摘要
用采自云南华山松疱锈病3个典型发病点的华山松植株样品,组成健康株组和感病株组进行ISSR-PCR反应体系的建立和引物筛选.构建的华山松ISSR-PCR最优反应体系为:反应总体积25μL,其中含10×Buffer2.5μL,Taq DNApolymerase 0.3μL(终浓度1.5 U),Primer2.0μL(终浓度2.0μmol/L),DNA Template 5.0μL(终浓度1.5 ng/μL),Mg2+2.5μL(终浓度2.5 mmol/L),dNTP2.5μL(终浓度250μmol/L),ddH2O10.2μL.扩增反应程序为:94℃预变性5 min,94℃变性30 s,47℃左右退火45 s,72℃延伸2 min,40个循环,最后在72℃F继续延伸10 min.通过初筛和中试筛选出扩增反应清晰、扩增稳定的10个引物作扩大实验,在健康株组和感病株组的两个DNA pool之间有明显的PCR扩增条带多态性.筛选出的ISSR引物可提供研究华山松种群差异的遗传标记.
A study was carried out to set up ISSR-PCR rection system and to screen out fight 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, MgC12 2.5 mM, dNTP 250 μM, and ddH20 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.
出处
《西南林学院学报》
2007年第2期41-45,共5页
Journal of Southwest Forestry College
基金
国家自然科学基金资助项目(30260089)
云南省森林培育学重点学科资助项目
