ISSR Analysis Of Vitis L. Germplasm Resources

The genetic diversity of 91 samples was analyzed by Inter-Simple Sequence Repeat (ISSR) markers in Vitis L. in this thesis. The purpose of this paper is to classify grape germplasm resource and identify with the different names of varieties of the same name by an efficient molecular marker and provide a theoretical guidance of sustained development for grape hybrid breeding and grape industry. The result was as follows:1. Efficient method of total DNA extraction from grape was developed. The total DNA was extracted from leaves by modified CTAB. The results showed that the high quality and purity of total DNA meet the requirements of ISSR analysis in Vitis L. The method took less time, was simply carried out and effectively reduced contaminations in the process of DNA extraction.2. The ISSR-PCR reaction system was optimized. Reaction system of ISSR-PCR was optimized with the total DNA in Kyoho and Vitis davidii Fo?x. The best reaction system was: added 2μl 10×Buffer, 20 ng total DNA, 0.20 mmol/L dNTP, 0.35μg mol/L primer, 2.0 U TaqDNA polymerase, 15.5μL ddH2O. The best process was: 1.5 min predenaturation at 94℃; 40 s denaturation at 94℃; 1 min annealing; 1.5 min extension at 72℃; The cycle repeats forty times and ends in 5 min extension at 72℃, save it at 4℃.3. The ISSR-PCR amplified reaction was carried out. 15 primers from 100 arbitrary primers were selected. The 91 germplasm resources were amplified by ISSR-PCR. The result was: 214 polymorphism bands among 237 amplified bands were obtained; the rate of polymorphism was 90.2%.4. The diversity of grape germplasm resource was analyzed. The similarity coefficient matrix of amplified bands was and analyzed by SPSS 13.0. The result was that the genetic similarity coefficient among 91 samples ranged from 0.546 to 0.853. 91 samples were classified into three kinds, Eurasian grape, euramerican hybrid grape and wild grape.'Minbei green grape'belonged to Eurasian grape;'Taiwan-Kyoho'is similar to'Tian Qiyuan';'Japan- Kyoho'was probably the excellent generation of giant grape.5. The botany characteristics and clustering results of samples was compared. Results show that: the color of grape's peel and resistance was probably related to its domestication and cultivation, higher level of domestication and cultivation, much weaker level of peel color and resistance.