栓皮栎ISSR反应体系的建立  

Establishment of ISSR Reaction System in Quercus variabilis

作　　者：李娜[1] 张存旭[1] 崔晓燕[1] 蒋鸿刚 郭乐[1] 刘伟[1] 

机构地区：[1]西北农林科技大学林学院,陕西杨陵712100

出　　处：《北方园艺》2014年第13期93-97,共5页Northern Horticulture

基　　金：林业公益性行业专项资助项目(21004011);西北农林科技大学唐仲英育种基金资助项目

摘　　要：以栓皮栎叶片为试材,采用单因素试验设计对栓皮栎ISSR反应条件进行系统优化,建立了栓皮栎ISSR的最佳反应体系和扩增程序。结果表明：PCR反应体系总体积为25μL,其中10×Buffer 2.5μL,30ng模板DNA,1.5UTaq聚合酶,2.5mmol/L Mg2＋,0.6μmol/L引物,0.3mmol/L dNTPs;扩增程序：94℃预变性1min;94℃变性30s,51.5～59.0℃退火60s,72℃延伸2min,35个循环;最后72℃延伸5min。在此条件下从100条ISSR引物中筛选出12条多态性好、稳定性高的引物对陕西省天然群体共22个栓皮栎个体的DNA进行扩增,共得到166个位点,其中142个为多态位点,多态位点百分率为85.54%,Nei指数为0.2928,Shannon信息指数为0.4381,表明栓皮栎的遗传多样性处于较高水平。ISSR reaction conditions of Quercus variabilis were optimized systematically via single-factor experiment by using the tender leaves of Quercus variabilis as test materials,resulting in establishment of optimal ISSR reaction system and thermal cycling conditions of Quercus variabilis.The results showed that the optimal ISSR reaction system included a total volume 25μL containing 2.5μL 10×Buffer,30ng template DNA,1.5UTaq DNA polymerase,2.5mmol/L MgCl2,0.6μmol/L primer,0.3mmol/L dNTPs.Thermal cycling conditions were as follows：denaturation 1min at 94℃;35cycles of 30sat 94℃,60sat 51.5～59.0℃,2min at 72℃;and a final extension of 5min at 72℃at the end of the amplification.Based on the optimized reaction system,12primers with superior stability and polymorphism were selected from 100ISSR primers.166bands were amplified in 22individuals of the natural population in Shaanxi Province,among which 142were polymorphic loci,the percentage of which was 85.54%,Nei＇s index 0.2928and Shannon＇s information index was 0.4381,indicating the relatively higher genetic diversity within population of Quercus variabilis.

关 键 词：栓皮栎 ISSR 体系优化 遗传多样性 

分 类 号：S792.189[农业科学—林木遗传育种]