| 摘要: |
| 无患子(Sapindus mukorossi)是我国长江以南地区传统的重要绿化树种,其果皮富含皂苷,种仁富含油脂,为新型木本油料树种之一。为了获得基于KOD FX高保真DNA聚合酶试剂盒的无患子ISSR-PCR的最佳反应体系,该文采用正交优化设计相结合的方法,研究了引物浓度、dNTPs浓度、KOD FX酶、模板DNA浓度和Mg2+浓度对无患子ISSR-PCR反应体系的影响,并在此基础上对退火温度进一步优化,最终确立了适合无患子的ISSR-PCR反应的最佳体系和程序,即20 μL PCR反应体系中,引物0.5 μmol·L-1、dNTPs 0.05 mmol·L-1、KOD FX酶 0.06 U·μL-1、模板DNA浓度1.0 ng·μL-1和Mg2+ 1.0 mmol·L-1。当以UBC841为引物时, PCR扩增程序为94 ℃预变性2 min, 98 ℃变性10 s, 48.6 ℃退火30 s, 68 ℃延伸90 s, 35个循环, 68 ℃延伸7 min,4 ℃保存。这一优化的ISSR-PCR反应体系的建立,为无患子种质资源的遗传多样性、亲缘关系及遗传结构研究、种质创新与分子辅助育种等奠定了良好的基础。 |
| 关键词: 无患子 ISSR PCR反应体系 正交设计 |
| DOI：10.11931/guihaia.gxzw201405042 |
| 分类号: |
Fund project:收稿日期: 2014-07-21修回日期: 2014-10-11
基金项目: 国家林业公益性科研专项(201404104,200804032); 浙江省重大科技专项重点农业项目(2011C12015)。
作者简介: 刁松锋(1989-),男,安徽亳州人,硕士,主要从事林木遗传育种研究,(E-mail)stanfordiao@yeah.net。*通讯作者: 姜景民,博士,研究员,从事林木遗传育种和种质资源研究,(E-mail)zzzyjiang@yeah.net。 |
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| Detection and establishment of ISSR-PCR system for multipurpose tree species Sapindus mukorossi |
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DIAO Song-Feng1,2, SHAO Wen-Hao1, JIANG Jing-Min1*, LUAN Qi-Fu1
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1.1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, China;2.2. No-timber
Forestry Research and Development Center, Chinese Academy of Forestry, Zhengzhou 450003, China
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| Abstract: |
| Sapindus mukorossi is a traditional and important virescencetree species in the south part of China with rich saponins in peel and rich oil in seed. This tree species is one of the newly-developed woody oil species that were approved by the State Forestry Administration of China. For optimizing ISSR-PCR reaction system of S. mukorossi, based on KOD FX High-fidelity DNA Polymerase Kit, orthogonal design experiments were conducted. The main factors affecting ISSR-PCR amplification, such as suitable concentration of primer, dNTPs, KOD FX DNA polymerase, DNA template and 2×PCR buffer for KOD FX were studied. Furthermore, the annealing temperature was optimized on the base of the above tests. An ideally ISSR-PCR reaction system was established, namely 25 μL reaction system containing primer 0.5 μmol·L-1,dNTPs 0.05 mmol·L-1,KOD FX DNA polymerase 0.06 U·μL-1、DNA template 1.0 ng·μL-1 and Mg2+ 1.0 mmol·L-1. The optimal PCR amplification program was: The profile of ISSR-PCR was an initial denaturation step for 2 min 94 ℃,followed by 35 cycles of 30 s at 98 ℃ for denaturation, 30 s at 48.6 ℃ for annealing, 90 s at 68 ℃ for extension, finally extension at 68 ℃ for 7 min and holding the samples at 4 ℃. This optimized ISSR-PCR reaction system would provide the basis for the analysis of genetic diversity, genetic structure, germplasm innovation and molecular assisted selection in S. mukorossi. |
| Key words: Sapindus mukorossi ISSR PCR reaction system orthogonal design experiments |
