摘要
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。
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 henzyme digestion,8 UEcoRI,6 U MseI,30 times diluted pre-amplification products and 10μL2×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.
作者
蒲小剑
田久胜
田新会
杜文华
PU Xiao-jian;TIAN Jiu-sheng;TIAN Xin hui;DU Wen-hua(College of Grassland Science ,Gansu Agricultural University/Key Laboratory for Grassland Ecosystem of Ministry of Education/Pratacultural Engineering Laboratory of Gansu Province/Sino-U. S. Centers for Grazing Land Ecosystem Sustainability ,Lanzhou 730070 ,China;College of Plant Protection ,Lanzhou 730070, China)
出处
《草原与草坪》
CAS
CSCD
2018年第2期89-96,共8页
Grassland and Turf
基金
国家自然科学基金(31360577
31760702)
甘肃省草地畜牧业可持续发展创新团队项目(2017C-11)
现代农业产业技术体系建设专项资金资助(CARS-40-09B)