摘 要 ：菰是一种稻族野生资源，水生或沼生，在我国有着广泛的分布。野生菰群体不仅是一种重要的育种基因资源，同时还有着巨大的生态作用。为建立适宜于菰的ISSR反应体系，以菰DNA基因组为模板对ISSR反应体系中的主要影响因子进行优化。采用单因素变量法在12个不同梯度水平上设计正交实验针对体系中dNTPs浓度、Mg2+浓度、Taq聚合酶浓度、引物浓度、模板DNA浓度及引物退火温度等6个因子进行优化，确定了在20 μL ISSR反应体系中各组分的最适因素水平分别为：3.0 mM Mg2+（10×Buffer），0.5 mM dNTPs、1.0 μM引物浓度、0.24 U·μL-1Taq聚合酶、1.5 ng·μL-1DNA模板以及55℃退火温度。应用该优化体系对80个菰材料进行扩增，证实了该体系的适用性和稳定性，为菰遗传资源的鉴定、评价与利用提供了技术支撑。
Abstract:Wild rice Zizania latifolia Griseb (Turcz), an aquatic/wetland genus, is a member of tribe Oryzeae widely distributed across China. Z. latifolia is not only an important genetic resource for crop improvement but also contributes significantly to the ecosystem. However, studies on Z. latifolia have been slow because of the lack of methodology and knowledge in all aspects. In this work, we first tried to develop and establish an optimized ISSR-PCR reaction system for genotyping of Z. latifolia using the single factor variable method in which 6 factors (dNTPs, Mg2+, Taq polymerase, primer, template DNA and primer annealing temperature) at 12 different concentrations or gradients were tested. We finally set up an optimized 20 μL reaction system as follows: 0.5 mM dNTPs, 3.0 mM Mg2+ (10× Buffer), 0.24 U·μL-1Taq polymerase, 1.0 μM primer，1.5 ng·μL-1DNA template and annealing temperature at 55℃. The optimized ISSR system was successfully applied in the amplification of 80 Z. latifolia individual samples, thus, the adaptability and stability of the system were testified.