禾本科作物基因组AFLP分析体系的建立及优化

Establishment and Optimization of AFLP System for Gramineous Crop Genome

以禾本科作物小麦为出发材料,通过优化AFLP选择性扩增步骤的4个限制性因素:Mg2+浓度、dNTP浓度、引物浓度和预扩产物稀释倍数,确立AFLP最佳反应体系为:预扩增产物稀释20倍吸取2μL做为选择性扩增模板,混合1.5 mmol/L MgCl2,0.2 mmol/L dNTPs,0.4μmol/L选扩引物1、U Ex Taq进行选择性扩增。应用该体系分析水稻和玉米两大类禾本科作物基因组DNA样品,同样取得理想结果,扩增出清晰可辨且适量的电泳条带。该技术体系为AFLP分子标记技术在禾本科作物遗传分析中的广泛应用奠定了基础。

After studying the four factors of selective amplification, Mg^2＋ concenctration, dNTP concenctration, primer concenctration and dilution times of pre-amplified product, the optimized system of AFLP for wheat was built：taking 2 μL of 20 times dilution of preamplification products as amplification template, adding 0.4 μmol/L of selective primer combinations, 1.5 mmol/L of MgCl2, 0. 2 mmol/L of dNTPs and 1 U of Ex Taq polymerase. The AFLP system was confirmed by analyzing the genomic DNA of maize and rice. The results laid a foundation for broadly using the AFLP technology in genetic analysis of gramineous crops.