Establishment Of SSR Reaction System And Analysis Of Genetic Diversity Of Grape Cultivars

Grape is one of the oldest economic fruit trees,cultivation has a long history and broad geographical distribution,the species is abundant resources,With modern agricultural science and the rapid development,growing area of cultivation,however,cultivars have become increasingly narrow,the grape understanding of the genetic characteristics of grapes due to a high degree of heterozygosity,inbred inhibition,a relatively long period of infancy,and its system was inadequate.In the late 1950s,it began to strengthen the genetics of grapes.In recent years,due to the development of molecular biology,molecular marker technology has been in the grape genetics research has achieved remarkable results.In this paper,SSR reaction system of grape was established,and the genetic diversity of 39 grape cultivars was analyzed using SSR marker in order to offer basis for variety identification and cultivars relationship,for the purpose of variety identification and plant breeder's rights protection.The main results from this study are as follows:(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 SSR reaction systemsuch as Taq DNA polymerase,Mg²⁺,dNTP,DNA and primer were screened.suitable reaction system and program of the SSR analysis for grape genomic DNA was settled.The following was the 20ul-reaction system with 20～30ng template DNA,0.2 mmol/L dNTP,1U TaqDNA polymerase,1.5mmol/L MgCl₂,0.5umol/L Primer,The program was that predenature in 95℃for 4 min,denature 95℃in for 50s,renature 50-60℃in for 1min,elongate 72℃in for 1min30s,cycling number in 30,holding in 72℃for 7 min.(3)The system was used for the amplification reaction in the experiment in all the material tested,twenty two random primers were selected for the amplification reaction in the experiment.of them twelve primers showed high efficient amplification and were used for all materials in amplification tested.the bands were clear and more polymorphic.A total of one hundred fifty five bands were obtained by amplification of the polymorphic primer.among which one hundred forty nine bands(96.13%)were found to be polymorphic.of them special bands are eight(5%),amplified fragments are between 50-1000bp,The number of amplified band per palmer pair ranged from 5 to 24,with an average of 12.9,the most bands are amplified by the primer VrZAG67,for 24,the lest are VrZAG47,only 5 amplification bands.(4)By using SSR,39 cultivars have been ampified,calculation of the similarity coefficient with NTSYS genetic similarity coefficients among all grape varieties ranged from 0.374 to 0.974,Cluster analysis was made with unweighted pair group method for arithmetic averages UPGMA using genetic similarity coeffieientc,at the genetic distance of 0.660,The 39 materials were clustered into 3 groups.the first group include Greaca and Manicure Finger,the second including Muscat Hamburg.Hongqitezao.Tamina. Meiguiyu.Hongmeigui and so on.the third including Jingyun.Augusta.Wuhezaohong. Jufeng etc.(5)DNA fingerprints of Jufeng,Gorbv,Greaca and other cultivars have been established firstly.Awailing of different panels of primers,according to DNA fingerprints and specific bands 39 cultivars can be distinguished.(6)The feasibility of using SSR in identifying cultivars and analyzing purifityin my paper,the results showed that the genetic polymorphism of grape was obvious,the cultivars,even if have the same cross parenthood,can be derided,such as Jingyun and Augusta,they have the same cross parenthood of Italy and Queen of vineyard.