菌草SRAP-PCR体系优化及引物筛选

杨东升; 王志鹏; 卢倩倩; 曹皓然; 丁大力; 安琪; 郝水源

菌草SRAP-PCR体系优化及引物筛选

SRAP-PCR System Optimization and Primer Selection for Juncao Studies

摘要

摘要:
目的为优化菌草SRAP-PCR的最佳反应体系，获得菌草SRAP多态性引物。
方法本试验以巨菌草基因组DNA为模板，采用单因素试验和正交试验设计相结合的方法，对影响SRAP-PCR体系的引物、Mg²⁺、模板DNA、dNTPs和Taq DNA聚合酶5个因素进行优化分析，并利用4个菌草材料筛选SRAP多态性引物组合。
结果菌草SRAP-PCR最佳体系为：引物1.0 μmol·L^–1、Mg²⁺ 2.50 mmol·L^–1、DNA用量2.5 ng·μL^–1、dNTPs 325 μmol·L^–1、Taq DNA聚合酶0.40 U，总体积20 μL。各因素对菌草SRAP反应体系的影响程度大小顺序为：引物＞DNA＞Taq DNA聚合酶＞dNTPs＞Mg²⁺。从35对引物中筛选出多态性丰富、条带清晰的SRAP引物组合18对，多态性位点为3～10个，平均多态性比率为77.36%。
结论优化的菌草SRAP-PCR体系稳定可靠，为后续菌草SRAP遗传多样性分析、品种保护利用等研究奠定了基础。

Abstract:
Objective SRAP-PCR system optimization and polymorphic SRAP primers identification for studying Juncao were conducted.
Method Genomic DNA extracted from Juncao was used as a template in a combined single-factor and orthogonal experimentation to optimize the primer, Mg²⁺, template DNA, dNTP, and Taq DNA polymerase for SRAP-PCR analysis. A set of polymorphic SRAP primers was selected using 4 varieties of Juncao as targets.
Result The optimized SRAP-PCR analysis required the concentrations of primer at 1.0 μmol·L^–1, Mg²⁺ at 2.50 mmol·L^–1, template DNA at 2.5 ng·μL^–1, dNTPs at 325 μmol·L^–1, and Taq DNA polymerase at 0.35 U. These parameters affected the SRAP amplification efficiency in the order of primer＞template DNA＞Taq DNA polymerase＞dNTPs＞Mg²⁺. From 35 pairs of SRAP primers, 18 with rich polymorphism and clear bands were selected. They had 3 to 10 polymorphic loci with an average polymorphism ratio of 77.36%.
Conclusion The optimized SRAP-PCR system was highly stable and reliable. It could be applied satisfactorily for analyzing genetic diversity, studying germplasm conservation, and broadening utilization of Juncao.

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