麻栗坡兜兰ISSR引物筛选及反应体系的优化

目的]对麻栗坡兜兰ISSR引物进行筛选,并建立一个稳定性高、重现性好、适合麻栗坡兜兰ISSR反应体系。方法]以麻栗坡兜兰DNA为模板,分别对Mg2+、dNTP、引物、Taq DNA聚合酶、DNA等PCR反应成分进行优化,并用优化的体系对25条兰科中报道的ISSR引物进行筛选。结果]确立了麻栗坡兜兰最适ISSR-PCR反应体系:在25μl反应体系中,Mg2+2 mmol/L、引物0.4 mmol/L、dNTP 0.20 mmol/L、DNA 1.5μl、Taq酶1.4 U。利用该体系,共筛选得到10条ISSR引物用于麻栗坡兜兰分析,并对21份麻栗坡兜兰材料进行扩增,获得了较好的扩增效果。结论]该体系稳定可靠,为今后ISSR标记在兜兰属植物的种质鉴定、遗传多样性等方面的广泛应用奠定了重要基础。

Selection of ISSR Primers and Optimization of Reaction System in Paphiopedilum malipoense

Objective ] ISSR primers were selected and PCR system was optimized in order to establish ISSR reaction system with high stability and good reproducibility. Method] Using single-factor test, Mg2., dNTP, primer concentration, Taq DNA polymerase and DNA dosage were optimized. Using the optimized system, primers were screened out of 25 primers. Result] The optimized reaction system was as the following： in a 25 μl reaction system, 10 × PCR Buffer 2.5 μl, Mg2＋2 mmol/L, the primers 0.4 mmol/L, dNTP 0.20 mmol/L, DNA 1.5 μl and Taq enzyme 1.8 U. Using this system, 10 primers were screened and 21 materials ofPaphiopedilum malipoense were amplified. The profile was clear and the polymorphism was high. Conclusion] The system was stable and reliable, and laid an important foundation for wide application of the ISSR marker in the pocket orchid genus plant identification, genetic diversity for the future.