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[目的]建立适合南药益智的扩增片段长度多态性(AFLP)扩增体系来研究不同地理居群益智遗传多样性。[方法]利用植物基因组试剂盒法提取高质量的益智基因组DNA,采用单因素和正交试验对AFLP过程中的酶切和PCR相关影响因素进行优化,并对适合益智AFLP分析的引物组合进行筛选。[结果]实验结果表明最佳酶切反应体系:模板DNA 0.6μg,酶量20 U,酶切时间2 h;最佳AFLP-PCR选择性扩增反应体系(总体积为25μL):10×PCR Buffer(不含Mg2+)2.5μL,d NTPs 2μL,Mg2+(25mmol/L)1.5μL,引物(10 pmol/μL)各2.0μL,Taq酶(5 U/ml)0.5μL,模板稀释25倍2μL。利用建立的最佳扩增体系从64对引物中筛选获得8对选择性引物适合益智AFLP分析。[结论]建立了稳定的AFLP-PCR体系,为研究益智遗传多样性的AFLP分析奠定了基础。
[Objective] The research aimed to establish an amplified fragment length polymorphism (AFLP) amplification system suitable for Southern medicine to study the genetic diversity of different geographical populations. [Method] The plant genomic kit method was used to extract high quality genomic DNA. The single factor and orthogonal experiments were used to optimize the factors influencing enzyme digestion and PCR in AFLP. The primer combinations suitable for AFLP analysis Screening. [Result] The results showed that the optimal reaction conditions were as follows: template DNA 0.6μg, enzyme 20 U, digestion time 2 h; optimal AFLP-PCR selective amplification reaction system (total volume 25μL) 2.5 μL of Buffer (without Mg2 +), 2 μL of dNTPs, 1.5 μL of Mg2 + (25 mmol / L), 2.0 μL of primer (10 pmol / μL) and 0.5 μL of Taq enzyme (5 U / mL). Eight pairs of selective primers were screened from 64 pairs of primers for the optimal AFLP analysis using the established amplification system. [Conclusion] The establishment of a stable AFLP-PCR system laid the foundation for the study of AFLP analysis of genetic diversity in puzzle.