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本研究利用L16(45)正交试验设计,对影响ISSR-PCR反应体系的5个影响因素(Mg~(2+)浓度,d NTPs浓度,Taq DNA聚合酶浓度,引物浓度及模板DNA含量)进行优化。并在50~60℃范围内摸索引物UBC845的最适退火温度,并建立了花吊丝竹的最佳ISSR-PCR反应体系:2.5 mmol/L Mg~(2+),0.25 mmol/L d NTPs,1.50 U Taq DNA聚合酶,0.5μmol/L引物,80 ng模板DNA,2μL 10x Buffer,9.5μL dd H2O,ISSR-PCR扩增程序为:94℃预变性5 min;94℃变性45 s;52.7℃退火30 s,72℃延伸90 s,35个循环;72℃延伸10 min,4℃保存。利用优化的反应体系从100条引物中筛选出16条多态性较好的引物,该体系的建立有助于花吊丝竹种质资源遗传多样性分析和指纹图谱的构建。
In this study, five factors affecting the ISSR-PCR reaction system (Mg 2+, d NTPs, Taq DNA polymerase, primer concentration and template DNA content) were designed using L16 (45) orthogonal design. optimize. The best annealing temperature of primer UBC845 was determined in the range of 50 ~ 60 ℃, and the optimal ISSR-PCR reaction system was established: 2.5 mmol / L Mg 2+, 0.25 mmol / L d NTPs, 1.50 U Taq DNA polymerase, 0.5μmol / L primer, 80ng template DNA, 2μL 10x Buffer, 9.5μL ddH2O, ISSR-PCR amplification program: 94 ℃ denaturation 5 min; 94 ℃ denaturation 45 s; 52.7 ℃ Annealing 30 s, 72 ℃ extension 90 s, 35 cycles; 72 ℃ extended 10 min, 4 ℃ preservation. Sixteen primers were selected from 100 primers based on the optimized reaction system. The establishment of this system is helpful to the genetic diversity analysis and fingerprinting of Sorbonne.