Establishment and Optimization of ISSR-PCR Reaction System for Yushu Lamiophlomis rotata (Benth.) Kudo(PDF)

《北方园艺》[ISSN:1001-0009/CN:23-1247/S]

Title:: Establishment and Optimization of ISSR-PCR Reaction System for Yushu Lamiophlomis rotata (Benth.) Kudo

Author(s):: WANG Jing; ZHANG Xiao-ming; QIAO Feng; JIN Lan; CHEN Zhi; LUO Gui-hua; Key Laboratory of Education Ministry on Environment and Resources of Qinghai and Tibet Plateau，School of Life and Geographical Sciences，Qinghai Normal University，Xining，Qinghai 810008

Keywords:: Lamiophlomis rotate(Benth.) Kudo; ISSR-PCR; reaction system; orthogonal design; optimization; primes screening

Abstract:: To establish the optimized Inter-Simple Sequence Repeat Polymerase Chain Reaction (ISSR-PCR) reaction system of Yushu Lamiophlomis rotata (Benth.) Kudo and screen primers，the orthogonal design test was used to optimize the ISSR-PCR amplification system on Lamiophlomis rotata(Benth.) Kudo by five factors such as Mg²⁺，DNA Taq polymerase，dNTP，DNA template and primer at four levels.The results showed that the optimal reaction system (25 μL) was as following：10×PCR Buffer Tango，75 ng template DNA，0.75 μmol/L primer，0.5 U Taq DNA polymerase，1.25 mmol/L MgCl₂ and 0.05 mmol/L dNTP.Then，the optimal annealing temperature of primers was investigated and 8 effective primes (811,818,825,826,834,835,836,840) were selected out.In this study，the ISSR-PCR reaction system for Yushu Lamiophlomis rotata(Benth.) Kudo could provide reliable reaction，clear bands and abundant polymorphisms that facilitate the further research of mating system and genetic diversity.

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Last Update: 2014-08-25