Establishment and Optimization of ISSR Reaction System of Broussonetia papyriferaz

LIAO Sheng-xi; CUI Kai; ZHANG Peng; WANG Hai-ying; CUI Yong-zhong; YUAN Shou-qian

The effects of five factors, such as the concentration of Taq DNA polymerase, primers, dNTPs, Mg2+ and DNA template, on ISSR-PCR reaction were optimized by orthogonal tests. Thus a suitable ISSR-PCR reaction system for Broussonetia papyrifera was established. The optimized ISSR-PCR system for B. papyrifera in 20 μL reaction mixture contained 0.07 U·μL-1 Taq DNA polymerase, 0.35 μmol·L-1 ISSR primers, 0.3 mmol·L-1 dNTPs, 2.0 mmol·L-1 Mg2+ and 1.2 ng·μL-1 DNA template. The suitable PCR protocol is preliminary denaturation at 94℃ for 5 min, 40 cycles of denaturation at 94℃ for 30 s, anneal at 46℃ for 45 s, extended at 72℃ for 2 min. This study could provide some references for the genetic diversity analysis of B. papyrifera.

1. Research Institute of Resources Insects, Chinese Academy of Forestry, Kunming 650224, Yunnan, China

2. Faculty of Natural Resources, Southwest Forestry University, Kunming 650224, Yunnan, China

Received Date: 2012-08-10

Abstract: The effects of five factors, such as the concentration of Taq DNA polymerase, primers, dNTPs, Mg2+ and DNA template, on ISSR-PCR reaction were optimized by orthogonal tests. Thus a suitable ISSR-PCR reaction system for Broussonetia papyrifera was established. The optimized ISSR-PCR system for B. papyrifera in 20 μL reaction mixture contained 0.07 U·μL-1 Taq DNA polymerase, 0.35 μmol·L-1 ISSR primers, 0.3 mmol·L-1 dNTPs, 2.0 mmol·L-1 Mg2+ and 1.2 ng·μL-1 DNA template. The suitable PCR protocol is preliminary denaturation at 94℃ for 5 min, 40 cycles of denaturation at 94℃ for 30 s, anneal at 46℃ for 45 s, extended at 72℃ for 2 min. This study could provide some references for the genetic diversity analysis of B. papyrifera.

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Reference (17)

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Establishment and Optimization of ISSR Reaction System of Broussonetia papyriferaz

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