MgCl₂, 1U Taq polymerase in 25 μl reaction solution. The reaction program was devised for one cycle at 94℃ 5 minutes and followed by 40 cycles, each with 50 seconds at 94℃, 50 seconds at 36℃,90 seconds at 72℃, and a final extension at 72℃ for 8 minutes.3. 20 primers were screened from 100 ten-bp arbitrary primers. A total of 136 DNA fragments ranging from 0.20- 2.0kbp were amplified, among which 120 ( 88.2% ) was polymorphic,using these 20 primers. The average number of DNA band produced by each primer was 6.8. The result of genetic similarity analysis for 23 cultivars showed that the Nei's coefficient ranged from 0.4386～0.8936, and the average Nei's coefficient was 0.7668.