Establishment of ISSR-PCR Reaction System and Primer Selection for Genetic Analysis on Susceptibility of Pinus armandii to Cronartium ribicola

A study was carried out to set up ISSR-PCR rection system and to screen out right primers for identifying the Pinus armandii plants infected by Cronartium ribicola through parallel reaction between the infested group and the healthy one both sampled from 3 blister rust prevalent sites in Yunnan Province. It was showed that the optimal ISSR-PCR reaction system was in 25 μL total volume, including 2.5 μL 10×buffer mix kit, Taq DNA polymerase 1.5U, primer 2.0 μM, genomic DNA template 30 ng, MgCl₂ 2.5 mM, dNTP 250 μM, and ddH₂O 10.2 μL. The optimum amplification condition was:pre-denature at 94℃ for 5 min, denature at 94℃ for 30 s, annealing at 47℃ for 45 s, extension at 72℃ for 2 min, 40 cycles, last extension at 72℃ for 10 min. 10 ISSR primers were preliminarily screened out by which clear and steady polymorphism amplification could be obtained between the susceptive and the healthy genomic DNA pools. These ISSR primers might provide the further researches with genetic markers on identifying the differences among Pinus armandii populations.