Optimization of SSR-PCR reaction system and primer sceening in Malus crabapple

doi:10.3969/j.issn.1000-2006.201801006

HUANG Bin, NI Zhouxian, ZHOU Ting, ZHANG Wangxiang, XU Li'an

Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 2018, Vol. 42 ›› Issue (03) : 51-57.

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Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 2018, Vol. 42 ›› Issue (03) : 51-57. DOI: 10.3969/j.issn.1000-2006.201801006

Optimization of SSR-PCR reaction system and primer sceening in Malus crabapple

HUANG Bin, NI Zhouxian, ZHOU Ting, ZHANG Wangxiang, XU Li'an^*

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Abstract: 【Objective】 To explore the optimal PCR conditions for SSR molecular markers of Malus crabapples, orthogonal design was used to optimize the SSR-PCR system, and SSR primers suitable for Malus crabapples were screened using the optimized system. 【Method】Six diploid Malus crabapple DNA templates were used as material. DNA template concentration, Taq enzyme concentration, primer concentration, Mg^{2 +} ion concentration, and dNTP concentration were optimized by the L₁₆(4⁵)orthogonal test and the optimum reaction system for SSR-PCR of crabapples was established. 【Results】 The results showed that 15 μL of optimized system was composed of DNA template 5 mg/L, Taq enzyme 1.25 U, primer 0.3 μmol/L, Mg²⁺ 2 mmol/L, dNTP 0.25 mmol/L and 1×Buffer 1.5 μL. Fifteen pairs of SSR primers from Malus with clear bands, abundant polymorphisms, and good repeatability were screened out using the optimization system. Its polymorphic information content was greater than 0.25. 【Conclusion】 Orthogonal test results achieved the purpose of optimization. The 15 polymorphic primers screened by the obtained system could be directly applied to the SSR molecular marker experiment for Malus crabapples, which provided an effective tool for identification, classification and genetic breeding of Malus crabapples.

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HUANG Bin, NI Zhouxian, ZHOU Ting, ZHANG Wangxiang, XU Li'an. Optimization of SSR-PCR reaction system and primer sceening in Malus crabapple[J]. Journal of Nanjing Forestry University (Natural Sciences Edition）. 2018, 42(03): 51-57 https://doi.org/10.3969/j.issn.1000-2006.201801006

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