百山祖冷杉的ISSR分析优化和遗传多样性初步研究

Optimization of inter simple sequence repeats (ISSR) analysis as applied to preliminary study of genetic variation in Abies beshanzuensis M. H. Wu.

以百山祖冷杉DNA为材料,分析了DNA浓度、Mg2+浓度、dNTP浓度、Taq酶的含量以及退火温度对ISSRPCR扩增结果的影响,经过优化实验建立了百山祖冷杉ISSRPCR最佳反应体系.25μL的PCR反应液含有的组分和终浓度分别为约40ng模板DNA,1.2UTaq酶,0.4μmol/L引物,1.5mmol/LMgCl2,0.2mmol/LdNTPs,2%去离子甲酰胺.利用优化反应体系从100个ISSR引物中,筛选出了13个稳定性高、重复性好的引物,对40个百山祖冷杉个体材料的DNA进行正式扩增,共扩增得到91条带,其中39条为多态性条带,多态位点百分率(PPB)为42.86%.百山祖冷杉的3个群体遗传多样性比较分析表明,人工辅助授粉产生的实生苗群体(PPB=37.36%)遗传多样性明显高于老树群体(PPB=16.48%)和嫁接苗群体(PPB=20.88%),这说明了人工辅助授粉繁育措施能有效提高百山祖冷杉的遗传变异水平.

Factors which affect the ISSR analysis of Abies beshanzuensis,such as annealing temperature and concentrations of template DNA,Taq DNA polymerase,Mg^2+ and dNTP,etc. were examined. The optimal ISSR-PCR reaction system (25μL) in our experiments was as the following: 1×Taq buffer,40 ng template DNA,1.2 U Taq DNA polymerase,0.4μmol/L primer,2.0mmol/L MgCl_2,0.2mmol/L dNTPs and 2% formamide. Of the 100 primers subjected to screening, 13 produced highly reproducible and polymorphic bands. Using these primers, 91 discernible DNA fragments were generated with 39(42.86%)being polymorphic bands, indicating the existence of low genetic variation in the species. The level of genetic variation in population originated from seedlings (PPB=37.36%) was higher than that in population of old trees (PPB=16.48%) and population of graft-originated (PPB=20.88%), which suggested that cross-pollination by human beings was helpful to increasing genetic diversity of A. beshanzuensis.