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【目的】建立大果油茶的ISSR-PCR适宜扩增反应体系和程序,为其深入研究奠定基础。【方法】以大果油茶嫩叶片为供试材料,采用改良的SDS法提取其基因组DNA,并对其ISSR反应体系条件进行筛选及优化。【结果】提取的基因组DNA纯度和完整性较好,OD260/OD280值在1.8～2.0之间,DNA无降解现象,完全可以满足ISSR-PCR扩增要求。在25.0μLISSR-PCR反应体系中,各组分的适宜浓度配比为:40ng/μL模板DNA1.0μL、2.5mmol/LdNTPs2.0μL、5U/μLTaqDNA聚合酶0.2μL、10μmol/L引物1.0μL、10×PCR Buffer2.5μL(含有Mg2+),加ddH2O至25.0μL。PCR反应程序为:94℃预变性5min;94℃变性40s,52℃和53℃退火40s,72℃延伸90s,40个循环;最后72℃延伸7min。由此可获得带型丰富和清晰可辨的DNA指纹图谱。【结论】建立的ISSR-PCR反应体系可用于研究大果油茶遗传变异和多样性。
【Objective】 The objective of this study was to establish an appropriate amplification reaction system and program for ISSR-PCR of Camellia oleifera, which laid the foundation for its further study. 【Method】 The leaves of Datong Camellia oleifera were used as tested materials, and the genomic DNA was extracted by modified SDS method. The conditions of ISSR reaction system were selected and optimized. 【Result】 The purity and integrity of the extracted genomic DNA was good. OD260 / OD280 values ranged from 1.8 to 2.0. No DNA degradation was observed, which fully met the requirements of ISSR-PCR amplification. In the 25.0μLISSR-PCR reaction system, the suitable concentration of each component was: 40μL / μL template DNA1.0μL, 2.5mmol / LdNTPs2.0μL, 5μL / μL Taq DNA polymerase 0.2μL, 10μmol / L primer1.0μL, 10μL × PCR Buffer 2.5μL (containing Mg2 +), plus ddH2O to 25.0μL. The PCR reaction program was: denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 40 s, annealing at 52 ° C and 53 ° C for 40 s, extension at 90 ° C for 90 s at 72 ° C, and extension at 72 ° C for 7 min. This results in a rich and legible DNA fingerprint. 【Conclusion】 The established ISSR-PCR reaction system can be used to study the genetic variation and diversity of Camellia oleifera.