正交设计优化莲雾ISSR-PCR反应体系

摘要

An optimization experiment was conducted on the waxapple cultivars, Nong Ke NO 1. Genomic DNA was extracted from the young leaves using the modified CTAB method. Orthogonal design was applied to optimize ISSR-PCR amplification of the waxapple with 5 factors, including Mg2+ , dNTPs, primer, Taq DNA polymerase and template DNA, at 4 levels. An optimal reaction system (25 μL) was established, i. e. , 2. 0 μL 10X buffer (Mg2+ free), 3. 0 mmol · L-1 Mg2+ , 0. 2 mmol · L-1 dNTPs, 0. 4 μnol · L-1 primer, 1. 5 U Taq DNA polymerase and 30 ng template DNA. Waxapple cultivars, Nong Ke NO 1 and Nong Ke NO 2, were used to challenge the optimized reaction system and select primers. The results indicated that the system was stable and 12 primers were selected.%以莲雾品种‘农科一号’为试材,采用经改良的CTAB法提取莲雾叶片的DNA,探讨了Mg2+、dNTPs、引物浓度,Tuq聚合酶、模板DNA含量对莲雾ISSR PCR的影响.建立了总体积为25 μL的莲雾ISSR-PCR反应体系:Mg2+浓度为3.0 mmol·L-1、dNTP浓度为0.2 mmol·L-1、primer浓度为0.4 μmol·L-1、Taq DNA聚合酶1.SU、模板DNA含量为30 ng,并含2.0μL 10×PCR Buffer(Mg2+ free).应用该反应体系对‘农科一号’和‘农科二号’进行ISSR-PCR扩增,共筛选出12条扩增稳定、多态性丰富的ISSR引物.