枫香ISSR-PCR扩增条件的优化

对浙江省天台山枫香自然种群的DNA进行提取和ISSR-PCR扩增条件的优化。分析了Mg2+浓度、4×dNTP浓度、BSA浓度、模板DNA用量、引物用量、Taq DNA聚合酶用量等对反应结果的影响。建立了适用于枫香ISSR分析最适宜的反应体系:10μl PCR反应体积中,1×Taq酶配套缓冲液(10 mmol.L-1Tris-HCl,pH值9.0,50 mmol.L-1KCl,0.1%TritonX-100),2.0 mmol.L-1Mg2+,0.15 mmol.L-14×dNTP,2 mg.mL-1BSA,10 ng模板DNA,6 pmol引物,0.9 U TaqDNA聚合酶,引物UBC808的最适退火温度为52.4℃。用此反应体系对天台山枫香自然种群进行了遗传多样性分析,结果显示其多态位点百分率为84.26%,Nei指数为0.2665,Shannon信息指数为0.4044,其遗传多样性处于较高水平。

The optimization of ISSR-PCR conditions of Liquidambar formosana

The optimal conditions of ISSR amplification of Liquidambar formosana from Tiantai Mountain in Zhejiang Province was screened.After the analysis of the concentration Mg2+,4×dNTP,BSA,template DNA dosage,primer dosage,and unit of Taq DNA polymerase,the optimal conditions of ISSR-PCR was determined as follows: 1×Taq buffer(10 mmol·L-1 Tris-HCl,pH9.0,50 mmol·L-1 KCl and 0.1% Triton X-100),2.0 mmol·L-1 Mg2+,0.15 mol·L-1 4×dNTP,2.0 mg·mL-1 BSA,10 ng template DNA,6 pmol primers and 0.9 U Taq DNA polymerase;the suitable annealing temperature of UBC808 primer in the ISSR-PCR reaction system was 52.4℃.Using the optimal amplification,91 loci were produced in L.formosana population of Tiantai Mountain.The polymorphic loci percentage was 84.26%,Nei's gene diversity was 0.2665,and Shannon's information index was 0.4044.The genetic diversity of L.formosana was in a high level.