无患子(Sapindus mukorossi )是我国长江以南地区传统的重要绿化树种,其果皮富含皂苷,种仁富含油脂,为新型木本油料树种之一。为了获得基于 KOD FX 高保真 DNA 聚合酶试剂盒的无患子 ISSR-PCR 的最佳反应体系,该文采用正交优化设计相结合的方法,研究了引物浓度、dNTPs 浓度、KOD FX 酶、模板 DNA浓度和 Mg2＋浓度对无患子 ISSR-PCR 反应体系的影响,并在此基础上对退火温度进一步优化,最终确立了适合无患子的 ISSR-PCR 反应的最佳体系和程序,即20μL PCR 反应体系中,引物0．5μmol.L-1、dNTPs 0．05 mmol.L-1、KOD FX 酶0．06 U.μL-1、模板 DNA 浓度1．0 ng.μL-1和 Mg2＋1．0 mmol.L-1。当以 UBC841为引物时,PCR 扩增程序为94℃预变性2 min,98℃变性10 s,48．6℃退火30 s,68℃延伸90 s,35个循环,68℃延伸7 min,4℃保存。这一优化的 ISSR-PCR 反应体系的建立,为无患子种质资源的遗传多样性、亲缘关系及遗传结构研究、种质创新与分子辅助育种等奠定了良好的基础。%Sapindus mukorossi is a traditional and important virescencetree species in the south part of China with rich saponins in peel and rich oil in seed.This tree species is one of the newly-developed woody oil species that were approved by the State Forestry Administration of China.For optimizing ISSR-PCR reaction system of S .mukorossi , based on KOD FX High-fidelity DNA Polymerase Kit,orthogonal design experiments were conducted.The main fac-tors affecting ISSR-PCR amplification,such as suitable concentration of primer,dNTPs,KOD FX DNA polymer-ase,DNA template and 2×PCR buffer for KOD FX were studied.Furthermore,the annealing temperature was op-timized on the base of the above tests.An ideally ISSR-PCR reaction system was established,namely 25 μL reaction system containing primer 0.5 μmol.L-1 ,dNTPs 0.05 mmol.L-1 ,KOD FX DNA polymerase 0.06 U.μL-1 、DNA tem-plate 1.0 ng.μL-1 and Mg2+ 1.0 mmol.L-1 .The optimal PCR amplification program was:The profile of ISSR-PCR was an initial denaturation step for 2 min 94 ℃,followed by 35 cycles of 30 s at 98 ℃ for denaturation,30 s at 48.6 ℃ for annealing,90 s at 68 ℃ for extension,finally extension at 68 ℃ for 7 min and holding the samples at 4℃.This optimized ISSR-PCR reaction system would provide the basis for the analysis of genetic diversity,genetic structure,germplasm innovation and molecular assisted selection in S .mukorossi .
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