菊花iPBS分子标记的建立及优化

Establishment and Optimization of iPBS Molecular Markers in Chrysanthemum

菊花是一种重要的观赏花卉,反转录转座子在其遗传多样性形成中发挥着重要作用.iPBS分子标记技术是一种基于反转录转座子的分子标记技术,建立此标记体系可用于检测菊花品种的多样性.本研究对菊花iPBSPCR体系进行优化,得到的最佳反应体系为:引物浓度0.40μmol/L,dNTPs浓度0.40 mmol/L,Mg2+浓度1.80mmol/L,Taq酶浓度0.10U,DNA模板用量60ng,反应总体积20.00μL.反应程序为:95℃预变性3min;95℃变性15s;48℃退火1min;68℃复性1min,30个循环;最后72℃延伸5min.依据上述iPBS-PCR反应体系和程序,从25个iPBS引物中筛选得到2个多态性较高的引物,对8个菊花品种进行iPBS-PCR扩增,结果显示所选用引物扩增效果较好,可以应用于菊花种质资源材料的iPBS分子标记分析.

Chrysanthemum is an important ornamental flower,retrotransposons playing an important role in the formation of genetic diversity.IPBS molecular marker technology is a molecular marker technology based on retrotransposon.The labeling system was established to detect the diversity of chrysanthemum varieties.In this study,the chrysanthemum iPBS-PCR system was optimized.The optimal reaction system was:primer concentration 0.40μmol/L,dNTPs concentration 0.40 mmol/L,Mg2+concentration 1.80 mmol/L,the concentration of Taq enzyme was 0.10 U,the amount of DNA template was 60 ng,and the total reaction volume was 20.00μL.The reaction procedure was:pre-denaturation at 95℃for 3 min;denaturation at 95℃for 15 s;annealing at 48℃for 1 min;renaturation at 68℃for 1 min,30 cycles;and finally extension at 72℃for 5 min.According to the above iPBS-PCR reaction system and program,two primers with higher polymorphism were screened from 25 iPBS primers,and iPBS-PCR amplification of 8 chrysanthemum varieties shows that the selected primers have better amplification effect.iPBS molecular markers can be applied to analyze the chrysanthemum germplasm resources.