摘要
A single factor design was applied to optimize five factors influencing SRAP system, including Taq DNA polymerase, template DNA concentration, dNTPs, primer and Mg2＋, each at four levels. The optimal SRAP-PCR system for Lonicera caerulea L. was 20 ktL SRAP-PCR amplification reaction solution containing 2.0 μL 10×PCR buffer, 1.0 U Taq DNA polymerase, 30 ng template DNA, 0.2 mmol·L-1 dNTPs, 2.0 mmol·L-1 Mg2＋ and 0.2μmol·L-1 primer. The suitable amplification procedure consisted of an initial denaturation at 94℃ for 5 min; denaturation at 94℃ for 1 min, annealing at 35℃ for 1 rain, extension at 72℃ for 90 s and in total five cycles; denaturation at 94℃ for 1 min, annealing at 50℃ for 1 min, extension at 72℃ for 90 s and in total 35 cycles; extension at 72℃ for 8 rain; preservation at 4℃. The procedures and systems could meet the demand for SRAP amplification of Lonicera caerulea L. and would play an important role in Lonicera caerulea L. germplasm identification and genetic diversity analysis.
A single factor design was applied to optimize five factors influencing SRAP system, including Taq DNA polymerase, template DNA concentration, dNTPs, primer and Mg2＋, each at four levels. The optimal SRAP-PCR system for Lonicera caerulea L. was 20 ktL SRAP-PCR amplification reaction solution containing 2.0 μL 10×PCR buffer, 1.0 U Taq DNA polymerase, 30 ng template DNA, 0.2 mmol·L-1 dNTPs, 2.0 mmol·L-1 Mg2＋ and 0.2μmol·L-1 primer. The suitable amplification procedure consisted of an initial denaturation at 94℃ for 5 min; denaturation at 94℃ for 1 min, annealing at 35℃ for 1 rain, extension at 72℃ for 90 s and in total five cycles; denaturation at 94℃ for 1 min, annealing at 50℃ for 1 min, extension at 72℃ for 90 s and in total 35 cycles; extension at 72℃ for 8 rain; preservation at 4℃. The procedures and systems could meet the demand for SRAP amplification of Lonicera caerulea L. and would play an important role in Lonicera caerulea L. germplasm identification and genetic diversity analysis.
基金
Supported by Postdoctoral Science-research Foundation (LBH-Q09170)
