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为更好地将ISSR标记应用于番石榴种质研究,以“维邦2号”番石榴DNA为筛选体系试验材料,采用单因子试验对ISSR-PCR反应中Mg~(2+)浓度、dNTPs浓度、引物浓度、Taq酶浓度、DNA浓度进行了优化;选用引物UBC810对9份番石榴种质进行PCR反应,选取“南宁本地番石榴实生2号”DNA作为模板对8条ISSR引物进行PCR反应,对优化扩增体系进行验证。结果表明,番石榴20μL最适ISSR-PCR反应体系为1.0mmol/L Mg~(2+),0.25mmol/L dNTPs,0.8μmol/L引物,0.2U Taq酶,10ng的DNA(2.0μL 10×Buffer,加入适量ddH2O至20μL)。验证试验得到的扩增产物条带多态性丰富,且特异性强、重复性好。试验确立的最适ISSR-PCR反应体系适用于番石榴的ISSR分子标记。
In order to better apply the ISSR marker to the study of guava germplasm, we used single factor test to test the effect of Mg 2+ concentration in ISSR-PCR , DNTPs concentration, primer concentration, Taq enzyme concentration and DNA concentration were optimized. Nine primers were used to detect the germplasm of guava germplasm. The primers of “Nanning native Guava 2” Primer PCR reaction, to optimize the amplification system validation. The results showed that 20μL optimal ISSR-PCR reaction system for guava was 1.0mmol / L Mg 2+, 0.25mmol / L dNTPs, 0.8μmol / L primer, 0.2U Taq enzyme and 10ng DNA Buffer, add appropriate amount of ddH2O to 20 μL). The amplification products obtained by the validation experiment are rich in band polymorphism with strong specificity and good repeatability. The optimal ISSR-PCR reaction system established by the experiment is suitable for the ISSR molecular marker of guava.