不同PCR程序对甜菜DAMD引物扩增效果的影响
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摘要
为了开发甜菜品种高效鉴定的DAMD-PCR分子标记技术,优化相应的检测技术流程。利用6种DAMD引物,以8个不同甜菜品种的DNA为模板,分别采用常规PCR扩增程序和touchdown PCR(TD-PCR)扩增程序对模板进行扩增。扩增产物利用8%的非变性聚丙烯酰胺凝胶电泳进行分离,经银染后观察不同PCR程序对甜菜DAMD引物扩增效果的影响。结果表明,TD-PCR扩增程序扩增效果明显好于常规PCR扩增程序,条带明亮、清晰,非特异性条带数量明显减少。推荐在甜菜DAMD引物的扩增中使用TD-PCR扩增程序。
In order to develop the DAMD-PCR molecular marker technology for high efficiency identification of sugar beet varieties, the corresponding detection process was optimized. A total of 6 DAMD primers were selected for amplification of DNA in 8 different sugar beet varieties based on both standard and touchdown PCR programs.The PCR products were separated by native PAGE. After silver stain, the effects of DAMD-PCR amplification by different PCR programs were analyzed. The results suggested that touchdown PCR had better amplification efficiency than that of standard PCR program, and the bands were bright, clear and the number of non-specific bands was significantly reduced. It is recommended for DAMD-PCR in term of sugar beet varieties identification.
In order to develop the DAMD-PCR molecular marker technology for high efficiency identification of sugar beet varieties, the corresponding detection process was optimized. A total of 6 DAMD primers were selected for amplification of DNA in 8 different sugar beet varieties based on both standard and touchdown PCR programs.The PCR products were separated by native PAGE. After silver stain, the effects of DAMD-PCR amplification by different PCR programs were analyzed. The results suggested that touchdown PCR had better amplification efficiency than that of standard PCR program, and the bands were bright, clear and the number of non-specific bands was significantly reduced. It is recommended for DAMD-PCR in term of sugar beet varieties identification.
引文
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[2]邹奕,吴则东,兴旺,等.甜菜种质资源遗传多样性研究进展[J].中国糖料,2018,40(5):73-76,80.
[3]邹奕,马龙彪,江伟,等.ISSR分子标记技术在甜菜育种中的应用[J].中国糖料,2018,40(3):75-77,80.
[4]王茂芊,张惠忠,吴则东,等.SRAP标记对甜菜抗根腐病品种的遗传多样性分析[J].中国农学通报,2017,33(11):9-13.
[5]刘华君,聂志勇,林明,等.利用ISSR标记分析20份甜菜品种的遗传多样性[J].中国糖料,2017,39(3):1-4.
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[7]马龙彪,吴则东,王茂芊,等.甜菜育种的研究进展及未来发展展望[J].中国糖料,2018,40(6):62-65.
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[10]胡建斌,张帆,李贞煌,等.黄瓜DAMD反应体系的建立及种质遗传资源研究[J].西北植物学报,2010,30(4):652-658.
[11]Métais I,Aubry C,Hamon B,et al.Assessing common bean genetic diversity using RFLP,DAMD-PCR,ISSR,RAPD AND AFLPmarkers[J].Acta Horticulturae,2001,546(546):459-461.
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[13]Ince A G,Karaca M,Onus A N.Development and utilization of diagnostic DAMD-PCR markers for Capsicum accessions[J].Genetic Resources and Crop Evolution,2009,56(2):211-221.
[14]Karaca M,Ince A G,Ay S T,et al.PCR-RFLP and DAMD-PCR genotyping for Salvia species[J].Journal of the Science of Food&Agriculture,2008,88(14):2508-2516.
[15]兴旺,童乐怡,孙燕红,等.甜菜DAMD-PCR体系的建立及优化[J].中国农学通报,2017,33(23):6-9.
[16]孙燕红,马龙彪,兴旺,等.甜菜DAMD引物的筛选及不同凝胶系统对扩增产物多态性的影响[J].中国农学通报,2018,34(16):42-45.
[17]符德欢,朱高倩,郭佳玉,等.改良CTAB法提取重楼属3种药用植物干燥根茎DNA[J].中药材,2017,40(6):1295-1299.
[18]郭培国,刘文杰,李海洋,等.一种快速有效检测SSR标记的非变性聚丙烯酰胺凝胶的银染方法[J].广州大学学报:自然科学版,2016,15(4):8-12.
[19]杨绍林,王先宏,李苏洁,等.应用降落PCR和正交设计优化甘蔗“割手密”SSR-PCR反应体系[J].分子植物育种,2016,14(2):431-436.
[20]李志强,何德,李翠新.基于兼并引物的PCR扩增特定基因的效果比较[J].湖北民族学院学报:自然科学版,2015,33(2):170-173.
[21]Murty S G,Patel F,Punwar B S,et al.Comparison of RAPD,ISSR,and DAMD markers for genetic diversity assessment between accessions of Jatropha curcas L.and its related species[J].Journal of Agricultural Science&Technology,2018,13(13):1007-1022.
[22]Purayil F T,Robert G A,Gothandam K M,et al.Genetic variability in selected date palm(Phoenix dactylifera L.)cultivars of United Arab Emirates using ISSR and DAMD markers[J].Biotech,2018,8(2):109.
[23]Xiong F,Liu J,Jiang J,et al.Molecular profiling of genetic variability in domesticated groundnut(Arachis hypogaea L.)based on ISJ,URP,and DAMD markers[J].Biochemical Genetics,2013,51(11-12):889-900.
[24]Misra S,Srivastava AK,Verma S,et al.Phenetic and genetic diversity in Indian Luffa(Cucurbitaceae)inferred from morphometric,ISSR and DAMD markers[J].Genetic Resources and Crop Evolution,2017,64(5):995-1010.
[25]Bhattacharyya P,Kumaria S,Tandon P.Applicability of ISSR and DAMD markers for phytomolecular characterization and association with some important biochemical traits of Dendrobium nobile,an endangered medicinal orchid[J].Phytochemistry,2015,117(306):306-316.
[2]邹奕,吴则东,兴旺,等.甜菜种质资源遗传多样性研究进展[J].中国糖料,2018,40(5):73-76,80.
[3]邹奕,马龙彪,江伟,等.ISSR分子标记技术在甜菜育种中的应用[J].中国糖料,2018,40(3):75-77,80.
[4]王茂芊,张惠忠,吴则东,等.SRAP标记对甜菜抗根腐病品种的遗传多样性分析[J].中国农学通报,2017,33(11):9-13.
[5]刘华君,聂志勇,林明,等.利用ISSR标记分析20份甜菜品种的遗传多样性[J].中国糖料,2017,39(3):1-4.
[6]Srivastava S,Pathak A,Kumar R,et al.Genetic diversity of sugar beet genotypes evaluated by microsatellite DNA markers[J]Journal of Environmental Biology,2017,38(5):777-783.
[7]马龙彪,吴则东,王茂芊,等.甜菜育种的研究进展及未来发展展望[J].中国糖料,2018,40(6):62-65.
[8]Touzet P,Villain S,Buret L,et al.Chloroplastic and nuclear diversity of wild beets at a large geographical scale:Insights into the evolutionary history of the Beta section[J].Ecology&Evolution,2018,8(5):2890-2900.
[9]Heath D D,Lwama G K,Devlin R H.PCR primed with VNTR core sequences yields species specific patterns and hypervariable probes[J].Nucleic Acids Research,1993,21(24):5782-5785.
[10]胡建斌,张帆,李贞煌,等.黄瓜DAMD反应体系的建立及种质遗传资源研究[J].西北植物学报,2010,30(4):652-658.
[11]Métais I,Aubry C,Hamon B,et al.Assessing common bean genetic diversity using RFLP,DAMD-PCR,ISSR,RAPD AND AFLPmarkers[J].Acta Horticulturae,2001,546(546):459-461.
[12]王掌军,王建设,刘玲,等.直接扩增甜瓜小卫星DNA指纹图谱[J].华北农学报,2006,21(3):77-80.
[13]Ince A G,Karaca M,Onus A N.Development and utilization of diagnostic DAMD-PCR markers for Capsicum accessions[J].Genetic Resources and Crop Evolution,2009,56(2):211-221.
[14]Karaca M,Ince A G,Ay S T,et al.PCR-RFLP and DAMD-PCR genotyping for Salvia species[J].Journal of the Science of Food&Agriculture,2008,88(14):2508-2516.
[15]兴旺,童乐怡,孙燕红,等.甜菜DAMD-PCR体系的建立及优化[J].中国农学通报,2017,33(23):6-9.
[16]孙燕红,马龙彪,兴旺,等.甜菜DAMD引物的筛选及不同凝胶系统对扩增产物多态性的影响[J].中国农学通报,2018,34(16):42-45.
[17]符德欢,朱高倩,郭佳玉,等.改良CTAB法提取重楼属3种药用植物干燥根茎DNA[J].中药材,2017,40(6):1295-1299.
[18]郭培国,刘文杰,李海洋,等.一种快速有效检测SSR标记的非变性聚丙烯酰胺凝胶的银染方法[J].广州大学学报:自然科学版,2016,15(4):8-12.
[19]杨绍林,王先宏,李苏洁,等.应用降落PCR和正交设计优化甘蔗“割手密”SSR-PCR反应体系[J].分子植物育种,2016,14(2):431-436.
[20]李志强,何德,李翠新.基于兼并引物的PCR扩增特定基因的效果比较[J].湖北民族学院学报:自然科学版,2015,33(2):170-173.
[21]Murty S G,Patel F,Punwar B S,et al.Comparison of RAPD,ISSR,and DAMD markers for genetic diversity assessment between accessions of Jatropha curcas L.and its related species[J].Journal of Agricultural Science&Technology,2018,13(13):1007-1022.
[22]Purayil F T,Robert G A,Gothandam K M,et al.Genetic variability in selected date palm(Phoenix dactylifera L.)cultivars of United Arab Emirates using ISSR and DAMD markers[J].Biotech,2018,8(2):109.
[23]Xiong F,Liu J,Jiang J,et al.Molecular profiling of genetic variability in domesticated groundnut(Arachis hypogaea L.)based on ISJ,URP,and DAMD markers[J].Biochemical Genetics,2013,51(11-12):889-900.
[24]Misra S,Srivastava AK,Verma S,et al.Phenetic and genetic diversity in Indian Luffa(Cucurbitaceae)inferred from morphometric,ISSR and DAMD markers[J].Genetic Resources and Crop Evolution,2017,64(5):995-1010.
[25]Bhattacharyya P,Kumaria S,Tandon P.Applicability of ISSR and DAMD markers for phytomolecular characterization and association with some important biochemical traits of Dendrobium nobile,an endangered medicinal orchid[J].Phytochemistry,2015,117(306):306-316.