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[Objective] To establish and optimize the ISSR-PCR system satiable for Nerviliae fordii(Hance) Schltr. [Methods]Five factors affecting the ISSR-PCR system reaction of N. fordii were optimized by orthogonal test,which were d NTP,template DNA,primer,Mg2 +and Taq DNA polymerase. Single factors in the test were analyzed by new multiple range method. [Results] The optimal reaction system for N. fordii was as follows: the 20 μL total reaction volume included 2 μL 10 × PCR buffer,225 μmol / L d NTP,60 ng template DNA,0. 4μmol /L primer,2. 5 mmol /L Mg2 +,1. 0 U Taq DNA polymerase,and 20 μL total reaction volume. A total of 16 ISSR primers were screened from 100 ISSR primers,which was stable and had abundant polymorphism. [Conclusions] The established ISSR-PCR reaction system for N. fordii was stable and reliable,and could be used for the genetic differentiation analysis of N. fordii.
[Objective] To establish and optimize the ISSR-PCR system satiable for Nervilia fordii (Hance) Schltr. [Methods] Five factors affecting the ISSR-PCR system reaction of N. fordii were optimized by orthogonal test, which were dNTP, template DNA , primer, Mg2 + and Taq DNA polymerase. Single factors in the test were analyzed by the new multiple range method. [Results] The optimal reaction system for N. fordii was as follows: the 20 μL total reaction volume included 2 μL 10 × PCR buffer, 225 μmol / L d NTP, 60 ng template DNA, 0.4 μmol / L primer, 2.5 mmol / L Mg 2+, 1.0 U Taq DNA polymerase, and 20 μL total reaction volume. primers were screened from 100 ISSR primers, which was stable and had abundant polymorphism. [Conclusions] The established ISSR-PCR reaction system for N. fordii was stable and reliable, and could be used for the genetic differentiation analysis of N. fordii.