Orthogonal Optimization of ISSR Reaction System for Chickpea

doi:10.11733/j.issn.1007-0435.2015.06.025

Acta Agrestia Sinica ›› 2015, Vol. 23 ›› Issue (6): 1303-1309.DOI: 10.11733/j.issn.1007-0435.2015.06.025

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Orthogonal Optimization of ISSR Reaction System for Chickpea

JIN Xiao-li, TIAN Xin-hui, DU Wen-hua

College of Grassland Science, Gansu Agricultural University/Key Laboratory of Grassland Ecosystem, Ministry of Education/Sino-U.S Centers for Sustainable Development of Grassland and Animal Husbandry, Lanzhou, Gansu Province 730070, China

Received:2014-07-07 Revised:2014-12-14 Online:2015-12-15 Published:2016-02-01

鹰嘴豆ISSR反应体系的正交优化

靳晓丽, 田新会, 杜文华

甘肃农业大学草业学院/草业生态系统教育部重点实验室/中-美草地畜牧业可持续研究中心, 甘肃兰州 730070

通讯作者: 杜文华
作者简介:靳晓丽(1986-),女,河南安阳人,硕士研究生,主要从事草种质资源及育种栽培方面的研究,E-mail:jinfei221.good@163.com
基金资助:
国家自然科学基金(31360577);公益性行业(农业)科研专项(201003019);教育部博士点基金(20136202110005);现代农业产业技术体系建设专项资金(CARS-40-09B)资助

Abstract

Abstract:

The establishment of the chickpea ISSR reaction system would provide theoretical bases and technical references for the analysis of chickpea diversity, the construction of the gene map and the localization of important traits for chickpea genotypes using ISSR markers.The DNA template for chickpea (Cicer arietinum) was extracted using CTAB method. An ISSR-PCR amplification system for chickpea was optimized by visual analysis and an orthogonal design L₁₆ (4⁵) involving 4 levels of 4 main factors (concentrations of Mg²⁺, Primer, Taq DNA polymerase, and dNTPs). The annealing temperature of the primer UBC826 was optimized based on the optimal ISSR-PCR amplification system. The results indicated that the concentrations of Mg²⁺ and primer affected the PCR amplification significantly, while the concentrations of dNTPs and Taq DNA polymerase had no effects on it. The suitable ISSR-PCR amplification system (25 μL) for chickpea included 3 mmol·L^-1 Mg²⁺, 0.2 mmol·L^-1 dNTP, 0.24 μmol·L^-1 primer and 2 U Taq DNA polymerase. The suitable annealing temperature for the primer UBC 826 was 56℃.

Key words: Chickpea, ISSR, Reaction system, Orthogonal design, Optimization

摘要：

鹰嘴豆(Cicer arietinum)ISSR反应体系的建立可以为鹰嘴豆种质资源遗传多样性分析、遗传图谱构建和基因定位提供理论依据和技术参考。本研究以CTAB法提取的鹰嘴豆DNA为模板,采用L₁₆(4⁵)正交设计直观分析法,对鹰嘴豆ISSR-PCR反应中的4个主要因素(Mg²⁺浓度,引物浓度,TaqDNA聚合酶浓度,dNTPs浓度)在4个水平上进行优化,并在最优ISSR-PCR反应体系的基础上对引物UBC826的最适退火温度进行筛选。结果表明:Mg²⁺和引物浓度对PCR扩增结果有显著影响,dNTPs和Taq酶的浓度变化对PCR扩增结果无显著影响。鹰嘴豆ISSR-PCR优化反应体系(25 μL)为:3 mmol·L^-1 Mg²⁺,0.2 mmol·L^-1 dNTP,0.24 μmol·L^-1引物,2 U Taq DNA聚合酶。UBC826最适退火温度为56℃。

关键词: 鹰嘴豆, ISSR, 反应体系, 正交设计, 优化

CLC Number:

Q341

JIN Xiao-li, TIAN Xin-hui, DU Wen-hua. Orthogonal Optimization of ISSR Reaction System for Chickpea[J]. Acta Agrestia Sinica, 2015, 23(6): 1303-1309.

靳晓丽, 田新会, 杜文华. 鹰嘴豆ISSR反应体系的正交优化[J]. 草地学报, 2015, 23(6): 1303-1309.

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Orthogonal Optimization of ISSR Reaction System for Chickpea

鹰嘴豆ISSR反应体系的正交优化

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