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Bulletin of Botanical Research ›› 2021, Vol. 41 ›› Issue (2): 312-320.doi: 10.7525/j.issn.1673-5102.2021.02.019

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Establishment and Optimization of SSR-PCR Reaction System for Casuarina

Zhen LI1,2, Chong-Lu ZHONG1, Yong ZHANG1(), Yong-Cheng WEI1, Jing-Xiang MENG1   

  1. 1.Institute of Tropical Forestry,Chinese Academy of Forestry,Guangzhou 510520
    2.Nanjing Forestry University,Nanjing 210037
  • Received:2020-07-03 Online:2021-03-20 Published:2021-01-05
  • Contact: Yong ZHANG E-mail:zhangyonggritf@caf.ac.cn
  • About author:LI Zhen(1991—),male,Ph.D candidates,engaged in research of forest germplasm resources and evaluation.
  • Supported by:
    A Specific Program for National Non-profit Scientific Institutions(CAFYBB2018ZB003);National Natural Science Foundation of China(31770716)


In order to obtain the optimal reaction medium of SSR-PCR from casuarina, 24 somaclonal clones from 4 species including Casuarina equisetifoliaC.junghuhnianaC.glauca and C.cunninghamiana, were selected as experimental materials. L16(45) orthogonal design was used to determine the optimum concentrations of four factors(primer, Mg2+, dNTP, Taq polymerase) within four concentration levels in the SSR-PCR reaction system of Casuarina respectively, and visual analysis and analysis of variance were used as evaluation. The results showed as follows: The three factors(primer, Mg2+, dNTP) had significant differences on the SSR-PCR reaction systems(P<0.01) respectively, and the order of significance was primer>Mg2+>dNTP, while Taq polymerase had no significant difference. The two optimal SSR-PCR reaction systems(10 μL) of Casuarina were determined, including system 6, which consisted of 1×PCR buffer, 2ng template DNA, 0.5 μmol·L-1 primer, 1.5 mmol·L-1 Mg2+, 0.1mmol·L-1 dNTP, 0.5 U Taq polymerase, and system 15, which contained 1×PCR buffer, 2 ng template DNA, 0.5 μmol·L-1 primer, 1.75 mmol·L-1 Mg2+, 0.2 mmol·L-1 dNTP, 1.25 U Taq polymerase. The system 6 was better than system 15. 60℃ was determined as the optimal annealing temperature of the fluorescent primers M26 and M36.

Key words: SSR-PCR reaction system, orthogonal design, Casuarina

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