均匀设计优化兴安落叶松ISSR-PCR反应体系

Optimization of ISSR-PCR Amplification System for Larix gmelinii by Uniform Design

采用均匀设计方法,对影响兴安落叶松ISSR-PCR体系的MgCl2、dNTPs、引物、TaqDNA聚合酶和模板5个组分的浓度进行了U16(45)和U12(35)两轮优化,建立了适合于兴安落叶松的ISSR-PCR反应体系。该优化的20μL反应体系包含MgCl23.0 mmol/L、dNTPs 0.15 mmol/L、引物0.5μmol/L、TaqDNA聚合酶1.0U、模板20ng/20μL。并进一步进行梯度退火试验,找到了最适的兴安落叶松ISSR退火温度为56.5℃。利用所确立的体系对部分兴安落叶松种质进行扩增的结果清晰可靠,多态性好。

Uniform design was applied to optimize ISSR-PCR system of Larix gmelinii. Five main factors influencing on ISSR-PCR, including MgCl2, primer, dNTP, Taq DNA polymerase and DNA template were examined using uniform design U16 （45） and U12 （35）. A 20μL ISSR-PCR system suitable for Larix gmelinii was established. The ISSR-PCR amplification reaction system was made up of 3.0 mmol/ L MgCI2, 0. 15 mmoL/L dNTPs, 0.51μmol/L primer, 1.00 U Taq DNA polymerase and 20ng/μL template. Annealing temperature was further researched and found that the optimal annealing temperature for ISSR-PCR reaction was 56. 5℃. Application of this optimum reaction system to fingerprint some Lar/x gmelinii accessions produced clear polymorphic patterns.