油松SSR-PCR引物筛选及反应体系的建立

Selection of primers and establishment of SSR-PCR reaction system on Pinus tabulaeformis Carr.

油松是我国特有的重要乡土针叶树种，开发合适的油松PCR-SSR引物并建立优化的反应体系，是开展油松天然群体和种子园人工群体遗传研究的基础．该研究以油松总DNA为材料，分析了Taq聚合酶、样本浓度、Mg^2＋浓度、dNTP浓度以及引物浓度对PCR-SSR扩增结果的影响，筛选出扩增条带清晰、多态性丰富的SSR引物12对，建立了稳定的、可重复的油松PCR-SSR最佳反应体系及PCR扩增参数．研究结果表明：在15pLSSR-PCR反应体系中，样本最适宜浓度为30ng，Mg^2＋的最适浓度为0．25mmol/L，dNTP最适浓度为0．2mmog/L，单引物的最适浓度均为250nmol／L；Taq聚合酶在15止反应体系中宜加入0．375U．统计利用所选12对引物，对辽宁兴城油松种子园内49个建园无性系进行了SSR-PCR反应，通过6％的变性聚丙稀酰胺凝胶电泳检测，每对引物扩增产物在100—250bp之间的等位谱带数最大为10，最小为5，不同无性系间DNA谱带多态性丰富,油松SSR-PCR引物筛选及反应体系的建立，为今后利用SSR标记技术开展油松种子园的父本分析及选择性受精研究提供了一个标准化程序和强有力的工具.

Taking Pinus tabulaeformis Cart. as the material, the effects of primer pairs, whole DNA concentration, Mg^2＋ and Taq polymerase concentration and dNTP concentration on SSR reaction system were studied. Twelve primer pairs with abundant polymorphism bands were selected and the SSR-PCR reaction system on P. tabulaeformis was established too. The results showed that in 15μL PCR reaction： 30 ng DNA, 0.25 mmol/L Mg^2＋, 0.2 mmol/L dNTP, 250 nmol/L Primer（F）, 250 nmol/L Primer（R）, 0.375 U Taq polymerase were the best. Detected by 6 % polyacrylamide gel, polymorphism among 49 clones of P. tabulaeformis in the Seed Orchard of Xingcheng, Liaoning Province was abundant, the length of amplification products was 100-250 bp, the number of alleles per locus varied from five to ten. The work provides a powerful tool for paternity analysis in P. tabulaeformis seed orchards.