Optimization of ISSR-PCR reaction system of carrot by orthogonal design

• Published in: Molecular plant breeding Vol. 11; no. 6
• Main Authors: Wu Chunyan, Jilin Agricultural University, Changehun (China), Department of Horticultural, Chen Henan, Jilin Agricultural University, Changehun (China), Department of Horticultural, Li Jing, Jilin Agricultural University, Changehun (China), Department of Horticultural
• Format: Journal Article
• Language: Chinese
• Published: 01.12.2013
• Subjects: ADN; DNA; http://www.fao.org/aos/agrovoc#c_2347; http://www.fao.org/aos/agrovoc#c_34079; http://www.fao.org/aos/agrovoc#c_5993; PCR; PLANTE; PLANTS
• ISSN: 1672-416X

为获得适于胡萝bISSR-PCR的反应体系，本研究以胡萝卜为试验材料，采用新型植物基因组DNA提取试剂盒提取胡萝b嫩叶DNA，利用正交试验设计方法，对ISSR．PCR的5个影响因素：Mg2＋、dNTP、TaqDNA聚合酶、引物和模板DNA进行了研究。结果得到胡萝bISSR．PCR的最佳反应体系：25μL的反应体系中含有1.5mmol／LMg2＋、0.15mmol／LdNTP、1.5UroeDNA聚合酶、0.2μmol／L引物、50ng模板DNA。利用优化的反应体系，对最佳反应次数进行了筛选，结果表明优化的反应体系适宜的循环次数是35次。该体系可以为胡萝b遗传多样性分析、基因定位等提供理论依据。 To obtain the optimum ISSR-PCR reaction system of carrot, the carrot genomic DNA was extracted from young leaf using new plant genomic DNA extraction kit. And five factors, including Mg2＋, dNTP, Taq DNA polymerase, primers and template DNA were researched to establish the optimum reaction system by orthogonal design, that is