摘要
为探讨萝卜抽薹紧密连锁基因,通过寻找萝卜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个重要影响因素的最优水平,探索适合其AFLP的反应体系。结果表明,400ngDNA用EcoRI(10U/μL)和MseI(10U/μL)各0.2μL进行酶切10min,完全满足预扩增和选择性扩增反应的要求,节省了试验成本和酶切时间;Mg2+的浓度是dNTPs浓度的5倍,扩增循环数为35,扩增效果最佳。模板稀释倍数对电泳条带结果影响较大,在预扩增反应中模板稀释5倍,选择性扩增反应中模板稀释倍数为20时反应效果最好,扩增条带带纹清晰。通过该优化体系初步筛选出13个清晰、多态性高的AFLP引物,并在5种不同生态型的16份萝卜材料上进行了验证,条带清楚,多态性强。该优化体系可用于萝卜抽薹紧密连锁基因的研究分析。
The study was conducted to study the genes linked to bolting trait in radish.The optimal level of 6 key factors of AFLP analysis system were found out such as the digestion time of DNA,the different levels of concentration of each enzyme,Mg 2＋,dNTPs,the dilution ratio of response template and the number of amplification cycles.The optimization system of AFLP markers linked to bolting trait in radish was established.The result showed that 400ng DNA were digested with 0.2μL EcoR I（10U/μL） and 0.2μL Mse I（10U/μL） for 10 min.The requirements of the pre-amplification and selective amplification reaction were met completely.The cost of experiment and the digestion time was saved.The concentration of Mg 2＋ was about 5 times as dNTPs ’ and the number of the selective amplification cycles was 35.The best effect of amplification reaction was obtained.The results of electrophoresis strips were affected by the dilution ratio of response template significantly.The 5 times dilutions of Ligation products and the 20 times dilutions of the pre-amplification samples were used as the response templates of the pre-amplification and selective amplification reaction respectively.The response effect was the best and the electrophoresis strips were clear.13 primers were screened preliminary with stable amplification and rich polymorphism through the optimization system of AFLP.And the applicability of the optimization system of AFLP makers was verified with 16 radish varieties which belonged to 5 ecotypes.Its electrophoresis strips were clear and polymorphism was rich.The optimization system could be used as the research analysis of genes linked to bolting trait in radish.
出处
《中国农学通报》
CSCD
2012年第34期166-171,共6页
Chinese Agricultural Science Bulletin
基金
山东省现代农业产业技术体系项目"萝卜抽薹机理与抑制先期抽薹效应研究"(621140)
山东省农业重大应用技术创新项目"大白菜
萝卜
甘蓝春夏设施栽培关键技术研究"(6210y5)
