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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (34): 166-171.

所属专题： 园艺

• 林学 园艺 园林 • 上一篇    下一篇

萝卜抽薹性研究的AFLP体系建立与优化

张美霞 林多   

  • 收稿日期:2012-04-05 修回日期:2012-05-23 出版日期:2012-12-05 发布日期:2012-12-05
  • 基金资助:

    萝卜抽薹机理与抑制先期抽薹效应研究 大白菜、萝卜、甘蓝春夏设施栽培关键技术研究

Construction and Optimization of AFLP Analysis System for Bolting Trait in Radish

  • Received:2012-04-05 Revised:2012-05-23 Online:2012-12-05 Published:2012-12-05

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摘要：

摘要：本研究以萝卜‘春雪总太’为优化材料，对AFLP体系的酶切时间，相关酶、Mg2+、dNTPs的用量，模板稀释倍数以及扩增循环数等6个重要影响因素进行摸索和优化，建立了适合萝卜抽薹紧密连锁基因研究的AFLP分子标记体系。结果表明：在20μL酶切体系中，将400ng的样品DNA用各0.2μLEcoR I和MseI 37℃双酶切10min， 65℃灭活10min；向 5μL酶切产物中加入EcoR I接头和MseI接头各0.5μL，用0.5μL的T4连接酶在20μL的体系中22℃连接过夜；向5μL稀释5倍后的连接产物中加入预扩引物各0.6μL，Mg2+1μL，dNTPs1.5μL,Taq酶0.2μL，ddH2O补齐至15μL，预扩增35个循环；取5μL稀释20倍后的预扩增产物，其他成分用量同预扩增相同，用ddH2O补齐至15μL进行选择性扩增。并将此优化体系在秋白、秋青、春白、秋红、水萝卜五种生态型的16份萝卜材料上进行了验证，电泳条带清楚，多态性强。

关键词: 移栽, 移栽

Abstract:

Abstract: Taking radish ‘Chun xue zong tai’ as materials, six key factors of AFLP analysis system such as the digestion time of DNA, the different levels of concentration of each enzyme , Mg2+, dNTPs, the dilution ratio of response template and the number of amplification cycles were studied. The optimization system of AFLP makers linked to bolting trait in radish was established. The result showed: 400ng radish DNA were digested with 0.2μL EcoR I and 0.2μL Mse I at 37℃ for 10min and then 65℃for 10 min. Then 5μL digestion products were made a ligation mixture with 0.5μL EcoR I adapter, 0.5μL Mse I adapter and 0.5μL T4 DNA Ligase at 22℃ for overnight in 20μL.The pre-amplification system in 15μL was set up with 5μL 5-times dilutions of Ligation products,10μmol/L pre-selective amplification primers 0.6μL respectively, Mg2+1μL, dNTPs1.5μL,Taq DNA Polymerase0.2μL and ddH2O 4.6μL by 35 cycles. After diluting the pre-amplification samples by 20 times, the selective amplification system was set up with 5μL the pre-amplification dilution products and selective amplification primers 0.6μLrespectively, the concentration of other components was the same as the pre-amplification system in 15μL. The applicability of the optimization system of AFLP makers was verified with 16 radish varieties, which belonged to five ecotypes. Its electrophoresis strips were clear and polymorphism was rich.