分蘖洋葱ISSR-PCR扩增体系的建立

Establishment of optimal ISSR-PCR reaction system in potato onion

以分蘖洋葱幼嫩叶片DNA为试材,采用单因素试验,对ISSR-PCR扩增体系中的退火温度、模板DNA量、Mg2+浓度、dNTP浓度、Taq酶含量以及引物浓度等各因素进行优化。结果表明,在反应体系为25μL反应液中,包含DNA模板40 ng(2μL),10×PCR Buffer 3μL,Mg2(+2.5 mmol.L-1)2μL,dNTPs(2.5 mmol.L-1)3μL,引物(5 mmol.L-1)3μL,Taq酶(5 U.μL-1)0.4μL,退火温度为56℃时,分蘖洋葱ISSR-PCR扩增获得最佳效果,初步建立分蘖洋葱叶片最佳ISSR-PCR扩增体系,为ISSR分子标记技术应用于分蘖洋葱种群遗传多样性分析、遗传图谱构建、核心种质资源保存利用等奠定分子生物学的理论基础和技术支撑。

Total genomic DNA was extracted from potato onion leaves for ISSR-PCR. With the single factor method, the effect of six ISSR marker factors on PCR reaction system was investigated. The results showed that the optimal ISSR-PCR reaction system of potato onion was established as the following： reaction volume （total 25 μL） including template DNA 40 ng, 10x PCR Buffer 3 μL, 2.5 mmol. L1 Mg2＋ 2 μL, 2.5 mmol. L-1 dNTP 3 μL, 5 U μL-1 Taq polymerase 0.4 μL and 5 mmol L-1 primer 3 pL, and 56 ℃ was the best annealing temperature. This study provided a technological and theory basis for construction of cultivar fingerprinting and genetic linkage map and applying of core germplasm in potato onion using ISSR molecular marker method.