中文摘要：

目的针对台湾金线莲ISSR反应的特点,建立一个稳定性高、重现性好,适合金线莲ISSR遗传差异分析的反应体系。方法确立金线莲ISSR初始反应体系并对引物进行初筛;设定反应体系中各因子的不同浓度并进行PCR扩增,依据稳定性高、重现性好的原则,对该反应体系进行调整与优化,最终建立稳定可靠的ISSR反应体系。结果首次建立了金线莲ISSR反应的最适体系（25μL）：1×PCR buffer,2 mmol/L MgCl2,200μmol/L dNTP Mixture,0.2μmol/L引物,1 U Taq酶,DNA模板约20 ng;扩增程序：94℃充分变性7 min,94℃变性30 s,52℃退火45 s,72℃延伸2 min,45个循环后,最后72℃延伸10 min。结论所建立的金线莲ISSR反应体系具有稳定性高、重现性好、标记位点清晰、检测多态性能力强等特点,可以较好地应用于野生金线莲的居群差异研究及经长期组培繁殖后金线莲无菌苗的遗传变异研究。

英文摘要：

Objective To establish a stable,reproducible,and suitable reaction system of Anoectochilus formosanus for ISSR analysis of genetic differences according to the ISSR-PCR characters in plants of Anoectochilus Bl.Methods The initial ISSR reaction system in plants of Anoectochilus Bl.was established and then the primers were screened.After that,PCR amplifications were carried out until the different concentrations of the factors in the ISSR reaction system were designed,and based on the principle of high stability and reproducibility,the stable and reliable ISSR reaction system was eventually established after a series of adjustment and optimization of the reaction system.Results The optimal ISSR reaction system in plants of Anoectochilus Bl.was reported for the first time,and the volume of 25 μL contained approximately 20 ng template DNA,1 U Taq DNA polymerase,0.2 mmol/L deoxyribonucleotide triphosphate（dNTP）,2 mmol/L MgCl2,200 μmol/L dNTP Mixture,and 0.2 μmol/L primer within 1× reaction buffer [10 mmol/L Tris-HCl（pH 8.3）,50 mmol/L KCl].The reaction mixtures were denatured at 94 ℃ for 7 min and subjected to 45 cycles of 30 s at 94 ℃,45 s at 52 ℃,2 min at 72 ℃,and a final extension step of 10 min at 72 ℃ and eventually stored at 4 ℃.Conclusion The ISSR-PCR systems established for studying on the plants of Anoectochilus Bl.show the characters of stable reaction system,better repeatability,clear marker site,and reliable abundant polymorphisms.It is suitable for the study on genetic diversity in plants of Anoectochilus Bl.in difference wild populations and genetic variation of its sterile seedlings after long time tissue culture.