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植物研究 ›› 2006, Vol. 26 ›› Issue (1): 93-97.doi: 10.7525/j.issn.1673-5102.2006.01.022

• 论文 • 上一篇    下一篇

萝卜基因组DNA的RAMP-PCR体系优化

龚义勤;李 培;王明霞;赵丽萍;柳李旺*   

  1. 南京农业大学作物遗传与种质创新国家重点实验室,南京农业大学园艺学院,南京 210095
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2006-01-20 发布日期:2006-01-20
  • 通讯作者: 柳李旺
  • 基金资助:
     

Optimization of RAMP-PCR reaction system in genomic DNA of radish (Raphanus sativus)

GONG Yi-Qin;LI Pei;WANG Ming-Xia;ZHAO Li-Ping;LIU Li-Wang*   

  1. National Key Laboratory of Crop Genetics and Germplasm Enhancement; College of Horticulture, Nanjing Agricultural University, Nanjing 210095
  • Received:1900-01-01 Revised:1900-01-01 Online:2006-01-20 Published:2006-01-20
  • Contact: LIU Li-Wang
  • Supported by:
     

摘要: 以萝卜为材料,对基因组DNA的RAMP分析体系中的Mg2+、dNTPs和引物浓度进行优化。分别设计3个浓度梯度:Mg2+为 0.75、1.5、3.0 mmol·L-1;dNTPs为 0.05、0.15、0.3 mmol·L-1;引物为 0.065、0.2、0.4 μmol·L-1,并对合适退火温度进行研究。筛选出的RAMP优化体系为(20 μL):dNTPs 0.15 mmol·L-1,Mg2+ 1.5 mmol·L-1,引物0.2~0.4 μmol·L-1,DNA 10 ng,Taq E 0.8U;PCR扩增程序为94℃ 3 min,94℃ 1 min,45℃ 1 min,72℃ 1.5 min,42个循环, 72℃ 8 min。运用此体系,进行引物组合筛选,并对7个萝卜品种的遗传多样性与品种鉴定进行RAMP标记分析。

关键词: 萝卜, RAMP标记, 体系优化, 遗传多样性

Abstract: Genomic DNA of Radish was analyzed by optimizing the concentration of Mg2+, dNTPs and primer in Random Amplified Microsatellite Polymorphism (RAMP) system. Three different concentration gradients were set respectively, Mg2+ 0.75,1.5,3.0 mmol·L-1;dNTPs 0.05,0.15,0.3 mmol·L-1;primer 0.065,0.2,0.4 μmol·L-1, and the suitable annealing temperature was also screened. An optimization of RAMP reaction system for radish genomic DNA is(20 μL): dNTPs 0.15 mmol·L-1,Mg2+ 1.5 mmol·L-1,primer 0.2~0.4 μmol·L-1,DNA 10 ng,Taq E 0.8U.The amplification protocol was: 94℃ 3 min,42 cycles for 1 min at 94℃,1 min at 45℃, 1.5 min at 72℃, followed by a final extension of 8 min at 72℃. The suitable system was applied in screening the primer combinations, and the genetic diversity of seven radish varieties was analyzed in RAMP marker system.

Key words: radish, RAMP marker, system optimization, genetic diversity

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