东方百合试管苗ISSR-PCR体系优化及引物筛选

Optimization of ISSR-PCR Reaction System and Primer Selection for Test Tube Seedlings of Oriental Lily

为了对东方百合杂交育种获得的大量杂种后代进行分子标记早期鉴定,本研究采用单因素试验方法对模板DNA、Mg2+、Taq DNA聚合酶、d NTPs、引物等因子设置不同浓度梯度,建立了东方百合试管苗叶片ISSR-PCR最佳反应体系。反应体系为20μL,内含1×PCR Buffer、模板DNA 20 ng、Mg2+2.0 mmol/L、Taq DNA聚合酶0.7 U、d NTPs 0.3 mmol/L、引物0.4μmol/L。PCR扩增程序采用Touchdown-PCR程序。利用优化后的体系对46条ISSR引物进行筛选,最终筛出3A37、3A61、UBC840、UBC841、UBC844等5条多态性高,重复性好的引物,且引物序列以二核苷酸重复序列为主。本研究可为利用ISSR分子标记开展东方百合杂种真实性鉴定提供技术方法和支持。

In order to originally identify the molecular marker of massive hybrid offspring which derived from oriental lily hybrid breeding, the effects of various concentrations on DNA template, Mg^2＋, Taq DNA polymerase, dNTPs and primers were investigated by single factor experimental method. The optimal ISSR-PCR reaction system of the oriental hybrid lily tube seedlings was established. Moreover, the total reaction system contained solution of 20μL, including 1×PCR Buffer, 20 ng DNA template, 2.0 mmol/L Mg^2＋, 0.7 U Taq DNA polymerase, 0.3 mmol/L dNTPs and 0.4 μmol/L primers. Finally, touchdown PCR program was applied in the PCR amplification procedure. The optimized system was used to screen the 46 ISSR primers, and the results indicated that 5 primers of 3A37, 3A61, UBC840, UBC841 and UBC844 have high polymorphism and reproducible properties. It was also found that primer sequence is mainly of dinucleotide repeat sequences. These findings create a new technological method which has potential applications in identification of hybrid offspring by the way of ISSR molecular marker.