【作者】 罗明明；

【导师】 王永清； 罗楠；

【作者基本信息】 四川农业大学 ， 果树学， 2005， 硕士

【摘要】 本试验利用RAPD技术对四川农业大学脱毒中心种植的29个葡萄品种之间的亲缘关系进行了遗传多样性分析,结果表明: 1.本研究使用了一种改良CTAB法完全适用于葡萄属基因组DNA的提取,得到的DNA量大,产率达到202—366ng/mg（鲜叶）质量高,OD₍260/OD₂₈₀的比值在1.73—1.86之间,不必去除RNA就能用于RAPD的分析,符合RAPD的要求。 2.优化了RAPD反应体系。所得最优反应体系为:反应体积25μl,其中模板DNA稀释5倍,引物浓度1.5gmol/L,Taq DNA聚合酶1.25U,dNTP 0.25mmol/L,MgCl₂1.5mmol/L,Buffer 25mmol/L,加去离子水补充总体积至25μl。反应程序为94℃预变性2min,94℃变性1min,35℃复性1min,72℃延伸2min,40个循环后,在72℃保持8分钟。 3.从40个随机引物中筛选出8个多态性好的随机引物,用这8个引物对29个葡萄品种进行扩增,8个引物共扩增出了126条DNA带,平均每个引物15.75条,其中111条具有多态性,占总条带数的88.1%,每个引物可扩增出3-34条多态性带,平均每个引物13.9条多态性带。 4.利用计算机软件NTSYSpc（2.10版）统计RAPD数据,用Genetic Distance计算材料间遗传距离。‘京玉’与‘红宝石无核’的遗传距离最小,只有0.1093,相似系数达到90%;‘优无核’与‘早红提’的遗传距离最大为0.8462,相似系数为15%。按照UPGMA法进行聚类分析,将供试的29个葡萄品种分成了五类,第一类有‘峰后’、‘黑蜜’、‘农科3号’、‘无核早红’、‘8612’、‘高妻’和‘红富士’;第二类有‘超藤’、‘美人指’、‘优选皮奥奈’、‘黄金指’、‘京玉’、‘红宝石无核’、‘蜜汁’、‘弗蕾无核’、‘克瑞森无核’、‘皇家秋天’、‘超级无核’、‘维纳斯Ⅱ’、‘青提’、‘维多利亚’、‘红高’、‘京秀’、‘农科2号’、‘维纳斯Ⅰ’;第三类是‘农科1号’;第四类是‘优无核’、‘莫丽莎无核’;第五类为‘早红提’。更多还原

【Abstract】 In this study, the genetic diversity of 29 grape varieties cultivated in Sichuan Agricultural University was studied by random amplified polymorphic DNA(RAPD).The relationship of the 29 grape varieties was analyzed. The results were as follows:1. The improved CTAB method developed in this study was the best one to extract vitis genomic DNA. DNA quality was high, and OD260/OD280 of DNA was 1.73-1.86. And DNA without getting rid of RNA can be used for RAPD amplification.2. The suitable reaction system and amplification procedure were found. The reaction mixture contained diluted 10 times template DNA in a 25 μL reaction volume with 2.5 μL lOxreaction buffer, 1.5mmol/L MgCl2, 250μmol/L dNTP and 1.25U Taq DNA polymerase and 1.5μmol/L primer covered with a drop of mineral oil. Amplification was performed in a PT-100 thermal cycler programmed for 40 cycles (94 ℃ for 1 min; 35 ℃ for 1 min; 72 ℃ for 2 min) followed by an extension at 72 ℃ for 8 min.3. 126 repeatable loci (including 111 polymorphic loci) were amplified using 8 random primers in 29 grape resources. The percentage of polymorphic bands was 88.1%,13.9 polymorphic bands per primer were amplified. 3-34 repeatable loci were amplified with each primer. As analyzed by NTSYSpc version 2.10, the genetic distance between ’Jinyu’ and ’Ruby Seedless’ was the smallest(0.1093). The distance between ’Superior Seedless’ and ’Zhaohongti’ was the biggest (0.8462). UPGMA clustering analysis based on genetic similarity showed that 29 materials could be classified into 5 groups. 7 materials (’Fenghou’、 ’Heimi’、 ’Nongke Ⅲ’ , ’Wuhezaohong’、 ’8612’ 、’Gaoqi ’and ’Beni Fuji’) belonged to group Ⅰ ; 18 materials (’Caoteng’、 ’Manicure Finger’、 ’Youxuanpiaolai’、 ’Huangjin Finger’、 ’Jinyu’、 ’Ruby Seedless’, ’Mitsushiru’、 ’Flame Seedless’、 ’Crimson Seedless’、 ’Autumn Royal’、 ’Super Seedless’、 ’Venus Ⅱ’ 、 ’Centenial Seedless’、 ’Victoria’、 ’Benitaka’、 ’Jinxiu’、 ’Nongke Ⅱ’、 ’Venus Ⅰ ’) belonged to group Ⅱ; Nongke I was in group Ⅲ; 2 materials (’Superior Seedless’ and ’Melissa Seedless’) belonged to group Ⅳ; Zaohongti belonged to group Ⅴ.更多还原