| Many species population and variety groups of grape(vitis) have been formed in the long history of grape evolution and culturing.Because of great variation within the specis,easily nature cross between species and wide generation in all the world,taxonomy and identification of grapes are very difficult, The study was undertaken to identify grape (vitis) material of wide used varieties by random amplified polymorphic DNA (RAPD).The aim is to establish new analysis system of RAPD for grape cultivar, and to draw DNA fingerprinting for identification of species and varieties, and to select molecular markers with special segment in grape cultivars.the main results as below: 1.On the basis of conventional methord CTAB,the antioxidant (pvp 20%) and combination physic(β-Mercaptoethanol) were used to wipe off hydroxybenzene in grape leaves and the high density of salt were used to separate amylose , this way is rapid and high efficient to extract DNA and the DNA extracted can be used in RAPD amplification from grape leaves. Spring leaves are the best materials to extract DNA. 2.The different PCR program and the different ingredient concentration in RAPD reaction system such as Taq DNA polymerase ,Mg2+,　dNTP,DNA and primer were screened.suitable reaction system and program of the RAPD analysis for grape genomic DNA was settled.The following was the 25ul –reaction system with 1ul (diluted 15 times)template DNA,0.8 mmol/L dNTP,1U TaqDNA polymerase, 2.0mmol/L MgCl2 ,8pmol Primer,The program was that predenature in 94℃for 4 min,denature 94℃in for 45s,renature 37℃in for 55s,elongate 72℃in for 80s,cycling number in 40,holding in 72℃for 10 min. 3.The system was used for the amplification reaction in the experiment in all the material tested ,one hundred random primers were selected from OPERON Company for the amplification reaction in the experiment.of them thirty primers showed high efficient amplification and were used for all materials in amplification tested.the bands were clear and more polymorphic .A total of two hundredand sixteen bands were obtained by amplification of the polymorphic primer.among which one hundred and twenty-one bands(56.01%)were found to be polymorphic. 4.47 cultivars have been analysed by statistics software according to similar coefficient. When the LD is 13.5, the studied cultivars can be classified into the following five categories: ○1 including Kyoto,Benizuiho,Ryo Ho and so on; ○2 including Muscat Hamburg,Hongqitezao,Fenghuang51#,etc;○3 including Manicure Finger, Rui bier,Exotie,Red globe; ○4 including interspecific crossing cultivars such as Kangke,Nijiala,Katawa,Gold finger;○5 including some seedless cultivars such as Centennial seedless,crimson seedless. 5. DNA fingerprints of JingXiu, Hongqitezao,Fenghuang51# and other cultivars have been established firstly. Awailing of different panels of primers, according to DNA fingerprints andspecific bands 47 cultivars can be distinguished. 6. The feasibility of using RAPD in identifying cultivars and analyzing purifity Using a set of primers to detect polymorphism of genome,RAPD can detect all the region of genome and can detect the little difference of cultivar, in my paper,the results showed that the genetic polymorphism of grape was obvious ,the cultivars,even if have the same cross parenthood,can be devided,such as Jingyu and Augusta,they have the same cross parenthood of Italy and Queen of vineyard,Benizuibo and Ryo Ho have the same cross parenthood of Gold(4×) and Heichao.
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