番茄叶片DNA提取、SSR-PCR反应体系优化及引物筛选  

DNA Extraction, Optimization of SSR-PCR Reaction System and Primer Screening of Tomato Leaf

作　　者：芮文婧 王晓敏[1,2,3] 张倩男[1] 张学琴 吕原[1] 胡学义 高艳明 李建设 Rui Wenjing;Wang Xiaomin;Zhang Qiannan;Zhang Xueqin;Lv yuan;Hu Xueyi;Gao Yanming;Li Jianshe(School of Agriculture, Ningxia University, Yinchuan, 750021;Ningxia Facility Horticulture （Ningxia University） Technology Innovation Center, Yinchuan, 750021;Ningxia Modem Faciliry Horticulture Engineering Technology Research Center, Yinchuan, 750021)

机构地区：[1]宁夏大学农学院,银川750021 [2]宁夏设施园艺(宁夏大学)技术创新中心,银川750021 [3]宁夏现代设施园艺工程技术研究中心,银川750021

出　　处：《分子植物育种》2018年第7期2230-2236,共7页Molecular Plant Breeding

基　　金：宁夏回族自治区农业育种专项(NXNYYZ20150303);宁夏大学研究生创新试验(GI2017039);宁夏自治区农业育种专项(NXNYYZ20150301)共同资助

摘　　要：为了进一步开发利用番茄种质资源和开展分子标记辅助选择育种,本研究利用高通量组织研磨机研磨微量叶片,通过改良CTAB法快速提取DNA,并利用L16(45)正交设计试验,综合采用直观量化分析和方差分析两种方法,分析Mg2+、d NTPs、Taq DNA聚合酶、引物、模板DNA浓度5个因素对番茄SSR-PCR扩增的影响,比较不同处理组合扩增效果的差异,最终筛选与验证最优的番茄SSR-PCR反应体系,并在此体系下对SSR引物进行筛选。结果表明番茄SSR-PCR最佳反应体系(20μL)为Mg2+3.0 mmol/L、d NTPs 0.4 mmol/L、Taq DNA聚合酶0.5 U、引物0.5μmol/L、模板DNA 40 ng;且利用优化后的反应体系,从540对SSR引物中筛选出373对(占69.07%)扩增条带清晰、多态性丰富的引物。该SSR-PCR体系的建立为番茄种质资源遗传多样性分析,品种鉴定及指纹图谱构建等研究提供了一个标准化的程序。In order to further develop and utilize tomato germplasm resources and carry out molecular marker- assisted selection breeding, few fresh tomato leaves were grinded by high throughput tissue grinding machine, and total DNA of tomato were extracted rapidly by a modified CTAB method. A research about the impacts of 5 reaction factors （Mg2＋, dNTPs, Taq DNA polymerase, primer concentrations and DNA template） on SSR-PCR ampli- fication of tomato was carried out by using an orthogonal experimental design of L16（45）, visual quantitative analysis and analysis of variance. The effect of different treatment combinations were compared, the optimized tomato SSR-PCR reaction system was selected and verified, and the SSR primers were screened under this system. The results showed that the optimal SSR-PCR reaction mixture was as follows： 20 μL total volume including 3.0 mmol/L Mg2＋, 0.4 mmol/L dNTPs, 0.5 U Tat/DNA polymerase, 0.5 p, mol/L primer, and 40 ng template DNA. 373 pairs of SSR primers with clear and rich polymorphic bands were screened from 540 pairs of primers （69.07%） using the optimized reaction system. The establishment of this optimal system for SSR-PCR would provide a standard protocol for the investigation on genetic diversity analysis of germplasm resources, cultivar identification, andfinger printing construction of tomato.

关 键 词：番茄 正交设计 SSR-PCR 体系优化 引物筛选 

分 类 号：S641.2[农业科学—蔬菜学]