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目的构建三桠苦ISSR反应体系,为三桠苦种质资源遗传多样性分析及分子鉴定奠定基础。方法通过设定影响三桠苦ISSR-PCR反应诸因子的不同梯度,检测不同反应体系的扩增效果,对条件进行优化,并筛选引物及退火温度,建立三桠苦ISSR-PCR最适反应体系,并用23个不同三桠苦样品验证体系稳定性。结果首次建立了三桠苦ISSR最佳反应体系(25μl):Master Mix 12.5μl(Mg2+浓度4 mmol/L,d NTP浓度0.4mmol/L)、引物0.6μmol/L、模板DNA 20ng。利用该体系从60条ISSR引物中筛选出了扩增条带清晰、多态性好的12条引物。结论所建立的三桠苦ISSR反应体系稳定可靠、标记位点清晰、检测多态性能力强,这一体系的建立及引物筛选可应用于三桠苦种质资源评估及遗传多样性分析的研究。
OBJECTIVE: To construct ISSR reaction system of Sanqi bitter, and lay the foundation for genetic diversity analysis and molecular identification of Sanhuang bitter germplasm resources. Methods The different ISSR-PCR reaction factors influencing the effect of ISSR-PCR were set up to detect the amplification effect of different reaction systems. The conditions were optimized and the primer and annealing temperature were selected to establish the ISSR-PCR optimum reaction system , And the stability of the system was verified with 23 different samples of triadimefon. Results The optimal ISSR reaction system (25μl) was established for the first time. Master Mix 12.5μl (Mg2 + concentration 4mmol / L, dNTP 0.4mmol / L), primer 0.6μmol / L and template DNA 20ng. Sixty ISSR primers were screened by this system. Twelve primers with clear amplification bands and good polymorphism were screened out. Conclusion The established ISSR reaction system was stable and reliable with clear marker loci and strong ability to detect polymorphisms. The establishment of this system and primer screening can be applied to the assessment of the germplasm resources and the analysis of genetic diversity .