Optimization of ISSR-PCR System on Zoysia spp.

doi:10.11733/j.issn.1007-0435.2009.01.010

›› 2009, Vol. 17 ›› Issue (1): 48-51.DOI: 10.11733/j.issn.1007-0435.2009.01.010

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Optimization of ISSR-PCR System on Zoysia spp.

WANG Zhi-yong^1,2, YUAN Xue-jun², GUO Hai-lin², LIU Jian-xiuCHENG Xiao-li², ZHOU Zhi-fang²

1. Department of Horticulture, Nanjing Agricultural Univeristy, Nanjing, Jiangsu 210095, China;
2. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, Jiangsu Province 210014, China

Received:2008-04-12 Revised:2008-12-15 Online:2009-02-15 Published:2009-02-15

结缕草属植物ISSR-PCR反应体系研究

王志勇^1,2, 袁学军², 郭海林², 刘建秀², 程晓丽², 周志芳²

1. 南京农业大学园艺学院, 中国, 南京, 210095;
2. 江苏省中国科学院植物所(南京中山植物园), 中国, 南京, 210014

通讯作者: 刘建秀,Author for correspondence,Email:turfunit@yahoo.com.cn
作者简介:王志勇(1979- ),男,江西景德镇市人,在读博士,研究方向为草坪植物种质评价与遗传育种Email:fafu301@yahoo.com.cn
基金资助:
国家自然基金项目(30571307);江苏省高技术项目(BG2006320)资助

Abstract

Abstract: The ISSR-PCR system was optimized and the effects of template DNA with different concentrations on the PCR amplification were studied by an orthogonal experimental design with 3 different levels of 4 factors,e.g.Mg²,dNTP,primers,and Taq DNA polymerase.The results show that each factor in different levels had significant effect on the amplification result of PCR and the orthogonal design was useful to optimize the ISSR-PCR system.The optimized PCR reaction system for Zoysia spp.was 10X Buffer,70 ng genomic DNA template,2.0 mmol·L^-1 Mg²⁺,150 μmol·L^-1 dNTP,0.3 μmol·L^-1 Primer,1.0 U Taq DNA polymerase with total reaction solution of 20 μL.The system was proved stable and credible according to the result of 11 Zoysia spp.and the result of this study could be useful to provide the basis in the analysis of genetic diversity,germplasm identification,and kinship of Zoysia spp.

Key words: Zoysia spp., ISSR-PCR, Orthogonal design, Systematic optimization

摘要： 以SDS法提取的结缕草(Zoysia spp.)叶片DNA为模板,应用正交试验设计,从Mg²、dNTP、引物和DNA聚合酶并通过比较不同浓度的模板DNA对PCR扩增的影响,确立结缕草属植物ISSR-PCR反应的最佳体系。结果表明:各因素不同浓度对PCR反应结果有显著影响,正交设计可以用于ISSR反应体系建立,该方法建立的结缕草属植物ISSR-PCR优化反应体系为10X Buffer7、0 ng模板DNA、Mg²+2.0 mmol·L^-1、dNTP 150μmol·L^-1、Primer 0.3μmol·L^-1、Taq DNA聚合酶1.0 U,总体积20μL;经对11份结缕草属植物进行检验,证明该体系稳定可靠。该体系的建立可为今后利用ISSR标记技术进行结缕草属遗传多样性研究、种质资源鉴定和亲缘关系分析等提供依据。

关键词: 结缕草, ISSR-PCR, 正交试验, 体系优化

CLC Number:

Q943.2

WANG Zhi-yong, YUAN Xue-jun, GUO Hai-lin, LIU Jian-xiuCHENG Xiao-li, ZHOU Zhi-fang. Optimization of ISSR-PCR System on Zoysia spp.[J]. , 2009, 17(1): 48-51.

王志勇, 袁学军, 郭海林, 刘建秀, 程晓丽, 周志芳. 结缕草属植物ISSR-PCR反应体系研究[J]. , 2009, 17(1): 48-51.

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Optimization of ISSR-PCR System on Zoysia spp.

结缕草属植物ISSR-PCR反应体系研究

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