Rapd Analysis Of Differnet Myrica Rubra Cultivars In Fujian Province

In this experiment 30 accessions of Myrica rubra cultivars in Fujian Province were studied. The method of leaf DNA extraction was used, and the RAPD reaction system and amplification program for Myrica rubra were established and optimized, and the genetic relationship and genetic diversity of Myrica rubra were studied by RAPD markers and clustering analysis. The results were as follows:1. The development of the method of leaf DNA extraction in Myrica rubra. Myrica rubra was rich in the secondary metabolites such as polysaccharides,polyphenols and pigments etc, which make it hard to obtain high quality genomic DNA from their tissues. The improved CTAB method was used to extract pure and high-quality genomic DNA from leaves of Myrica rubra for RAPD amplification. However by this method, we need to pay some attentions on some points. First, the fresh and tender leaves shall be used as the materials and triturating the leaves before the DNA extraction can effectively solve the problems that the times of treating with the samples are not accordant and the distinction of the qualities and outputs among the same group DNAs was big. This was one of the pivotal techniques of extracting high-quality genomic DNA of Myrica rubra. Secondly, in order to reduce the intervention of the polysaccharides, the detergent was used before the cell nucleus was split. Thirdly, adding 2% PVP andβ-Mercaptoethanol into the extraction buffer can effectively prevent the oxidization of the polyphenols and others.2. The optimization of RAPD reaction system in Myrica rubra. Based on the common RAPD reaction program and adjusting experiments, the optimal amplification program for Myrica rubra RAPD-PCR was described as follows: 94℃for 3min; 38 cycles at 94℃for 30s,37℃for 30s,72℃for 90s; 72℃for 7min. The RAPD amplification system was in a 20μL reaction mixture containing 2.0μL 10×PCR buffer,2.5mmol/L MgCl2,0.25mmol/L dNTPs,1.5μmol/L primer,40ng DNA and 1.0U Taq DNA polymerse.3. The analysis of RAPD polymorphic degree in Myrica rubra.14 primers were selected from 171 random primers. They were used to analyze the 30 accessions with RAPD, and 155 bands were generated, among which the number of polymorphic bands was 130, and the percentage of polymorphism was equaled to 83.8%. The average number of bands directed by each primer was 9.3.4. The clustering analysis of RAPD markers in Myrica rubra. The genetic distances among 30 accessions of Myrica rubra cultivars in Fujian Province for RAPD marks were between 0 and 1. The genetic relationship between Wangzi'anhai and Zaosheng' anhai was the closest while the genetic relationship between Tongzi and Wandao was the farthest.