南瓜ISSR-PCR反应体系的建立与优化  

Establishment and Optimization of ISSR-PCR Amplification System in Squash

作　　者：朱海生[1,2] 卢丽芳[3] 温庆放[1,2] 林义章[3] 

机构地区：[1]福建省农业科学院作物研究所,福州350013 [2]福建省农业科学院蔬菜研究中心,福州350013 [3]福建农林大学园艺学院,福州350002

出　　处：《分子植物育种》2014年第4期802-809,共8页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31101534);福建省农业科学院瓜类育种科技创新团队(CXTD-1-04);福建省农业科学院科技创新团队项目(CXTD-1-1304)共同资助

摘　　要：为获得南瓜ISSR最优扩增体系,为后续南瓜的遗传多样性分析和亲缘关系鉴定奠定基础,本研究以‘密本’南瓜为筛选体系材料,采用单因素试验,对ISSR反应体系的Mg2+、dNTP、Taq酶、引物和DNA浓度等5个因素进行优化。研究结果表明,南瓜ISSR 25μL最佳反应体系为:2.5μL 10×Buffer,2.0 mmol/L Mg2+,0.3mmol/LdNTP,1UTaq酶,0.48mmol/L引物,75ng的DNA。在此基础上,对筛选出的12条引物进行退火温度的优化,最终确定每条引物的最佳退火温度。利用该反应体系,选用引物891对85个南瓜品种进行所确立扩增体系的验证,结果显示扩增产物条带清晰明亮、多态性丰富,且特异性强、重复性好,表明本研究所确定的反应体系适用于南瓜的ISSR分子标记。In order to obtain the ISSR-PCR reaction system of Squash, We took an optimization experiment with single factor design which was concentrated on five factors of Mg2＋, dNTP, Taq DNA polymerase, primer and template DNA by using the squash ‘miben＇as the material of filter system to set up a good foundation of analysing the genetic diversity and relationship identification of Squash. The test result showed that the greatest 25 μL ISSR reaction system which contained 2.5 μL 10×Buffer, 2.0 mmol/L Mg2＋, 0.3 mmol/L dNTP, 1 U Taq DNA polymerase, 0.48 mmol/L primer, 75 ng template DNA. On this basis, optimized annealing temperature of 12 primers which have been screened, and ultimately determined the optimal annealing temperature of each primer.85 squashes were amplified with primer 891 under the optimized reaction conditions. The test result showed the amplification products which were clear and bright, abundant polymorphism, strong specificity and good repeatability. Accordingly, it was suitable for ISSR analysis of squash.

关 键 词：南瓜 ISSR 分子标记 PCR扩增 

分 类 号：S642.1[农业科学—蔬菜学]