Acta Agrestia Sinica ›› 2016, Vol. 24 ›› Issue (5): 1080-1086.DOI: 10.11733/j.issn.1007-0435.2016.05.023

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Optimization of SRAP-PCR System in Paspalum vaginatum and Primers Screening

Liu Yan1,2, Wang Yi1, Ma Jing-jing1, Shi Jing-ang1, Wang Zhi-yong3, Liu Jian-xiu1,2, Guo Hai-lin1   

  1. 1. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China;
    2. College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China;
    3. College of Agriculture, Hainan University, Haikou 570228, China
  • Received:2015-08-07 Revised:2016-03-04 Online:2016-10-15 Published:2017-01-13

海雀稗SRAP-PCR分子标记体系优化及引物筛选

刘燕1,2, 汪毅1, 马晶晶1, 史经昂1, 王志勇3, 刘建秀1,2, 郭海林1   

  1. 1. 江苏省中国科学院植物研究所(南京中山植物园), 江苏 南京 210014;
    2. 南京农业大学园艺学院, 江苏 南京 210095;
    3. 海南大学农学院, 海南 海口 570228
  • 通讯作者: 郭海林
  • 作者简介:刘燕(1990-),河南三门峡人,硕士研究生,研究方向为草坪草种质多样性及其新品种选育,E-mail:nianliuyan@163.com
  • 基金资助:

    江苏省第四期“333高层次人才培养工程”项目;江苏省植物迁地保护重点实验室项目(QD201301);江苏省农业自主创新项目(CX(14)2049);江苏省属公益类科研院所能力提升项目(BM2015019)资助

Abstract:

L9(34) orthogonal experiment design including four factors of Mg2+, dNTP, Primer, Taq DNA polymerase was applied to optimize SRAP-PCR amplification system of Paspalum vaginatum, and the influence of different concentration of template DNA on the amplification were compared. The optimization of SRAP-PCR reaction system in Paspalum vaginatum was established. The results showed that the optimal SRAP-PCR reaction system was 10×PCR buffer 1μL, template DNA 50 ng, Mg2+ 2.50 mmol·L-1, dNTP 150 μmol·L-1, primer 0.4 μmol·L-1 and TaqDNA polymerase 1.5 U in 10 μL total reaction system, respectively. Eighty-three pairs of primers with stable amplification and legible bands were screened form 100 SRAP primers through this system. Forty-four pairs of primers with rich polymorphism among 4 tested materials were screened from 83 pairs of primers. Optimization of SRAP-PCR system and primers screening in Paspalum vaginatum will provide technical assistance for the evaluation of genetic diversity, molecular marker assisted breeding and linkage map construction of Paspalum vaginatum.

Key words: Paspalum vaginatum, SRAP molecular marker, Orthogonal experiment, Optimization of system, Primers Screening

摘要:

以4个代表性海雀稗(Paspalum vaginatum)种质的叶片DNA为模板,采用L9(34)正交试验设计,对影响海雀稗SRAP-PCR反应的Mg2+、dNTP、引物和TaqDNA聚合酶的用量进行了优化,并比较了不同浓度模板DNA对扩增的影响,以确定适合海雀稗的SRAP-PCR最佳反应体系。结果表明:海雀稗SRAP-PCR最佳反应体系为10×PCR buffer 1 μL、模板DNA50 ng、Mg2+2.5 mmol·L-1、dNTP150 μmol·L-1、引物0.4 μmol·L-1、TaqDNA聚合酶1.0 U,总体积10 μL。利用该反应体系从100对引物中筛选出可扩增清晰条带的引物83对,在83对引物中选出多态性丰富的引物44对。SRAP-PCR反应体系的优化及引物筛选,为今后利用SRAP标记技术进行海雀稗遗传多样性研究、基因定位、遗传图谱的构建以及分子标记辅助育种提供技术支持。

关键词: 海雀稗, SRAP标记, 正交实验, 体系优化, 引物筛选

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