ISSR Reaction Optimization and Primer Selection for Species Authentication of <i>Fritillaria hupehensis</i> Hsiao et K. C. Hsia

JIANG Xiaogang; WANG Hua; GUO Kunyuan; ZHANG Meide

doi:10.19303/j.issn.1008-0384.2021.06.004

Volume 36 Issue 6

Jun. 2021

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Article Navigation > Fujian Journal of Agricultural Sciences > 2021 > 36(6): 642-650

JIANG X G, WANG H, GUO K Y, et al. ISSR Reaction Optimization and Primer Selection for Species Authentication of Fritillaria hupehensis Hsiao et K. C. Hsia [J]. Fujian Journal of Agricultural Sciences，2021，36（6）：642−650 doi: 10.19303/j.issn.1008-0384.2021.06.004

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JIANG X G, WANG H, GUO K Y, et al. ISSR Reaction Optimization and Primer Selection for Species Authentication of Fritillaria hupehensis Hsiao et K. C. Hsia [J]. Fujian Journal of Agricultural Sciences，2021，36（6）：642−650 doi: 10.19303/j.issn.1008-0384.2021.06.004

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ISSR Reaction Optimization and Primer Selection for Species Authentication of Fritillaria hupehensis Hsiao et K. C. Hsia

doi: 10.19303/j.issn.1008-0384.2021.06.004

Institute of Chinese Medicinal Materials, Hubei Academy of Agricultural Sciences/Enshi Comprehensive Test Station, National Traditional Chinese Medicine Industry Technology System/Chinese Medicinal Materials Sub-center, Hubei Agricultural Science and Technology Innovation Center, Enshi, Hubei　445000, China

Received Date: 2020-08-23
Rev Recd Date: 2021-05-24

Available Online: 2021-06-06

Publish Date: 2021-06-28

Abstract

Abstract

Objective The optimized ISSR-PCR reaction system and appropriate polymorphic primers were established to facilitate the authentication, breeding, and genetic studies on Fritillaria hupehensis Hsiao et K.C. Hsia. Method A U₁₂(4³) uniform design method combined with a single factor screening test was employed to optimize the dosages of 2×Taq Master Mix, template DNA, and primer applied for ISSR-PCR and followed by a gradient temperature experiment to determine the primer annealing temperature. Result The optimal ISSR reaction system used 10.5 μL of 2×Taq Master Mix, 0.8 μL of template DNA (40.0 ng), 2.2 μL (2.5 μmol·L⁻¹) of primers, and 6.5 uL of ddH₂O for a 5 min sequencing at 94 ℃ and 40 cycles at 94 ℃ for 0.75 min, 54.3–60.0 ℃ for 0.75 min, and 72 ℃ for 1.5 min, followed by 10 min at 72 ℃ prior to storage at 4 ℃. Six stable polymorphic primers, UBC848, UBC850, UBC853, UBC857, UBC859, and UBC866 with the optimal annealing temperatures of 59.3, 58.2, 56.9, 54.3, 59.3, and 60.0 ℃, respectively, were selected. On 12 F. hupehensis germplasms from various locations, the optimized ISSR-PCR yielded stable and reliable results that showed abundant genetic diversity. Conclusion The optimized ISSR-PCR reaction system could be satisfactorily applied for the resource authentication and genetic analysis on F. hupehensis.
- Fritillaria hupehensis Hsiao et K. C. Hsia,
- ISSR,
- molecular marker,
- system optimization

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References(25)

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