Establishment and Optimization of ISSR-PCR Reaction System for Iris lacteal

doi:10.11733/j.issn.1007-0435.2008.06.007

›› 2008, Vol. 16 ›› Issue (6): 580-584,589.DOI: 10.11733/j.issn.1007-0435.2008.06.007

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Establishment and Optimization of ISSR-PCR Reaction System for Iris lacteal

WANG Kang^1,2, DONG Kuan-hu², SUN Yan³, KANG Jun-mei¹, DONG Jie², MENG Lin⁴

1. Institute of Animal Sciences, CAAS, Beijing 100193, China;
2. School of Animal Science, Shanxi Agricultural University, Taigu, Shanxi 030801, China;
3. Institute of Grassland Science, China Agricultural University, Beijing 100193, China;
4. Beijing Research Development Center for Grass and Environment, BAAFSR Beijing 100097, China

Received:2008-03-10 Revised:2008-05-07 Online:2008-12-15 Published:2008-12-15

马蔺ISSR-PCR反应体系的建立与优化

王康^1,2, 董宽虎², 孙彦³, 康俊梅¹, 董洁², 孟林⁴

1. 中国农业科学院北京畜牧兽医研究所, 北京, 100193;
2. 山西农业大学动物科技学院, 太谷, 山西, 030801;
3. 中国农业大学动物科技学院草地研究所, 北京, 100193;
4. 北京市农林科学院草业与环境发展中心, 北京, 100097

通讯作者: 康俊梅,E-mail:kangjmei@yahoo.com.cn
作者简介:王康(1982- ),男,山西霍州人,硕士研究生,研究方向为草地资源与草地管理,E-mail:wangkangkang224@yahoo.com.cn.
基金资助:
北京市科学自然基金项目(6062012);院所基本科研业务费专项基金创新团队(ywf-td-3)

Abstract

Abstract: In this paper,two orthogonal experiments were conducted to optimize ISSR amplification system of Iris lacteal Pall.var.chinensis(Fisch.) Koidz.Results show that the optimum concentrations of reactants in 25 μl reaction mixture were as follows: dNTP 0.3 mmol/L,Taq DNA polymerase 1.5 U,Primers 0.4 μmol/L,Mg²⁺ 2.5mmol/L,1×buffer and 50 ng template DNA.The optimal annealing temperature of this primer was proposed by gradient PCR at 52℃ for ISSR-PCR reaction.The result provided a standardized ISSR-PCR program for the analysis of genetic diversity of I.lacteal var.chinensis.

Key words: Iris lacteal Pall.var.chinensis(Fisch.) Koidz, ISSR-PCR, Reaction system, Orthogonal optimization

摘要： 根据正交试验设计原理,设计探索正交试验和细调正交试验以确定马蔺(Iris lacteal Pall.var.chinensis(Fisch.)Koidz)ISSR-PCR反应体系中各成分的浓度,从dNTP、Taq酶、Pri mers、Mg²⁺4种因素对马蔺ISSR反应体系进行了优化,得到适合马蔺的ISSR-PCR最佳反应体系,即25μl的反应体系中含有dNTP 0.3 mmol/L、Taq酶1.5 U、Pri mers 0.4μmol/L、Mg²⁺2.5 mmol/L以及1×buffer和50 ng模板DNA。通过对马蔺ISSR-PCR最佳反应体系进行梯度退火试验,得到这条引物的最佳退火温度为52℃。这一体系的建立为今后利用ISSR标记技术研究马蔺遗传多样性奠定了基础。

关键词: 马蔺, ISSR-PCR, 反应体系, 正交优化

CLC Number:

S812
Q943

WANG Kang, DONG Kuan-hu, SUN Yan, KANG Jun-mei, DONG Jie, MENG Lin. Establishment and Optimization of ISSR-PCR Reaction System for Iris lacteal[J]. , 2008, 16(6): 580-584,589.

王康, 董宽虎, 孙彦, 康俊梅, 董洁, 孟林. 马蔺ISSR-PCR反应体系的建立与优化[J]. , 2008, 16(6): 580-584,589.

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Establishment and Optimization of ISSR-PCR Reaction System for Iris lacteal

马蔺ISSR-PCR反应体系的建立与优化

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