Establishment Of RAPD Analysis Techniques And It's Application Studies On Pepper Variety Resources

In this report , a simple and efficient protocol for RAPD assay in pepper was established through optimization in two main procedures-DNA extraction and PCR .Based on the protocol, 46 pepper varieties from homeland and abroad were analyzed by RAPD and clustering and their evolution relationship were also analyzed . The main results are as follows : The immature leave of pepper is suitable for the DNA extraction, with high yield and good quality . The DNA extraction from 0.5gram immature leave is sufficient for RAPD analysis .or the methods of pepper DNA extraction, the reagent-box method is the best and has highest yield, best quality, highest purity and simplest procedure, but higher cost. This method can be used when a lot of high quality DNA with few variety are needed . (D The optimization program of RAPD in pepper is : DNA 2 ul ,dNTP 0.5 ul,primer 1 ul,Buffer 2.5 ul ,Taq ployrase 0.5 U ,pure water 18.5 ul ,total volume 25 ul; PCR program : 95 XH predenaturation for 1 min ,36 annealing for 40s ,72 prolong for Imin20s ,94X denaturation 20s ,41times circle ,36 annealing for 1 min ,72 prolong 3 min ,4X1 for ever.Primer T11,Y14,D13 are very important in study of pepper genetic diversification ,variety identification ,finger map building and identification of their hybrid Fl .Cluster are analyzed based on RAPD .46 varieties can be divided into six groups .Luodianjiao from Guizhou province,ICPN9-20 from AVRDC ,Ziseguanshaongjiao from Liaonin province ,Jinsajianglaojia and Xiaomila from Yunnan province were divided into single group independly , others were clustered into a large group . The evolution of pepper based on RAPD is : The taper shaped pepper with small fruit ,many seeds ,hot chilli taste-fruit cluster type with small fruit ,hot chilli taste ,many fruit setting long shaped pepper with long fruit , chilli taste , easy to dehydration long shaped pepper with long fruit ,thick wall ,chilli taste , suitable for fresh market and dehydration -ox-horn shaped pepper with thick wall ,large fruit -lantern shaped pepper with thick wall ,large fruit, without hot taste.