Establishment and Optimization of ISSR Reaction System for Valeriana officinalis L. var. latifolia Miq of Guizhou Province

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.011

Abstract

Abstract: This study is to establish a stable and reliable ISSR system for Valeriana officinalis L.var. latifolia Miq. Based on the single factor experiment in 10 levels, L₁₆(4⁵)orthogonal design was applied to conduct combinatorial optimization in 4 levels of 4 major factors, and the results were analyzed by visual method, range analysis and variance method. The primer and the optimal annealing temperature were screened by optimized reaction system. The stability and reliability of this system were tested by 9 samples from different origins. The optimal ISSR conditions in the experiments were as following: in 25 μL reaction system containing 2.5 μL 10×Taq Buffer, 2.0 mmol/L Mg²⁺, 0.28 mmol/L dNTPs, 0.3 μmol/L primer, 1.75 U Taq DNA polymerase, 60 ng DNA template, and ddH₂O completed. The order of the effect by the factors was in Mg²⁺ > primer > dNTP s > Taq DNA polymerase. The optimal annealing temperature for each primer was determined, and 17 primers with distinct and stable amplified bands were screened. Stability test showed that the optimized system was stable and reliable. By this study we may reach the conclusion that the optimal ISSR reaction system for V. officinalis var. latifolia was generated, and the desired primers also were screened, which may provide the technical foundation for the application of ISSR molecular marker technology in the study of genetic diversity of V. officinalis var. latifolia.

Key words: Valeriana officinalis L. var. latifolia Miq, ISSR, single factor experiment, orthogonal design, optimization

Qian Zhiyao, Zhou Daotang, Huang Xiuping, Zhou Mei, Chen Zuyun, Qin Ronggui. Establishment and Optimization of ISSR Reaction System for Valeriana officinalis L. var. latifolia Miq of Guizhou Province[J]. Biotechnology Bulletin, 2015, 31(7): 69-75.

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