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目的构建三叶青ISSR反应体系,为三叶青种质资源遗传多样性分析、品种选育与分子鉴定奠定基础。方法用常规CTAB法、SDS法、改良CTAB法、试剂盒法提取三叶青叶片基因组DNA,采用正交设计试验L12(34),在4个主要因素的3个水平上进行ISSR体系构建,用12个不同种源三叶青样品验证体系稳定性。结果改良CTAB法提取的三叶青叶片基因组DNA质量最好,纯度高,试剂盒法次之,常规CTAB法与SDS法所提DNA质量较差。构建的ISSR最佳反应体系为:20μl反应体系中,Taq酶用量0.5U,模版DNA20ng,d NTPs0.20mmol/L,引物0.4μmol/L,2μl 10×Buffer缓冲液(Mg2+浓度1.5mmol/L)。结论采用正交设计方法构建的三叶青ISSR体系经检验,稳定可靠,可用于三叶青种质资源遗传分析。
OBJECTIVE: To construct the ISSR reaction system of Clover so as to lay the foundation for the genetic diversity, breeding and molecular identification of Clover. Methods The CTAB method, SDS method, modified CTAB method and kit method were used to extract the genomic DNA of Clover leaf. The orthogonal design L12 (34) was used to construct the ISSR system at three levels of four major factors. Twelve different provenances were used to verify the stability of the system. Results The genomic DNA extracted from CTAB improved CTAB with the highest quality and high purity, followed by the kit method. The quality of DNA extracted by conventional CTAB and SDS methods was poor. The optimal ISSR reaction system was as follows: 0.5U Taq enzyme, 20ng template DNA, 0.20mmol / L dNTPs, 0.4μmol / L primer and 2μl 10x Buffer buffer (Mg2 + 1.5mmol / L) . Conclusion The ISSR system constructed by orthogonal design has been proved to be stable and reliable, and can be used for genetic analysis of Grifola frondosa germplasm resources.