ISSR-PCR Optimization and Primer Selection for Analyzing <i>Exserohilum turcicum</i> Isolates Collected in Fujian

DAI Yu-li; GAN Lin; RUAN Hong-chun; YANG Jing-min; SHI Niu-niu; DU Yi-xin; CHEN Fu-ru; YANG Xiu-juan

doi:10.19303/j.issn.1008-0384.2019.07.009

Volume 34 Issue 7

Jul. 2019

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Article Navigation > Fujian Journal of Agricultural Sciences > 2019 > 34(7): 810-817

DAI Yu-li, GAN Lin, RUAN Hong-chun, YANG Jing-min, SHI Niu-niu, DU Yi-xin, CHEN Fu-ru, YANG Xiu-juan. ISSR-PCR Optimization and Primer Selection for Analyzing Exserohilum turcicum Isolates Collected in Fujian[J]. Fujian Journal of Agricultural Sciences, 2019, 34(7): 810-817. doi: 10.19303/j.issn.1008-0384.2019.07.009

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DAI Yu-li, GAN Lin, RUAN Hong-chun, YANG Jing-min, SHI Niu-niu, DU Yi-xin, CHEN Fu-ru, YANG Xiu-juan. ISSR-PCR Optimization and Primer Selection for Analyzing Exserohilum turcicum Isolates Collected in Fujian[J]. Fujian Journal of Agricultural Sciences, 2019, 34(7): 810-817. doi: 10.19303/j.issn.1008-0384.2019.07.009

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ISSR-PCR Optimization and Primer Selection for Analyzing Exserohilum turcicum Isolates Collected in Fujian

doi: 10.19303/j.issn.1008-0384.2019.07.009

1.
Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China
2.
Jian'ou Municipal Bureau of Agriculture and Rural Affairs, Jian'ou, Fujian 353100, China

Received Date: 2019-04-09
Rev Recd Date: 2019-07-03
Publish Date: 2019-07-20

Abstract

Abstract

Objective To determine the optimal reaction conditions and primers for the inter-simple sequence repeat (ISSR) PCR in analyzing isolates of Exserohilum turcicum, a pathogen of the northern corn leaf blight in Fujian. Method Conditions for the ISSR-PCR amplification including Taq polymerase dosage, concentrations of dNTPs, Mg²⁺ and template DNA and reaction cycle as well as annealing temperatures for selected primers were optimized using a single-factor method. Result For a 25 μL ISSR-PCR genetic diversity analysis on E. turcicum, the following conditions were applied:0.55 U of Taq polymerase, 0.30 mmoL·L^-1 of dNTPs, 1.30 mmoL·L^-1 of Mg²⁺, 100 ng of template DNA, and 10 pmol of primer under 94℃ for 4 min followed by 35 cycles of 45 s at 94℃, 45 s at 51.2-56.0℃, and 1.5 min at 72℃, and finally, at 72℃ for 10 min. Ten stable, polymorphic primers, i.e., UBC117, UBC118, UBC808, UBC835, UBC847, UBC855, UBC856, UBC857, UBC866, and UBC887, were selected from 56 ISSR primers, with their optimized annealing temperatures determined to be 55.6, 53.1, 51.2, 51.2, 56.0, 53.1, 53.1, 51.2, 51.2, and 51.2℃, respectively. Regardless of their geographical origins, the 21 E. turcicum isolates differed on DNA polymorphism indicating an abundance on genetic diversity among them. Conclusion The optimized ISSR-PCR reaction conditions and primers could be applied for genetic studies on E. turcicum in Fujian.
- Exserohilum turcicum,
- reaction conditions,
- inter-simple sequence repeat,
- northern corn leaf blight,
- genetic diversity

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References(30)

References

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