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真藓(Bryum argenteum)ISSR-PCR反应体系优化及引物筛选
马晓英, 韦伟, 臧程, 于晶
上海师范大学 生命与环境科学学院 植物种质资源开发协同创新中心, 上海 200234
摘要:
为了确定真藓最佳ISSR-PCR反应体系,采用PCR正交实验设计方法,对影响ISSR-PCR试验的模板DNA、引物、dNTPs、Mg2+、和TaqDNA聚合酶5个因素在4个水平上进行优化,并对100条引物逐一进行温度梯度PCR,筛选合适的引物并确定每个引物的最佳退火温度.结果显示优化的20 μL ISSR-PCR反应体系中包括20 ng/20 μL DNA模板、0.45 μmol/L引物、2.65 mmol/L Mg2+、0.4 U/20 μL Taq DNA聚合酶、0.45 mmol/LdNTPs.利用该体系最终筛选出50条扩增条带清晰,重复性好,多态性高的引物并确定其退火温度.这一体系的建立、引物的筛选及退火温度的确立为进一步利用ISSR分子标记技术对苔藓遗传多样性的研究提供理论基础.
关键词:  真藓  ISSR  正交设计  引物筛选  退火温度
DOI:10.3969/J.ISSN.1000-5137.2017.05.009
分类号:Q949
基金项目:国家自然科学基金(31570208);上海植物种质资源工程技术研究中心项目(17DZ2252700);上海师范大学校前瞻性预研项目(DYL201503)
Optimization of ISSR-PCR reaction system and selection of primers in Bryum argenteum
Ma Xiaoying, Wei Wei, Zang Cheng, Yu Jing
Development Center of Plant Germplasm Resources, College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234, China
Abstract:
In order to determine optimum ISSR-PCR reaction system for moss Bryum argenteum,the concentrations of template DNA primers,dNTPs,Mg2+ and Taq DNA polymerase were optimized in four levels by PCR orthogonal experimental method. The appropriate primers were screened from 100 primers by temperature gradient PCR,and the optimal anneal temperature of the screened primers were determined. The results showed that the optimized 20 μL ISSR-PCR reaction system was as follows:template DNA 20 ng/20 μL,primers 0.45 μmol/L,Mg2+2.65 mmol/L,Taq DNA polymerase 0.4 U/20 μL,dNTPs 0.45 mmol/L. Using this system,50 primers with clear bands,repeatability well and polymorphism highly were selected from 100 primers. The establishment of this system,the screened primers and the annealing temperature could provide a theoretical basis for further research on the genetic diversity of bryophytes using ISSR molecular markers.
Key words:  Bryum argenteum  ISSR  orthogonal design  primer screening  annealing temperature