红三叶AFLP体系的建立及优化

采用正交设计和单因素试验相结合的方法,以红三叶(Trifolium pratense)高感白粉病品种岷山红三叶与抗白粉病新品系(甘农PR1)杂交产生F1代作为试验材料,对红三叶AFLP反应中7个因素(DNA浓度,酶切时间,两酶(EcoRI,MseI)浓度,预扩增产物稀释倍数,2×PCR Master Mix用量,引物组合)进行优化试验。建立了适合红三叶稳定且多态性高的最佳AFLP反应体系:90ng/μL DNA模板、5h酶切、8UEcoRI、6UMseI、30倍稀释预扩增产物、10μL 2×PCR Master Mix。优化的AFLP反应体系在红三叶的验证中表现出良好的稳定性和重复性,为进一步进行红三叶种质鉴定、遗传图谱构建、基因定位和遗传多样性的分析奠定了技术基础。

Establishment and optimization on the AFLP reaction system of Trifolium pratense

Orthogonal and single factor experiment designs were applied for optimizing seven factors (DNA template,cutting time,concentration for EcoRI and MseI,dilution multiple of pre-amplification product,amount of 2 × PCR Master Mix and primer combination) in the AFLP system on Trifolium pratense.The results showed that a reliable AFLP reaction system for T.pratense with desirable repeatability and polymorphic bands was established.In a total volume of 20μL AFLP system,it contained 90 ng/μL DNA template,5 h enzyme digestion,8 U EcoRI,6 U MseI,30 times diluted pre-amplification products and 10 μ L2 × PCR Master Mix.The optimal reaction system was furtherly verified in T.pratense varieties with good stability and repeatability.This optimized AFLP reaction system would provide the basis for germplasm evaluation of T.pratense,the establishment of genetic maps,gene localization and analysis of genetic diversity.