玉树地区独一味ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Yushu Lamiophlomis rotata(Benth.) Kudo

为了建立玉树地区独一味的简单重复序列区间聚合酶链式反应(ISSR-PCR)体系并筛选出引物,采用Mg2+、DNATaq酶、dNTP、模板DNA和引物进行5因素4水平正交实验,对独一味ISSR-PCR反应体系进行筛选。结果表明:最佳反应体系(25μL)为:10×PCR Buffer,75ng模板DNA,0.75μmol/L引物,0.5UTaq DNA聚合酶,1.25 mmol/L MgCl2,0.05 mmol/LdNTP。然后确定了引物的最佳退火温度,并筛选出811、818、825、826、834、835、836、840这8条丰富多态稳定扩增的引物。该研究建立的玉树地区独一味ISSR反应体系具有稳定、清晰以及重复性好等特点,可为该区独一味的繁育系统和遗传多样性的进一步研究提供参考。

To establish the optimized Inter-Simple Sequence Repeat Polymerase Chain Reaction （ISSR-PCR） reaction system of Yushu Larniophlomis rotata （Benth.） Kudo and screen primers, the orthogonal design test was used to optimize the ISSR-PCR amplification system on Larniophlomis rotata （Benth.） Kudo by five factors such as Mg2＋ ,DNA Taq polymerase, dNTP,DNA template and primer at four levels. The results showed that the optimal reaction system （25 μL） was as following：10×PCR Buffer Tango ,75 ng template DNA,0. 75 μmol/L primer,0. 5 U Taq DNA polymerase, 1.25 mmol/L MgCl2 and 0. 05 mmol/L dNTP. Then,the optimal annealing temperature of primers was investigated and8 effective primes （811,818,825,826,834,835,836,840） were selected out. In this study, the ISSR-PCR reaction system for Yushu Lamiophlomis rotata （Benth.） Kudo could provide reliable reaction,clear bands and abundant polymorphisms that facilitate the further research of mating system and genetic diversity.