Establishment of ISSR-PCR Reaction System of Illicium verumand Selection of Primers
Journal of West China Forestry Science(2013)
Abstract
Based on summarizing related researches of ISSR-PCR systems of other tree species,experiments were designed and conducted to establish the ISSR-PCR reaction systems and to screen for primers.The experiment took leaves of Illicium verum from 6 individuals of 2 populations from Yunnan Wenshan and Guangxi as materials and genomic DNA of I.verum leaf as the template.A reaction system was established to screen ISSR primer library and optimize specific primers combining with orthogonal design.The results indicated that the optimized reaction system of 25 μL included 0.003~0.016 ng template DNA,0.5~1.0 U Taq DNA polymerase,0.2 mmol·L-1 dNTPs,2.5~3.0 mmol·L-1MgCl2,0.6~0.8 μmol l·L-1 primer.Amplifications were performed with the following programs: 94℃ for 5 min;40 cycles of 94℃ for 30 s,primer-dependent annealing temperature for 45 s,72℃ for 1 min;a 7-min extension at 72℃;4℃ conservation.Thirteen stable polymorphism primers were selected out.The research result would play an important role in further research of genetic diversity and identification of germplasm of I.verum.
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Key words
amplification,reaction system,Illicium verum,primer selection,DNA template,ISSR-PCR
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