珍稀濒危树种毛红椿微卫星DNA分离及SSR反应体系优化

本研究以江西宜丰种源毛红椿为材料，成功提取其基因组DNA。利用改良的链亲和素磁珠法亲和捕捉出毛红椿基因组微卫星DNA片断，并构建了富含微卫星的基因组文库。从构建的基因组文库中随机挑选了63个单克隆进行测序，其中50个单克隆成功测序，含有微卫星的单克隆有18个，并根据测序结果设计并合成SSR引物18对。利用所合成的引物优化SSR反应体系，对影响SSR反应的各个因子进行了探讨。确定了模板DNA浓度最适浓度为30ng；dNTP的最适浓度为0.3mmol·L-1；0.3μmol·L-1是引物在反应体系中的最合适浓度。建立了重复性好、稳定性好的SSR反应体系，为下一步进行毛红椿群体遗传结构和遗传变异研究提供了技术支持。

Isolation of microsatellite DNA from endangered tree species Toona ciliata var. pubescens and optimization of SSR reaction system

T. ciliata var. pubescen is an endangered tree species. The population genetic structure should be essential to conserve it effectively. Genomic DNA was successfully extracted from fresh leaves of Toona ciliata var. pubescens using CTAB method. Microsatellite DNA was isolated from genomic DNA with improved method of streptavitin beads.We got rid of some tedious and inefficient processes, such as gel extraction.The genomic library which enriched with microsatellite was constructed.After sequencing the positive clones,we found eighteen positive clones enriched with microsatellite.Eighteen SSR primer pairs were designed and synthesized.The factors which affected on the SSR reaction of Toona ciliata var. pubescens were studied.The results showed that the optimized concentration of DNA is 30ng ; the concentration of dNTP is 0.3 m mol·L-1;0.3μmol·L-1 is the optimized concentration of primers.Through above PCR system, repetitive and steady SSR reaction system was constructed.This is the base of studying the population genetic structure and genetic variation of Toona ciliata var. pubescens.