油松遗传多样性研究的ISSR-PCR体系优化与初步应用  

Optimization of the ISSR-PCR System for Genetic Diversity Research of Pinus Tabulaeformis Carr.and Its Preliminary Application

作　　者：郝真真[1] 王孟本[1] 

机构地区：[1]山西大学黄土高原研究所,山西太原030006

出　　处：《山西大学学报（自然科学版）》2009年第1期119-124,共6页Journal of Shanxi University(Natural Science Edition)

基　　金：国家自然科学基金(30570284)

摘　　要：以油松基因组DNA为模板，对影响ISSR-PCR的主要因素进行研究，建立了适宜于油松的IssR_PCR优化体系及扩增程序；并采用ISSR标记对山西灵空山和陕西洛南2个油松种群的遗传多样性进行了分析，同时将此结果与使用RAPD标记的研究结果进行了对比．结果表明，在20“L反应体系中，模板DNA、Mg”、dNTPs、TaqDNA聚合酶和引物5种主要成分的最适浓度分剐为40ng、2．5mmol·L^-1、0．20mmol·L^-1、1 U和0．5μm01．L^-1．扩增程序为：94℃预变性5min；94℃变性30S，最适温度（54～56℃）退火45s，72℃延伸2min，共计35个循环；循环结束后72℃延伸7min，4℃保存．山西灵空山和陕西洛南2个油松种群的Nei’S遗传多样性分别为0．3520和0．3514，前者的遗传多样性显著高于后者（P〈0．001）．使用ISSR标记与RAPD标记所获结果基本一致，但是ISSR标记优于RAPD标记．The genomic DNA of Pinus tabulaeformis Cart. was extracted as the inter simple sequence repeat （ISSR） template, and the influencing factors of ISSR-PCR were optimized.The the genetic diversity of two populations of this species, which were collected from Lingkong Mountain, Shanxi and Luonan, Shaanxi,was investigated using ISSR markers, and a comparison of the results from ISSR markers with those from RAPD markers was made. The results showed that the ontimal exneriment conditions for ISSRPCR were as follow：20 μL system containing 40 ng template DNA,2 μL 10×PCR Buffer （Mg^2＋ free） ,2.5mmol · L^-1 MgCl2,0.2 mmol · L^-1 dNTPs,IU Taq DNA polymerase; and 0.5 μmol · L^-1 primer. The optimal amplification program was 5 min of pre-denaturalization at 94℃ ; 35 cycles of 30s for denaturalization at 94 ℃ ,45s of anneal at 54-56℃ ,2 min of extension at 72 ℃ ; and 7 min of extension at 72℃ in the final cycle. The Nei＇s genetic diversity （h） of Lingkong Mountain and Luonan populations were 0. 352 0 and 0.351 4, respectively ; the former＇s was significantly higher than the former＇s （P〈0. 001）. The similar resuits were obtained from ISSR and RAPD markers,but ISSR was better than RAPD in terms of detecting genetic polymorphism.

关 键 词：油松 ISSR 优化 遗传多样性 

分 类 号：O943.2[理学]