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作 者 ：张涛, 杨青川, 毛培胜

期 刊 ：草地学报 2006年 14卷 4期 页码：333-337

关键词：紫花苜蓿；品种鉴定；RAPD；优化；

Keywords:Alfalfa, Verification of cultivar, RAPD, Optimize,

摘 要 ：以中苜1号紫花苜蓿(Medicago sativa L.cv.Chongmu No.1)为研究材料,通过对RAPD-PCR反应退火温度、反应体系中的模板DNA、Taq聚合酶、Mg²⁺、dNTP、引物用量的梯度处理,分别对各项单因子进行优化。结果表明,当退火温度为37℃,总反应体积25μl时,各反应物适宜用量为模板DNA80ng,Taq聚合酶1.5U,Mg²⁺6.25×10^-5mmol,dNTP0.3×10^-5mmol,随机引物0.4×10^-5mmol,缓冲液KCl1.25×10^-3mmol,扩增结果清晰、稳定,并且在不同实验室扩增结果具有较好的一致性。

Abstract:It is important to use superior cultivars for the breeding and extension of alfalfa to cultivate grassland for hay and for modifying environment. The RAPD reaction system for verifying alfalfa cultivars was applied to optimize by gradient quantity of the annealing temperature, template DNA, Taq polymerase, Mg²⁺,dNTP, and the primer in the Zhongmu No.1 alfalfa. The results show that when the annealing temperature reached 37℃ and the volume of the RAPD reaction system was 25 μl, the optimal amount of template DNA would be 80 ng; of Taq polymerase, 1.5 U; of Mg²⁺, 6.25×10^-5mmol; of dNTP, 0.3×10^-5mmol; of primer, 0.4×10^-5mmol; and of buffer KCl, 1.25×10^-3mmol. The electrophoretic patterns of amplifying in the optimized reaction system were similar in the 3 different types of PCR amplifiers.

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