ISSR Analysis Of Germplasm Resources Of Clausena Lansium(Lour.)

Wampee(Clausena lansium Lour. Skeels) belongs to Rutacease, consists of about 30 species all over the world. It is one of the most well-known special fruit tree in south china, where is the native land of Wampee, there are 11 species, mainly distributing in Fujian, Guangdong, Guangxi, and Hainan. Wampee has been cultivated in china for 1500 years old. Guangdong and Guangxi have carried some basic studies accordingly, including cultivation, management, morphologically, physiologically and so on, seldom from the angle of molecular biology. The research on both genetic diversity investigation and idioplasm evaluation systematically are still absent.In this study, inter-simple sequence repeat(ISSR) method was applied to detect genetic variation of cultivated Wampee from 40 samples. The results could provide scientific basis for making right protection strategy and help to foster new cultivars. The main results were as follow:1. Faced the fact that it is difficulty to extract DNA from Wampee which contains phenolic compound and quite a lot of sugar, the researcher used improved SDS method to extract DNA of Wampee, which meets ISSR requirements in quantity and purity. So far it is quite a proper way to extract DNA from Wampee.2. The technological system of ISSR-PCR reaction of Wampee has been built up. In the 20μL reaction system, there is 1.5 U of TaqDNAenzyme, 0.4μmol/L of primer, 300μmol/L of dNTP, 30 ng/reaction of template DNA, 2.0μL 10×Buffer solution. According to the system, the experimental steps are as follows. 5 min predenaturation at 94℃, 30 seconds denaturation at 94℃, 60 seconds annealing, 90 seconds extension at 72℃. The cycle repeats forty times and ends in 10 min extention at 72℃.3. A total of 15 ISSR primers were employed on 40 individuals of cultivated Wampee from 96 primers, and 165 scorable amplified polymorphic were detected, of which 100(60.6%) were polymorphic loci. And this research found that primers containing ( AG) n, (GA) n sequence resulted in higher levels of amplified polymorphism of Wampee.4. According to the clustering analysis of ISSR results, it proved that the genetic similarity index was 0.714～1.000. From the result, genetic diversity and similarity and genetic relationships were revealed.