水稻茄丝核菌SRAP引物筛选与体系优化

Optimization of SRAP system and primer screening of Rhizoctonia solani in rice

为了应用新型的SRAP分子标记技术分析水稻茄丝核菌基因多态性和探究基因多态性与采集来源及水稻品种之间的关系,以吉林省不同地理位置的3株水稻丝核菌为材料,从100对SRAP引物中筛出较理想的引物16对,并对影响SRAP反应的Mg2+、dNTPs、引物、Taq DNA聚合酶、模板DNA浓度及两次退火温度等条件进行了优化,建立了适宜于该菌的SRAP反应体系,25μL的反应体系包含2.5 mmol/L Mg2+、0.15 mmol/L dNTPs、0.8μmol/L引物、1.5 U Taq DNA聚合酶和80 ng模板DNA。利用24株水稻丝核菌对优化后的SRAP反应体系进行了验证,结果表明该反应体系稳定可靠,能够有效地用于水稻茄丝核菌的遗传多样性分析。

In order to apply the sequence-related amplified polymorphism （SRAP） to analyze gene poly- morphism of Rhizoctonia solani in rice and study the relationship among the gene polymorphism, collected site and rice varieties, 100 pairs of SRAP primers were screened by using 3 isolates from different regions in Jilin Province as materials. And 16 pairs were selected and the concentrations of Mg2 ~ , dNTPs, prim- ers, Taq DNA polymerase, template DNA and the two annealing temperatures which affect the SRAP re- actions of R. solani were optimized. Accordingly, a SRAP system suitable for R. solani was established. The optimum 25 txL reaction system contained 2.5 mmol/L Mg2 ＋ , 0.15 mmoL/L dNTPs, 0.8 txmol/L of each primer, 1.5 U Taq DNA polymerase and 80 ng template DNA. The optimum system was testified using 24 isolates of R. solani. The results showed that the SRAP primers and the system were steady and reliable, which could be applied to genetic diversity analysis of R. solani in rice.