枣选择性扩增微卫星体系的建立及优化

【目的】建立和优化枣的选择性扩增微卫星（SAM）技术体系,丰富枣群体遗传学研究的方法,同时为枣利用SAM法开发SSR引物提供技术参考和模板。【方法】以冬枣、大荔龙枣和金丝小枣为试材,采用CTAB法提取样品DNA,对SAM试验过程中的酶切-连接、抑制性扩增、预扩增、选择性扩增等关键因子进行研究。【结果】利用PstⅠ和MseⅠ双酶切系统,分步法进行酶切-连接是后续试验成功的关键,抑制性扩增（20.0μL体系）模板取酶切连接液2.0μL,预扩增和选择性扩增（20.0μL体系）的模板分别取上步扩增的稀释产物各2.0μL;抑制性扩增和预扩增反应中,ExTaq取0.5 U,反应25个循环,产物稀释20倍进行下一步扩增;选择性扩增采用前6个循环中退火温度梯度降低（每循环降低1℃）的反应程序,体系中可将ExTaq用量增加到1.0 U以保证酶的保真效果。【结论】试验利用优化后的反应体系,选用16个MseⅠadapter+NN primer和SSR primer（PCT6）组成引物对进行筛选,获得了谱带清晰、分辨率高、多态性丰富的电泳图谱。该研究结果为利用SAM技术开展枣的亲缘演化、遗传多样性分析及SSR引物开发等方面的研究打下了基础。

Establishment and Optimization of Selectively Amplified Microsatellite (SAM) Analysis System on Chinese Jujube (Zizyphus jujuba Mill.)

Objective] The objective of this study is to establish and optimize the SAM (selectively amplified microsatellite) analysis system, enrich the research methods in population genetics of Chinese jujube and provide reference and templets for SSR primer development using SAM in Chinese jujube. Method] High-quality genomic DNA was isolated by improved CTAB method from leaves of different jujube cultivars (Dongzao, Dalilongzao and Jinsixiaozao). The SAM reaction system was established by optimizing enzyme digestion and ligation reaction, suppression-amplification, pre-amplification and SAM selective amplification. Result] Two-step-enzyme digestion and ligation reaction was approved to be the key step in the whole system. In the suppression-amplification (the total volume was 20.0 μL), the suitable template volume was tested to be containing 2.0 μL of the production of digestion and ligation reaction, in pre-amplification and SAM selective-amplification (the total volume was 20.0 μL), the suitable template volume was 2.0 μL of the diluted production of the above amplification reaction. In the suppression-amplification and pre-amplification, 0.5 U ExTaq was used in the reaction mixture, and then the two amplifications were performed with 25 cycles, respectively. After reaction, the products were diluted 1:20 with TE buffer. In the SAM selective-amplification, the annealing temperature was performed by reducing 1℃ per cycle in the first 6 cycles, and by adding ExTaq to 1.0 U for the accuracy of the amplification. Conclusion] 16 pairs of Mse Ⅰ adapter +NN primer and SSR primer (PCT_6) were designed for the SAM selective-amplification, all of the 16 primer-combinations revealed sufficient amplified bands with high polymorphism. The established SAM reaction system could be used to study the population structure, genetic diversity and the SSR primer development in the population of Chinese jujube.