苜蓿假盘菌ISSR反应体系优化及指纹图谱构建

以北京苜蓿假盘菌(Pseudopeziza medicaginis (Lib.) Sacc.)菌株为试材,用ISSR-23引物序列为(AC)8T]研究PCR反应体系的主要成分及退火温度对假盘菌ISSR扩增的影响,以期为苜蓿假盘菌的深入研究奠定基础。结果表明:Primer、Mg²⁺、Taq酶和dNTP浓度对扩增均有影响,以Primer影响最为明显;优化的反应体系为:20μL反应体系中含0.1 ng模板DNA,2.0mmol/L MgCl₂、0.5μmol/L Primer、1U Taq DNA聚合酶、0.2 mmol/LdNTP,2μL 10×PCR Buffer和2.5%去离子甲酰胺;在此基础上,建立6个不同地理来源苜蓿假盘菌菌株的指纹图谱并分析其亲缘关系;对44条ISSR引物进行筛选,22条可产生清晰稳定的多态性条带,其中16条可将6个供试菌株完全分开,建立了苜蓿假盘菌指纹图谱;经聚类分析,在0.55遗传相似水平下,6个供试菌株分为两个遗传谱系,菌株的遗传谱系与其地理来源之间不表现相关性。

Optimization of ISSR Reaction System and Construction of Fingerprinting of Pseudopeziza medicaginis (Lib.) Sacc.

The effects of PCR reaction components and annealing temperature on ISSR(inter-simple sequence repeat) amplification of Pseudopeziza medicaginis(Lib.) Sacc.cv.Beijing were studied using primer ISSR-23(sequence:(AC)₈T).The results show that all of components,i.e.concentrations of primer,Mg²⁺,Taq DNA polymerases,and dNTP,affected the amplification results,but the effect of primer concentration was distinct.The optimal concentration of ISSR-PCR reaction system was 0.1 ng template DNA,2.0 mmol/L MgCl₂,0.5μmol/L primer,1U Taq DNA polymerase,0.2 mmol/L dNTP,2μL 10×PCR buffer,and 2.5% deionized formamide per 20μL reaction volume.The ISSR fingerprinting profiles of 6 P.medicaginis strains with different geographical originals were established with 22 primers screened out from 44 ISSR primers.The isolated P.medicaginis strains were clustered into 2 genetic lineages at 0.55 genetic similarity and the genetic lineages of 6 strains showed no obvious relation with their geographical originals.