刺葡萄ISSR分子标记体系的建立及种质资源聚类分析

Establishment of ISSR marker system and analysis of genetic diversity in Vitis davidii Fo?x.

【目的】建立刺葡萄(Vitis davidii Fo?x.)的ISSR分子标记体系以及分析刺葡萄种质资源的亲缘关系,为刺葡萄的保护和利用提供依据。【方法】以8份外缘葡萄品种或类型为对照,52份刺葡萄类型为材料,采用改良CTAB法提取植物基因组DNA,以基因组DNA为模板,用同一试验考察多个因素及水平的筛选方式对ISSR-PCR扩增反应体系中的Mg2+、d NTPs、引物、模板DNA的浓度及循环数进行优化,建立适用于刺葡萄的最佳ISSR-PCR反应体系;在优化的反应体系中进行多态性引物的筛选并进行分析,同时根据Jaccard遗传相似系数进行UPGMA聚类分析。【结果】采取改良CTAB法提取的刺葡萄DNA质量优于常规的CTAB法,在优化的ISSR-PCR反应体系中,从100条ISSR引物筛选出了13个具有多态性的引物,共检测到139个位点,其中多态性位点116个,多态位点百分率为83.45%。聚类结果显示,对照组8份外缘资源均在刺葡萄种群外,刺葡萄与华东葡萄的遗传距离较腺枝葡萄近;52份刺葡萄类型分为三大组群,两大江西刺葡萄组群及湖南与福建刺葡萄组群,而在湖南与福建刺葡萄种群中又分为4个群组。【结论】刺葡萄种质资源具有丰富的遗传多样性,ISSR分子标记可有效揭示刺葡萄种质资源的遗传多样性和亲缘关系,对刺葡萄种质资源的保护和利用有重要的意义。

【Objective】Vitis davidii Fo x. is a special wild resource in China, which belongs to the eastAsian population and is mainly distributed in Hunan, Jiangxi and Fujian provinces. It is typically charac-terized with the rawhide sting on the new tip, petiole and veins and strong resistance to humidity, high tem-perature and diseases. The fruits are rich in nutrition with abundant flavonoids（mainly anthocyanins）, res-veratrol, superoxide dismutase, vitamin C and other active ingredients, which is believed functionally ben-eficial to the health of human. Traditional research on the diversity of the V. davidii Fo x. 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 pro-tection and utilization.【Methods】52 samples of V. davidii Fo x.（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 were treated by CATB-free buffer（0.2 mol·L（-1）Tris-HCl,p H 8.0;50 mmol·L（-1）EDTA,p H 8.0;0.25 mol·L（-1）Na Cl） and ice bath for 30 min; 1 μL extract DNA（p H 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,p H8.0）. The concentration of Mg（2＋）,d NTPs,primer,DNA template and the number of amplification cycles suitable for ISSR-PCR amplification reaction of V. davidii Fox 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 Fox. The clustering figure was also established.【Results】The optimum ISSR-PCR reaction system was as follows： in the total reaction 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）d NTPs,0.6 μmol·L（-1）primer,30 ng DNA template, respectively. In optimization of ISSR-PCR reaction system, 13 primers from 100 arbitrary primers were selected. 116 polymorphism bands among 139 amplified bands were obtained with high quality, purification and stability. The rate of polymorphism 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 the control were outside the V. davidii Fox species,The genetic distance between the V. pseudoreticulata W.T. Wang and V. davidii Fox was near. All the 52 samples of V. davidii Fox could be divided into three subgroups.【Conclusion】Vitis germplasm resources had abundant genetic diversity. ISSR markers could effectively reveal the genetic diversity and genetic relationship of the V. davidii Fox. The results would be beneficial to the protection and and utilization of the species.