凤仙花属植物SSR-PCR反应体系的建立与优化  

Establishment and Optimization of SSR-PCR Reaction System of ImpatiensL.

作　　者：罗超 黄武略 朱佳鹏 冯志熙 刘应丽 黄海泉[1,2] 黄美娟[1,2] Luo Chao;Huang Wulue;Zhu Jiapeng;Feng Zhixi;Liu Yingli;Huang Haiquan;Huang Meijuan(College of Landscape Architecture,Southwest Forestry University,Kunming,650224;Research and Development Center of Landscape Plants and Horticulture Flowers,Southwest Forestry University,Kunming,650224)

机构地区：[1]西南林业大学园林园艺学院,昆明650224 [2]西南林业大学,园林园艺花卉研发中心,昆明650224

出　　处：《分子植物育种》2020年第17期5790-5799,共10页Molecular Plant Breeding

基　　金：国家自然科学基金资助项目(31560228,31860230);云南省重点研发计划(2018BB013);云南省中青年学术和技术带头人培养项目(2015HB046,2018HB024);云南省高校科技创新团队支持计划资助;国家林业局西南风景园林工程技术研究中心项目(2016-48);西南林业大学校重点项目(111124);云南省教育厅科学研究基金项目(2019Y0148)共同资助。

摘　　要：SSR-PCR反应体系的建立与优化是凤仙花属植物(Impatiens L.)SSR标记研究的基础,本研究以8种凤仙花属植物为试材,对Mg^2+、Taq酶、dNTPs浓度、DNA模板、引物浓度等5因素进行L16(4^5)正交试验,探讨适合凤仙花属植物的SSR-PCR反应体系,并对主要因素进行优化。结果表明:Mg^2+、Taq酶和dNTPs浓度3个因素对凤仙花属SSR-PCR扩增结果有显著影响,影响程度为Mg^2+>Taq酶>dNTPs浓度>DNA模板>引物浓度;20μL为最佳反应体系,其中,2.6 mmol/L(含10×PCR Buffer)的Mg^2+,1.6 mmol/L的dNTPs,2μmol/L的引物,60 ng的DNA,0.1 U的Taq酶,ddH2O补齐剩余部分。利用优化后的反应体系对同属54种材料进行PCR扩增,扩增产物在130~200 bp,具4个等位基因,目标条带清晰,多态性良好。该体系的建立可为今后利用SSR标记对凤仙花种质鉴定、遗传多样性分析、系统发育研究、遗传图谱构建、基因定位和分子标记辅助育种等研究提供了依据。The establishment and optimization of SSR-PCR reaction system is the basis of SSR marker study of Impatiens.In this study,8 species of Impatiens were used as materials,and the concentration of Mg^2+,Taq,dNTPs and DNA were determined.L16(45)orthogonal test was carried out by using five factors such as template and primer concentration to explore the SSR-PCR reaction system suitable for Impatiens and optimize the main factors.The results showed that the three factors of Mg^2+,Taq enzyme and dNTPs had significant effects on the SSR-PCR amplification results of Impatiens,and the degree of influence was Mg^2+>Taq enzyme>dNTPs concentration>DNA template>primer concentration.The optimal total reaction system is 20μL;the concentration of Mg^2+is 2.6 mmol/L(including 10×PCR Buffer);the concentration of dNTPs is 1.6 mmol/L;the concentration of primer is 2μmol/L,the amount of DNA template is 60 ng;the amount of Taq polymerase is 0.1 U,and the rest is performed with ddH2O supplement.The optimized reaction system was used for PCR amplification of 54 species of the same genus.The amplified product was 130~200 bp with 4 alleles,and the target bands were clear and the polymorphism was good.The establishment of this system can provide a basis for future research on germplasm identification, geneticdiversity analysis, phylogenetic research, genetic map construction, gene mapping and molecular marker-assistedbreeding of Impatiens balsamina using SSR markers.

关 键 词：凤仙花 SSR-PCR 体系建立 正交试验 

分 类 号：S681.1[农业科学—观赏园艺]