LI Na,ZHANG Cun-xu,LIU Wei,et al.Establishment of ISSRPCR Reaction System of Cornus walteri[J].Northern Horticulture,2013,37(22):114-119.
毛梾ISSR-PCR反应体系的建立
- Title:
- Establishment of ISSRPCR Reaction System of Cornus walteri
- 文章编号:
- 1001-0009(2013)22-0104-06
- Keywords:
- Cornus walteri; ISSR; single factor with two circular tests; system optimization; primer screening
- 分类号:
- S 792. 99
- 文献标志码:
- A
- 摘要:
- 以毛梾叶片为试材，通过单因素2次循环试验对毛梾ISSR反应体系进行优化，建立了毛梾ISSR-PCR的最佳反应体系和扩增程序。结果表明：PCR反应体系总体积为25 μL，其中10×Buffer 2.5 μL，模板DNA用量50 ng，Taq DNA聚合酶用量1.5 U，Mg2+浓度2.0 mmol/L，引物浓度0.6 μmol/L，dNTPs浓度0.15 mmol/L；扩增程序：94℃预变性5 min；94℃变性50s，48.8～59.1℃退火60 s，72℃延伸1.5 min，38个循环；最后72℃延伸10 min。在此基础上，从100条ISSR通用引物中筛选出17条多态性好、稳定性高的引物，并优化了它们的退火温度。这一优化体系的建立，为开展毛梾种质资源鉴定、遗传多样性及分子育种方面的研究提供了技术依据和参考。
- Abstract:
- Using leaves of Cornus walteri as material,ISSR reaction conditions of Cornus walteri were optimized systematically via single factor with two circular tests,and the optimal ISSR reaction system and thermal cycling conditions of Cornus walteri were established.The results showed that the optimal ISSR reaction system included a total volume 25 μL containing 2.5 μL 10×Buffer,50 ng template DNA,1.5 U Taq DNA polymerase,2.0 mmol/L MgCl2,0.6 μmol/L primer,0.15 mmol/L dNTPs.Thermal cycling conditions were as follows:denaturation 5 min at 94℃;38 cycles of 50 s at 94℃,60 s at 48.8～59.1℃,1.5 min at 72℃;and a final extension of 10 min at 72℃ at the end of the amplification.Based on the optimized reaction system,17 primers with superior stability and polymorphism were screened out from 100 ISSR primer candidates.Subsequently,annealing temperatures of selected primers were subjected to further optimization.As a consequence,the optimized system in this study may provide technological base and reference for identification of germplasm resources,genetic diversity and molecular breeding of Cornus walter.
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备注/Memo
第一作者简介：李娜（1988-），女，硕士研究生，现主要从事林木遗传育种学研究工作。E-mail：657874432@qq.com.
责任作者：张存旭(1961-)，男，博士，副教授，硕士生导师，现主要从事林木遗传育种学研究工作。E-mail：cxzhang@nwsuaf.edu.cn.
基金项目：国家林业公益性行业专项资助项目（200804010）；
陕西省教育厅科研计划资助项目（06JK153）。
收稿日期：2013-06-19