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采用浓度梯度设计法研究适合白及的SRAP-PCR反应体系。对影响SRAP-PCR的DNA模板、引物、dNTP、Mg2+、Taq酶、退火温度等6个因素进行优化。最终确定白及的SRAP-PCR优化体系:在20μL体系中,DNA模板30 ng、Mg2+2.5 mmol/L、dNTPs 0.25μmol/L、引物20μmol/L、Taq酶2.0U,扩增体系为:94℃预变性5 min;5个循环的94℃变性1 min,36℃退火1 min,72℃延伸1 min;之后35个循环:94℃变性1 min,48℃退火1 min,72℃延伸1 min;72℃最后延伸5 min。该体系的建立为野生白及种质资源遗传多样性的进一步相关遗传分析奠定了基础。
The concentration gradient design method was used to study the SRAP-PCR reaction system suitable for white and white. Six factors affecting DNA template, primer, dNTP, Mg2 +, Taq enzyme and annealing temperature of SRAP-PCR were optimized. Finally, the optimized system of SRAP-PCR was established: in 20μL system, DNA template was 30ng, Mg2 + 2.5mmol / L, dNTPs 0.25μmol / L, primer 20μmol / L, Taq 2.0U, 5 cycles of denaturation at 94 ° C for 1 min, annealing at 36 ° C for 1 min and extension at 72 ° C for 1 min; then 35 cycles of denaturation at 94 ° C for 1 min, annealing at 48 ° C for 1 min, extension at 72 ° C for 1 min ; Final extension at 72 ° C for 5 min. The establishment of this system laid the foundation for the further genetic analysis of the genetic diversity of wild white and germplasm resources.