华山松ISSR反应体系的优化

Optimization of ISSR Reaction System for Pinus armandii

为获得条带清晰、稳定和多态性高的华山松ISSR扩增结果,对引物、Mg^(2+)浓度和退火温度等因素进行了优化.确定了ISSR分析的最佳PCR条件:15μL反应体系中,10×PCR buffer反应缓冲液1.5μL,模板DNA 22.5 ng,Mg^(2+)2.2 mmol/L,d NTPs 0.23 mmol/L,引物0.93μmol/L,Taq DNA聚合酶0.027 U/μL.PCR反应程序为:经94℃预变性4min后,经过94℃变性30 s,Tm-4.8℃退火30 s,72℃延伸108 s共33个循环;循环结束后72℃延伸10 min.从44条ISSR引物中筛选出12条扩增稳定、多态性丰富的ISSR引物.对华山松ISSR实验优化体系的建立,为今后利用ISSR分子标记技术开展华山松种间遗传变异分析和构建遗传图谱等研究奠定了基础.

To obtain amplification result with clear,stable and rich polymorphism fragment of Pinus armandii,the affection ISSR-PCR by main reaction system elements,such as primer,Mg 2＋ and annealing temperature,was presented.The reaction system and amplified procedure suitable for P.armandii were established,the main elements include,15 μL amplification reaction mixture containing 1.5 μL 10× reaction buffer,22.5 ng template DNA,2.2 mmol/L Mg 2＋ ,0.23 mmol/L dNTPs,0.93 μmol/L primers,0.027 U/μL TaqDNA polymerase.The reaction program was devised for 4 min of predenaturalization at 94℃,33 cycles of 30 s for denaturalization at 94℃,30 s of anneal at Tm-4.8℃,108 s of extension at 72℃,and 10 min of extension at 72℃ after the final cycle.The 12 best primers were selected for ISSR-PCR system of P.armandii from 44 primers,whose results of ISSR amplification were clear and stable.The research on the optimization of ISSR reaction system for P.armandii will lay a foundation for studying interspecific genetic variation and genetic map construction of P.armandii using ISSR molecular marker technique in the future.