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中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (1): 32-36.doi: 10.11924/j.issn.1000-6850.2013-2119

• 林学 园艺 园林 • 上一篇    下一篇

文冠果ISSR反应体系的建立及优化

芦娟,柴春山,吴文俊,戚健莉   

  • 收稿日期:2013-08-06 修回日期:2013-09-23 出版日期:2014-01-05 发布日期:2014-01-05
  • 基金资助:
    甘肃省自然科学基金项目 “甘肃省文冠果资源遗传多样性的研究” (1010RJZA132)。

Optimization of ISSR-PCR Amplification in Xanthoceras sorbifolia Bunge

Lu Juan, Chai Chunshan, Wu Wenjun, Qi Jianli   

  • Received:2013-08-06 Revised:2013-09-23 Online:2014-01-05 Published:2014-01-05

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摘要： 为了建立文冠果ISSR-PCR最佳反应体系，从而为文冠果遗传多样性研究提供理论依据，从定西巉口林场文冠果人工林内选取健康嫩叶为试验材料，通过单因子试验研究了影响ISSR-PCR反应各因素的浓度，分析因子包括模板DNA用量、退火温度、dNTP浓度、Mg2+浓度、引物浓度及TaqDNA聚合酶用量等。通过研究，找出了各因子合适的条件，建立并优化了文冠果ISSR反应体系，即20μL总反应体系含模板DNA约30 ng，Mg2+2.5 mmol/L，引物0.2μmol/L，dNTP 0.20 mmol/L，Taq聚合酶1 U。试验结果表明，在此反应体系下可得到多样性好、条带清晰的图谱。

关键词: 甘蓝型油菜, 甘蓝型油菜, 杂交种纯度鉴定, SSR标记

Abstract: The aims were to establish an optimal reaction system of ISSR-PCR in Xanthoceras sorbifolia Bunge, and provide theoretical basis for its genetic diversity research. From X. sorbifolia artificial forest in Chankou forest farm of Dingxi City, picking health and tender leaves as experiment material, the concentrations of main factors influencing the effects of ISSR-PCR were tested by single factor experiments respectively, the tested factors including template DNA concentration, annealing temperature, dNTP concentration, template DNA concentration, annealing temperature, Mg2+ concentration, primer concentration and Taq DNA polymerase. Through test, this study had found suitable conditions for each factor, established and optimized X. sorbifolia’s reaction system of ISSR-PCR, namely the optimal reaction system for ISSR analysis in X.sorbifolia, included 30 ng template DNA, 2.5 mmol/L Mg2 + , 0.2μmol/L primer, 0.20 mmol/L dNTP and 1 U of Taq DNA polymerase in a total volume of 20μL. The study indicated that atlas with fine diversity and clear strap was gained.