短花针茅SSR-PCR反应体系的建立及引物筛选  

Optimization of SSR-PCR Reaction System and Primers Screening in Stipa breviflora

作　　者：赵磊[1] 党振华[1] 赵艳宁[1] 张颖[1] 牛建明[1,2] 任婧[1] 周俊梅[1] 

机构地区：[1]内蒙古大学生命科学学院,呼和浩特010021 [2]内蒙古大学中美生态能源与可持续性科学研究中心,呼和浩特010021

出　　处：《内蒙古大学学报（自然科学版）》2016年第1期58-64,共7页Journal of Inner Mongolia University：Natural Science Edition

基　　金：国家重点基础研究发展计划(973)项目(2012CB722201);国家自然科学基金(31460154)资助

摘　　要：以采集于我国内蒙古高原、黄土高原和新疆等地的6个短花针茅野生种群为材料,采用正交实验的方法对模板DNA、Mg2+、dNTPs、Taq DNA聚合酶和引物浓度进行5因素4水平的正交优化设计,依据同源物种紫花针茅的19对SSR引物筛选适用于短花针茅的SSR引物.结果表明:(1)短花针茅最佳SSR-PCR反应体系为(25μL):100ng模板DNA 1μL、2.0mmol/L的Mg2+2.5μL、100μmol/L dNTPs 2μL、2.0U Taq DNA聚合酶0.5μL、0.2μLmol/L上下游引物各1μL,其余用ddH2O补足.(2)建立了重复性和稳定性较好的SSR-PCR扩增程序:94℃预变性3min;94℃变性30s,退火温度与每个引物相对应,退火30s,72℃延伸30s,共30个循环;最后72℃延伸10min;4℃保存,扩增结束.(3)筛选出7对多态性较好的短花针茅SSR引物,同属植物基因组SSR引物的通用率为36.84%.本研究对于进一步探讨短花针茅种群遗传多样性提供参考价值.Establishing PCR reaction system and screening primers are important basic works to genetic diversity research. In current study,samples of Stipa breviflora （S. breviflora） from 6 plots located in Inner Mongolia plateau,the Loess plateau and Xinjiang were used,and an orthogonal ex- periment with 5 factors （template DNA,Mg2＋ ,dNTPs,Taq DNA polymerase,primers） in 4 levels was designed to test the PCR reaction system,while 19 primer pairs from homologous species Stipa purpurea were compared to screen the primer pairs work on S. breviflora. The results were as fol- lows：1） The best SSR-PCR reaction system for S. breviflora at a 25μ1 system was 100ng template DNA for 1μL,2.0mmol/L Mg2＋ for 2.5μL, 100μmol/L dNTPs for 2μL,2.0U Taq DNA polymer- ase for 0.5μL,0.2μmol/L primer for 1μL;2） the PCR amplification program for better repeatability and stability was a pre-degeneration for 3 min at 94℃ followed by degeneration for 30s at 94℃ and annealing for 30 s with annealing temperature corresponds to each primer, then with extension for 30s at 72℃. The program run for 30 cycles with a final extension for 10 min at 72℃. 3） 7 pairs of S. breviJlora SSR primers with good amplification results were selected. The amplification ratio within genus was 36. 84%. This study provide useful information for further investigation on S. breviflora genetic diversity.

关 键 词：短花针茅 SSR-PCR 体系优化 引物筛选 

分 类 号：Q347[生物学—遗传学]