Establishment of ISSR marker system and analysis of genetic diversity inVitis davidii Foëx.

Abstract: 【Objective】Vitis davidii Foëx. is a special wild resource in China, which belongs to the east Asian population and is mainly distributed in Hunan, Jiangxi and Fujian provinces. It is typically characterized with the rawhide sting on the new tip, petiole and veins and strong resistance to humidity, high temperature and diseases. The fruits are rich in nutrition with abundant flavonoids (mainly anthocyanins), resveratrol, superoxide dismutase, vitamin C and other active ingredients, which is believed functionally beneficial to the health of human. Traditional research on the diversity of the V. davidii Foëx. was mainlybased on morphological features and biological characteristics with low accuracy. Therefore, ISSR markersystem was established and employed to analyze the diversity of the species for providing the basis for protection and utilization.【Methods】52 samples of V. davidii Foëx. (41 from Hunan province, 1 from Fujianprovince, 10 from Jiangxi province) and 8 varieties or accessions of the other species of Vitis were used asthe materials. ISSR primers were selected at Canada Columbia University for PCR amplification. The ge⁃nomic DNA was extracted by modified CATB method. Firstly, the healthy leaves were swabbed with 75%alcohol and freezing liquid nitrogen and were stored in a refrigerator at -70 ℃. Then the dried leaves weretreated by CATB-free buffer (0.2 mol·L-1 Tris-HCl，pH 8.0；50 mmol·L^-1 EDTA，pH 8.0；0.25 mol·L^-1 NaCl) and ice bath for 30 min; 1 μL extract DNA (pH from 8.0 to 10.0) with 1.5 μL bromophenol blue solution (2.5 g·L^-1 bromophenol blue，2.5 g·L^-1 xylene cyanol，400 g·L^-1 saccharose) of each sample was taken for electrophoresis (0.8% agarose gel) with TE buffer (10 mmol·L^-1 Tris-HCl，1 mmol·L^-1 EDTA，pH8.0). The concentration of Mg2+，dNTPs，primer，DNA template and the number of amplification cycles suitable for ISSR-PCR amplification reaction of V. davidii Foëx were optimized using the multifactor and multilevel experiments. The PCR reaction cycling profile was modified as 94 ℃ for 5min，94 ℃ for 30 s，54-58 ℃ annealing for 1 min，72 ℃ for 5 min followed by 35 cycles ,and a final extension at 72 ℃ for 7 min.SPSS 17.0 software program was employed for clustering analysis of the V. davidii Foëx. The clustering figure was also established.【Results】The optimum ISSR-PCR reaction system was as follows: in the total re⁃action system with amount of 20.0 μL, there were 4 μL 10×PCR Buffer，1 UT Tag enzyme，2.5 mmol·L^-1 Mg2+，0.25 mmol·L-1 dNTPs，0.6 μmol·L^-1 primer，30 ng DNA template, respectively. In optimization ofISSR- PCR reaction system, 13 primers from 100 arbitrary primers were selected. 116 polymorphismbands among 139 amplified bands were obtained with high quality, purification and stability. The rate ofpolymorphism was 83.45%. The primers UBC807 and UBC834 had got the highest number of polymorphic loci (14 loci). According to genetic similarity coefficient clustering analysis, the 8 materials of thecontrol were outside the V. davidii Foëx species，The genetic distance between the V. pseudoreticulata W.T. Wang and V. davidii Foëx was near. All the 52 samples of V. davidii Foëx could be divided into threesubgroups.【Conclusion】Vitis germplasm resources had abundant genetic diversity. ISSR markers couldeffectively reveal the genetic diversity and genetic relationship of the V. davidii Foëx. The results wouldbe beneficial to the protection and and utilization of the species.