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杜仲SSR-PCR反应体系建立及引物筛选
朱晓敏,王弦云,陈龙灿,康向阳,王勤,束庆龙,张良富,曹翠萍
0
(安徽农业大学林学与园林学院;北京林业大学生物科学与技术学院)
摘要:
为了对杜仲种质资源的遗传多样性研究奠定理论和技术基础,采用正交试验设计和单因素分析,建立并优化了杜仲的SSR-PCR反应体系。结果表明,杜仲SSR-PCR 最适反应体系为正反向引物 (10 μmol?L-1) 各0.2 μL, Taq DNA聚合酶 (5 U?μL-1) 0.1 μL,dNTP (10 mmol?L-1) 0.2 μL,模板DNA(5~10 ng?μL-1)1.0 μL,Mg2+ (25 mmol?L-1) 0.6 μL,10×PCR缓冲液1.0 μL,HPLC超纯水6.7 μL,总体积10.0 μL。同时筛选出了3对多态性有效引物,研究结果证明了微卫星引物在不同物种间的通用性受物种间亲缘关系远近的影响,并初步揭示了杜仲的遗传多样性,表明了微卫星标记在遗传多样性研究上的优越性。
关键词:  杜仲  微卫星标记  PCR反应体系  引物筛选  遗传多样性
DOI:
基金项目:安徽高校省级自然科学重点项目 (KJ2011A115)
Establishment of SSR-PCR reaction system and primers screening for Eucommia ulmoides
ZHU Xiao-min,WANG Xian-yun,CHEN Long-can,KANG Xiang-yang,WANG Qin,SHU Qin-long,ZHANG Liang-fu,CAO Cui-ping
(School of Forestry Landscape Architecture, Anhui Agricultural University;College of Biological Sciences and Biotechnology, Beijing Forestry University)
Abstract:
To provide theoretic and technical support for molecular assessment of genetic diversity in germplasm resources of Eucommia ulmoides, simple sequence repeat-polymerase chain reaction (SSR-PCR) system was established through orthogonal test and optimized through single factor experiment. The results showed that the optimal SSR-PCR reaction system was a volume of 10.0 μL containing forward- and reversed-primer (10 μmol?L-1) 0.2 μL, respectively, Taq DNA polymerase (5 U?μL-1) 0.1 μL, dNTP (10 mmol?L-1) 0.2 μL, DNA(5-10 ng?μL-1)1.0 μL, Mg2+ (25 mmol?L-1) 0.6 μL, 10 × PCR buffer 1.0 μL and HPLC water 6.7 μL. Three SSR polymorphic primer pairs were selected. The results proved the limited transferability of SSR primers between distantly related species, revealed the genetic diversity of E. ulmoides preliminarily, and exhibited the advantages of SSRs for the investigation of genetic diversity.
Key words:  Eucommia ulmoides  SSRs  PCR reaction system  primer screening  genetic diversity

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