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Optimization of ISSR amplification conditions for Rhododendron fortunei

GU Qi-Ping1,2,JIN Ze-Xin2,LI Jun-Min2(1.Key Laboratory of the Three Gorge Reservoir Region's Eco-Environment,Ministry of Education,Southwest University,Chongqing 400715,China;2.Institute of Ecology,Taizhou University,Linhai 317000,China)  
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.
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