关键词: 夏蜡梅, ISSR, 优化, 遗传多样性

Abstract: It was necessary to optimize the ISSR amplification conditions to ensure the replication and stability in the PCR amplification. The ISSR amplification conditions of endangered plant Sinocalycanthus chinensis was optimized and the effect of 7 factors such as Mg²⁺ concentration, dNTP concentration, DNA templates dosage, Taq DNA polymerase dosage, BSA concentration, primer dosage and glycerol concentration on ISSR amplification were tested using single factor method. The optimal amplification conditions of ISSR for Sinocalycanthus chinensis were determined as follows: 1 Taq polymerase corresponding buffer (10 mmol·L^-1 Tris·HCl pH9.0，50 mmol·L^-1 KCl, 0.1%Triton X-100), 1.5 mmol·L^-1 MgCl₂, 0.75U Taq DNA polymerase, 20 ng template DNA, 6 pmol primer, 0.15 mmol·L^-1 dATP, dCTP, dGTP, dTTP for each in total 10 μL reaction volume. Using these optimal amplification conditions, 12 stable and repeatable ISSR primers were selected from total 100 primers. 156 loci were produced in total 10 Sinocalycanthus chinensis populations with 200 individuals. There were 114 polymorphic loci among them and the total polymorphic loci percentage was 73.08%. The polymorphic loci percentage in every population was quite different with a mean of 23.65%. The establishment of the PCR reaction conditions could settle favorable basis for the further study on the genetic diversity of Sinocalycanthus chinensis using ISSR molecular marker techniques.

Key words: Sinocalycanthus chinensis, ISSR, optimization, genetic diversity