LI Na,ZHANG Cun-xu,CUI Xiao-yan,et al.Establishment of ISSR Reaction System in Quercus variabilis [J].Northern Horticulture,2014,38(13):93-97.
栓皮栎ISSR反应体系的建立
- Title:
- Establishment of ISSR Reaction System in Quercus variabilis
- 文章编号:
- 1001-0009（2014）13-0093-05
- Keywords:
- Quercus variabilis; ISSR; system optimization; genetic diversity
- 分类号:
- S 792.189
- 文献标志码:
- A
- 摘要:
- 以栓皮栎叶片为试材，采用单因素试验设计对栓皮栎ISSR反应条件进行系统优化，建立了栓皮栎ISSR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，30 ng模板DNA，1.5 U Taq 聚合酶，2.5 mmol/L Mg2+，0.6 μmol/L引物，0.3 mmol/L ?dNTPs；扩增程序：94℃预变性1 min；94℃变性30 s，51.5～59.0℃退火60 s，72℃延伸2 min，35个循环；最后72℃延伸5 min。在此条件下从100条ISSR引物中筛选出12条多态性好、稳定性高的引物对陕西省天然群体共22个栓皮栎个体的DNA进行扩增，共得到166个位点，其中142个为多态位点，多态位点百分率为85.54％，Nei指数为0.2928，Shannon信息指数为0.4381，表明栓皮栎的遗传多样性处于较高水平。
- Abstract:
- ISSR reaction conditions of Quercus variabilis were optimized systematically via single-factor experiment by using the tender leaves of Quercus variabilis as test materials，resulting in establishment of optimal ISSR reaction system and thermal cycling conditions of Quercus variabilis.The results showed that the optimal ISSR reaction system included a total volume 25 μL containing 2.5 μL 10×Buffer，30 ng template DNA，1.5 U Taq DNA polymerase，2.5 mmol/L MgCl2，0.6 μmol/L primer，0.3 mmol/L dNTPs.Thermal cycling conditions were as follows：denaturation 1 min at 94℃；35 cycles of 30 s at 94℃，60 s at 51.5～59.0℃，2 min at 72℃；and a final extension of 5 min at 72℃ at the end of the amplification.Based on the optimized reaction system，12 primers with superior stability and polymorphism were selected from 100 ISSR primers.166 bands were amplified in 22 individuals of the natural population in Shaanxi Province,among which 142 were polymorphic loci,the percentage of which was 85.54％,Nei’s index 0.2928 and Shannon’s information index was 0.4381,indicating the relatively higher genetic diversity within population of Quercus variabilis.
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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等研究工作。E-mail：657874432@qq.com.