嵩草AFLP分析体系的建立及引物筛选  

Establishment of AFLP reaction system and screening of primer pairs on Kobresia

作　　者：郑红梅[1] 呼天明[2] 王佺珍[2] 张国云[2] 宋江湖[2] 胡晓宁[2] 

机构地区：[1]西北农林科技大学资源环境学院,陕西杨凌712100 [2]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2008年第3期33-38,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：科技部国际科技合作项目(2006DFA33630);西藏自治区科技攻关项目(2005011)

摘　　要：【目的】建立一套适用于嵩草的AFLP技术体系。【方法】参考前人在其他物种上的研究结果,对嵩草AFLP体系的影响因素(包括模板DNA的制备、MseⅠ/EcoRⅠ以及T4 DNA连接酶的浓度、反应时间以及反应体系的组成等)进行研究,建立适用于嵩草的AFLP分析体系,并对引物进行了筛选。【结果】用于酶切的嵩草基因组DNA模板以600 ng为宜,连接最适反应时间为10 h,预扩增产物最适稀释倍数为5倍。E-ACC+M-CAG、E-ACA+M-CAG和E-ACA+M-CTG 3对引物均适合于嵩草AFLP分析,其中以E-ACA+M-CAG引物组合效果最佳,扩增出101条多态性带,多态性带比率为99.02%。【结论】建立的AFLP技术体系可用于嵩草AFLP分析,用该体系可得到条带清晰多态性好的嵩草AFLP指纹图谱。适合嵩草种质资源研究的引物组合有E-ACC+M-CAG,E-ACA+M-CAG和E-ACA+M-CTG,其中E-ACA+M-CAG组合的效果最好。【Objective】 The study was intended to establish the AFLP reaction system of Kobresia.【Method】 Referring fo the tesearch results of other species,several main factors including the preparation of genomic DNA,concentration and time for reaction of Mse Ⅰ/EcoRⅠand T4 DNA ligase,ingredients for solution that influenced the results of the AFLP method were studied by using Kobresia germplasms,and primers were screened by the established AFLP system.【Result】 The usage of DNA template was 600 ng,The optimal time was 10 hours for ligation,5 times of dilution for the products of pre-amplification was suggested for selective amplification.The polymorphism bands of Kobresia AFLP system could be obtained from three Primer pairs E-ACC＋M-CAG、E-ACA＋M-CAG and E-ACA＋M-CTG,E-ACA＋M-CAG was the best in the studies of Kobresia genetic diversification.【Conclusion】 With the established system the clear polymorphic AFLP fingerprints of Kobresia were obtained,primer pairs such as E-ACC＋M-CAG,E-ACA＋M-CAG and E-ACA＋M-CTG which were suitable for the research of Korbresia were screened and E-ACA＋M-CAG was the best.

关 键 词：嵩草 AFLP体系 引物筛选 

分 类 号：Q948.12[生物学—植物学]