Abstract:

【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 MseⅠadapter +NN primer and SSR primer (PCT6) 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.

Key words: Chinese jujube (Zizyphus jujuba Mill.), selectively amplified microsatellite (SAM), system establishment, optimization