荷花品种ISSR-PCR反应体系的优化
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摘要
以荷花品种红苔莲的DNA为材料,引物UBC 811为单因素的固定引物,对影响ISSR-PCR扩增的重要因子进行单因素的优化设计,建立荷花品种ISSR-PCR最佳反应体系:25μL反应体系中含2.50μL 10×buffer、0.75U Taq DNA聚合酶、0.20mmol/L dNTPs、2.50mmol/L MgCl_2、0.25μmol/L引物、10~60ng/μL模板DNA。将最佳体系应用于21个荷花品种,重复性强,稳定性高。
The influencing factors of ISSR reaction system were studied by using genomic DNA of different lotus cultivars in Nelumbo nucifera Gaertn.ISSR-PCR optimized reaction systems were eatablished for lotus cultivars.With the total volume of 25μL were included 2.5μL 10× buffer,0.75 Uunit of DNA polymerase,0.2 mmol/L dNTPs,2.5 mmol/L MgCl_2,0.25μmol/L primer and 10-60 ng template DNA.Twenty-one different lotus cultivars in Nelumbo nucifera Gaertn.were used to check the stabilization of the optimized reaction system.The study results showed that the optimal reaction condition of lotus ISSR-PCR was stable,repeatable and effective.
The influencing factors of ISSR reaction system were studied by using genomic DNA of different lotus cultivars in Nelumbo nucifera Gaertn.ISSR-PCR optimized reaction systems were eatablished for lotus cultivars.With the total volume of 25μL were included 2.5μL 10× buffer,0.75 Uunit of DNA polymerase,0.2 mmol/L dNTPs,2.5 mmol/L MgCl_2,0.25μmol/L primer and 10-60 ng template DNA.Twenty-one different lotus cultivars in Nelumbo nucifera Gaertn.were used to check the stabilization of the optimized reaction system.The study results showed that the optimal reaction condition of lotus ISSR-PCR was stable,repeatable and effective.
引文
[1]陈俊愉主编.中国花卉品种分类学[M].北京:中国林业出版社,2001.
[2]王其超,张行言.中国荷花品种图志[M].北京:中国林业出版社,2005.
[3]张行言.中国荷花新品种图志Ⅰ[M].北京:中国林业出版社,2011.
[4]Zietkiewicz,E.,Rafalski,A.,Lubuda D.Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplified[J].Genome,1994,20:176-183.
[5]田艳伶,李志辉,杨模华,等.钩栗ISSR-PCR反应体系的建立与优化[J].中南林业科技大学学报,2015,35(2):32-37.
[6]任风鸣,胡开治,刘燕琴,等.传统中药金钱草ISSR-PCR反应体系的正交优化研究[J].中国中药杂志,2014,39(12):2 233-2 238.
[7]刁松锋,邵文豪,姜景民,等.多用途树种无患子ISSR-PCR体系建立与检测[J].广西植物,2015,35(6):899-904.
[8]Pradeep Reddy M,Sarla N,Siddiq E A.Inter simple sequence repeat(ISSR)polymorphism and its application in plant breeding[J].Euphytica,2002,128:9-17.
[9]桂腾琴,伍伟,卢鹏,等.荷花品种不同部位DNA提取的比较研究[J].种子,2016,35(7):41-44.
[10]邹喻苹,葛颂,王晓东.系统与进化植物学中的分子标记[M].北京:科学出版社,2001.
[11]桂腾琴,乔爱民,陆春连,等.梨品种ISSR-PCR反应体系的优化与应用[J].广东农业科学,2013,24(12):117-120.
[12]宋江华,赵颖,汪承刚,等.乌塌菜ISSR-PCR反应体系的建立及优化[J].植物研究,2013,33(2):238-242.
[13]唐辉,陈宗游,史艳财,等.正交设计优化地枫皮ISSR-PCR反应体系[J].中草药,2013,44(5):610-615.
[14]赵振华,严萍,焦旭雯,等.何首乌ISSR-PCR反应体系的建立与优化[J].时珍国医国药,2008,5(8):1 361-1 365.
[15]乔玉山,章镇,房经贵,等.李种质资源ISSR反应体系的建立[J].果树学报,2003,20(4):270-274.
[16]冯夏连,何承忠,张志毅,等.毛白杨ISSR反应体系的建立及优化[J].北京林业大学学报,2006,28(3):61-65.
[17]李立升.21个荷花品种遗传多样性的ISSR分析[D].郑州:河南农业大学,2011:20-20.
[18]范海艳,曹福祥,彭继庆,等.博白大果油茶ISSR-PCR反应体系的建立与优化[J].中南林业科技大学学报,2011,31(4):97-103.
[19]孙清信,陈坚,张辉,等.紫云英ISSR引物的筛选及PCR反应体系的优化[J].植物遗传资源学报,2012,13(5):870-878.
[20]姜静,杨传平,刘桂丰,等.桦树ISSR-PCR反应体系的优化[J].生态学杂志,2003,22(3):91-93.
[2]王其超,张行言.中国荷花品种图志[M].北京:中国林业出版社,2005.
[3]张行言.中国荷花新品种图志Ⅰ[M].北京:中国林业出版社,2011.
[4]Zietkiewicz,E.,Rafalski,A.,Lubuda D.Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplified[J].Genome,1994,20:176-183.
[5]田艳伶,李志辉,杨模华,等.钩栗ISSR-PCR反应体系的建立与优化[J].中南林业科技大学学报,2015,35(2):32-37.
[6]任风鸣,胡开治,刘燕琴,等.传统中药金钱草ISSR-PCR反应体系的正交优化研究[J].中国中药杂志,2014,39(12):2 233-2 238.
[7]刁松锋,邵文豪,姜景民,等.多用途树种无患子ISSR-PCR体系建立与检测[J].广西植物,2015,35(6):899-904.
[8]Pradeep Reddy M,Sarla N,Siddiq E A.Inter simple sequence repeat(ISSR)polymorphism and its application in plant breeding[J].Euphytica,2002,128:9-17.
[9]桂腾琴,伍伟,卢鹏,等.荷花品种不同部位DNA提取的比较研究[J].种子,2016,35(7):41-44.
[10]邹喻苹,葛颂,王晓东.系统与进化植物学中的分子标记[M].北京:科学出版社,2001.
[11]桂腾琴,乔爱民,陆春连,等.梨品种ISSR-PCR反应体系的优化与应用[J].广东农业科学,2013,24(12):117-120.
[12]宋江华,赵颖,汪承刚,等.乌塌菜ISSR-PCR反应体系的建立及优化[J].植物研究,2013,33(2):238-242.
[13]唐辉,陈宗游,史艳财,等.正交设计优化地枫皮ISSR-PCR反应体系[J].中草药,2013,44(5):610-615.
[14]赵振华,严萍,焦旭雯,等.何首乌ISSR-PCR反应体系的建立与优化[J].时珍国医国药,2008,5(8):1 361-1 365.
[15]乔玉山,章镇,房经贵,等.李种质资源ISSR反应体系的建立[J].果树学报,2003,20(4):270-274.
[16]冯夏连,何承忠,张志毅,等.毛白杨ISSR反应体系的建立及优化[J].北京林业大学学报,2006,28(3):61-65.
[17]李立升.21个荷花品种遗传多样性的ISSR分析[D].郑州:河南农业大学,2011:20-20.
[18]范海艳,曹福祥,彭继庆,等.博白大果油茶ISSR-PCR反应体系的建立与优化[J].中南林业科技大学学报,2011,31(4):97-103.
[19]孙清信,陈坚,张辉,等.紫云英ISSR引物的筛选及PCR反应体系的优化[J].植物遗传资源学报,2012,13(5):870-878.
[20]姜静,杨传平,刘桂丰,等.桦树ISSR-PCR反应体系的优化[J].生态学杂志,2003,22(3):91-93.