早熟砂梨ISSR-PCR反应体系的建立与优化

摘要

以我国南方主栽的早熟砂梨品种‘翠冠’Pyrus pyrifolia‘Cuiguan’为材料，对ISSR技术体系中的模板DNA浓度、Taq DNA聚合酶用量、引物浓度、dNTP浓度、Mg2+浓度、退火温度、PCR循环数等7个主要因素进行优化和筛选，建立了适合早熟砂梨的ISSR-PCR反应体系。最终反应体系为20μL体系中10×PCR buffer （不含Mg2+）2μL，模板 DNA浓度60 ng，TaqDNA聚合酶0.75 U，引物浓度1μmol/L，dNTP浓度90μmol/L， Mg2+浓度2.25 mmol/L。扩增程序为：预变性94℃5 m in，变性94℃45 s，退火45 s，72℃延伸1 min，共42个循环，然后72℃再延伸10 min，4℃保存，用1.5%琼脂糖凝胶电泳检测多态性。%　　To obtain 7 factors (DNA template dosage, Taq DNA polymerase dosage, primer concentration, dNTP concentration, Mg2+concentration, annealing temperature and PCR cycles ) of ISSR-PCR Reaction System in Pyrus pyrifolia ‘Cuiguan’, a precocious cultivar of southern China was been used as template. An optimum reaction system was been established. The final total volume was 20 μL, contains 2 μL 10×PCR buffer (Mg2+ free), 60 ng DNA template, 0.5 U Taq DNA polymerase, 1 μmol/L of primer concentration, 90 μmol/L of dNTP concentration, 2.25 mmol/L of Mg2+ concentration. Amplication procedure was as follows: initial-denaturing of 5 min at 94 ℃, followed by 42 cycles of 45 s for denaturing at 94℃, at annealing temperature for 45 s and 1 min at 72 ℃, after that 10 min extending at 72 ℃, and then keep at 4 ℃. Use 1.5% of agarose gel electrophoresis to test the genetic polymorphism.