云锦杜鹃ISSR扩增条件的优化  

Optimization of ISSR amplification conditions for Rhododendron fortunei

作　　者：顾奇萍[1] 金则新[2] 李钧敏[2] 

机构地区：[1]西南大学三峡库区生态环境教育部重点实验室,重庆400715 [2]台州学院生态研究所,浙江临海317000

出　　处：《广西植物》2007年第4期560-563,共4页Guihaia

基　　金：浙江省自然科学基金(Y504220);台州市科技局项目(044205)~~

摘　　要：以云锦杜鹃基因组DNA为研究对象,对影响ISSR-PCR扩增效果的一些因素,包括镁离子浓度、dNTP浓度、模板DNA含量、TaqDNA聚合酶量、BSA浓度、引物用量以及退火温度等进行筛选和优化,建立了稳定、可重复的最佳反应体系:10μLPCR反应体积中,1×Taq酶配套缓冲液(10mmol/LTris.HClpH9.0,50mmol/LKCl,0.1%TritonX-100),1.5mmol/LMgCl2,0.15mmol/LdNTP,0.45UTaqDNA聚合酶,2mg/mLBSA,12pmol引物,16ng模板DNA。利用所建立的优化反应体系从100个ISSR引物中共筛选出12个稳定性好、重复性高的引物,对5个居群共100个云锦杜鹃个体的DNA进行扩增,检测到170个位点,其中多态位点150个,多态位点百分率88.24%,5个居群的多态位点百分率平均为48.23%。云锦杜鹃ISSR反应体系的建立为利用ISSR分子标记技术研究云锦杜鹃的遗传多样性奠定了良好的基础。Factors which affect the ISSR-PCR amplification, such as Mg^2＋ concentration, dNTP concentration, template DNA dosage, Taq DNA polymerase units, BSA concentration and primer dosage, and annealing temperature were optimized and selected by using the genomic DNA of Rhododendron fortunei as material. The suitable ISSRPCR conditions BSA, 12 pmol primer, 16 ng template DNA. The suitable annealing temperature was 56.9℃. Based on these suitable conditions, 12 primers were selected and 170 DNA bands were produced in 5 populations with 100 individuals among which 150 loci were polymorphic. The total percentage of polymorphic loci was 88.24%, while the mean percentage of polymorphic loci of 5 Rh. fortunei population was 48.23%. The establishment of the PCR reaction conditions could settle favorable basis for the further study on the genetic diversity of Rh. fortunei by using ISSR molecular marker techniques.

关 键 词：云锦杜鹃 ISSR 优化 遗传多样性 

分 类 号：Q943[生物学—植物学]