WANG Jing,ZHANG Xiao-ming,QIAO Feng,et al.Establishment and Optimization of ISSR-PCR Reaction System for Yushu Lamiophlomis rotata (Benth.) Kudo[J].Northern Horticulture,2013,37(09):126-130.
玉树地区独一味ISSR-PCR反应体系的建立与优化
- Title:
- Establishment and Optimization of ISSR-PCR Reaction System for Yushu Lamiophlomis rotata (Benth.) Kudo
- 文章编号:
- 1001-0009(2013)09-0126-05
- Keywords:
- Lamiophlomis rotate(Benth.) Kudo; ISSR-PCR; reaction system; orthogonal design; optimization; primes screening
- 分类号:
- S 567.23+9
- 文献标志码:
- A
- 摘要:
- 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+、DNA Taq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（25 μL）为：10×PCR Buffer，75 ng模板DNA，0.75 μmol/L引物，0.5 U Taq DNA聚合酶，1.25 mmol/L MgCl2，0.05 mmol/L dNTP。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。
- Abstract:
- To establish the optimized Inter-Simple Sequence Repeat Polymerase Chain Reaction (ISSR-PCR) reaction system of Yushu Lamiophlomis rotata (Benth.) Kudo and screen primers，the orthogonal design test was used to optimize the ISSR-PCR amplification system on Lamiophlomis 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 and 8 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.
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备注/Memo
第一作者简介：王京（1987-)，男，安徽安庆人，在读硕士，现主要从事植物遗传多样性等研究工作。