目的:建立麻花秦艽的简单重复序列区间-聚合酶链反应(ISSR-PCR)最佳反应体系,为该药材的种质鉴定及遗传多样性等研究提供参考。方法:运用单因素试验和正交试验优选麻花秦艽的ISSR-PCR反应体系中DNA模板用量,Mg2+浓度,dNTPs浓度,TaqDNA聚合酶用量和引物浓度等参数,确定麻花秦艽每条引物的最佳退火温度。结果:ISSR-PCR最佳反应体系(20μL)为模板DNA1μL(36mg·L-1),10×PCR缓冲液(含Mg2+)1.75μL(20mmol·L-1),dNTPs0.8μL(10mmol·L-1),Taq酶0.1μL(0.5%)及引物0.6μL(10mmol·L-1);筛选出12条多态性高、扩增稳定的ISSR引物,UBC-809引物的最佳退火温度55.5℃。扩增出的7条带中多态性带为6条,多态性位点比率85.71%。结论:建立的麻花秦艽ISSR-PCR反应体系稳定可靠,位于500bp处的条带为非多态性条带,可作为该种属的特征性条带。 OBJECTIVE: To establish an optimal reaction system for simple repetitive sequence-Genomic DNA Polymerase Chain Reaction (ISSR-PCR) of Gentiana macrophylla, and to provide references for the research on germplasm identification and genetic diversity of Gentiana macrophylla. Methods: The optimum annealing conditions of each primer of Gentiana straminea were determined by single factor test and orthogonal test, such as DNA template dosage, Mg2 + concentration, dNTPs concentration, Taq DNA polymerase dosage and primer concentration in the ISSR-PCR reaction system of Gentiana macrophylla. temperature. RESULTS: The optimal ISSR-PCR reaction system (20μL) consisted of 1μL DNA template (36mg · L-1), 1.75μL 10μL PCR buffer (containing Mg2 +), 0.8μL dNTPs 1), 0.1|ÌL (0.5%) of Taq enzyme and 0.6|ÌL (10mmol¡¤L-1) of primer. Twelve ISSR primers with high polymorphism and stable amplification were screened out. The optimum annealing temperature of UBC-809 primer was 55.5¡æ . Among the 7 bands amplified, there were 6 polymorphic bands and the percentage of polymorphic loci was 85.71%. CONCLUSION: The established ISSR-PCR reaction system of Gentiana straminea is stable and reliable. The band at 500 bp is a non-polymorphic band that can be used as a characteristic band of this genus.