云锦杜鹃ISSR扩增条件的优化

Optimization of ISSR amplification conditions for Rhododendron fortunei

关键词:  云锦杜鹃  ISSR  优化  遗传多样性

Key words:  Rhododendron fortunei  ISSR  optimization  genetic diversity

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、Taq DNA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:10 μL PCR反应体积中,1×Taq酶配套缓冲液(10 mmol/L Tris·HCl pH9.0,50 mmol/L KCl,0.1% Triton X-100),1.5 mmol/L MgCl₂,0.15 mmol/L dNTP,0.45 U Taq DNA聚合酶,2 mg/mL BSA,12 pmol引物,16 ng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。

Abstract:

Factors which affect the ISSR-PCR amplification,such as Mg²⁺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 ISSR-PCR 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.