利用PCR仪快速提取甜菜基因组DNA
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摘要
为了寻找一种快速提取甜菜基因组DNA的方法,以甜菜干种子、幼苗、种仁以及甜菜叶片干粉为原料,利用PCR仪结合碱裂解法快速提取甜菜基因组DNA,利用微量分光光度计检测取DNA的浓度,并用甜菜SSR引物对提取的DNA进行扩增。结果表明,在4种材料中均检测到了DNA,干种子、幼苗、种仁以及叶片干粉中提取的DNA平均浓度分别为432、197、158、448 ng/μL,无论是DNA原液还是稀释到20 ng/μL的工作液,均能在SSR-PCR反应中扩增出清晰的条带。该方法提取甜菜基因组DNA简单、快速,仅需要Na OH和HCl两种药品,提取的DNA完全可以用于SSR-PCR反应,为快速鉴定甜菜品种纯度和真实性提供了技术支持。
In order to find a method for rapidly extracting genomic DNA from sugar beet, a PCR instrumentwas used in combination with an alkaline lysis method with dried seeds, seedlings and kernels of sugar beet aswell as dry powder of sugar beet leaf as raw materials, the concentration of extracted DNA was measured by amicro-spectrophotometer, and amplification of extracted DNA was conducted using the SSR primers of sugarbeet. The results demonstrated that DNA was detected in all materials, the mean concentration of extractedDNA from dried seeds, seedlings, kernels and leaf dry powder was 432 ng/μL, 197 ng/μL, 158 ng/μL and448 ng/μL, respectively, and clear bands were amplified in both DNA stock solution and diluted 20 ng/μLworking solution by SSR-PCR reaction system. This method is simple and rapid in extracting genomic DNAfrom sugar beet, and only requires two medicines Na OH and HCl. The extracted DNA can be completely usedfor SSR- PCR reaction, which provides technical support for the rapid identification of the purity andauthenticity of sugar beet varieties.
In order to find a method for rapidly extracting genomic DNA from sugar beet, a PCR instrumentwas used in combination with an alkaline lysis method with dried seeds, seedlings and kernels of sugar beet aswell as dry powder of sugar beet leaf as raw materials, the concentration of extracted DNA was measured by amicro-spectrophotometer, and amplification of extracted DNA was conducted using the SSR primers of sugarbeet. The results demonstrated that DNA was detected in all materials, the mean concentration of extractedDNA from dried seeds, seedlings, kernels and leaf dry powder was 432 ng/μL, 197 ng/μL, 158 ng/μL and448 ng/μL, respectively, and clear bands were amplified in both DNA stock solution and diluted 20 ng/μLworking solution by SSR-PCR reaction system. This method is simple and rapid in extracting genomic DNAfrom sugar beet, and only requires two medicines Na OH and HCl. The extracted DNA can be completely usedfor SSR- PCR reaction, which provides technical support for the rapid identification of the purity andauthenticity of sugar beet varieties.
引文
[1]Munthali M,Newbury H,Ford-Lloyd B.The detection of somaclonal variants of beet using RAPD[J].Plant cell reports,1996,15(7):474-478.
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[10]王茂芊,吴则东,王华忠.利用SRAP标记分析我国甜菜三大产区骨干材料的遗传多样性[J].作物学报,2011,37(5):811-819.
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[12]M?hring S,Salamini F,Schneider K.Multiplexed,linkage groupspecific SNP marker sets for rapid genetic mapping and fingerprinting of sugar beet(Beta vulgaris L.)[J].Molecular Breeding,2004,14(4):475-488.
[13]Cureton A,Burns M,Ford-Lloyd B,et al.Development of simple sequence repeat(SSR)markers for the assessment of gene flow between sea beet(Beta vulgaris ssp.maritima)populations[J].Molecular Ecology Notes,2002,2(4):402-403.
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[15]单志,吴宏亮,李成磊,等.改良SDS法提取多种植物基因组DNA研究[J].广东农业科学,2011,38(8):113-115.
[16]宋国立,崔荣霞.改良CTAB法快速提取棉花DNA[J].棉花学报,1998,10(5):273-275.
[17]谭君,杨俊品.玉米种子DNA快速提取及杂交种纯度的快速鉴定[J].分子植物育种,2009,7(4):811-816.
[18]郭景伦,赵久然,王凤格.适用于SSR分子标记的玉米单粒种子DNA快速提取新方法[J].玉米科学,2005,13(2):16-17,25.
[19]郭景伦,赵久然,辛景树,等.玉米单株幼芽DNA快速提取新方法[J].华北农学报,2005,20(1):38-40.
[20]李强,揭琴,刘庆昌,等.甘薯基因组DNA高效快速提取方法[J].分子植物育种,2007,5(5):743-746.
[21]黄东亮,覃肖良,廖青,等.高质量甘蔗基因组DNA的简便快速提取方法研究[J].生物技术通报,2010(5):101-106.
[22]郎需勇,刘伟霞,杨亮,等.一种快速提取棉花干种子基因组DNA的新方法[J].棉花学报,2014,26(1):87-94.
[23]吴则东,王茂芊,马龙彪,等.适于甜菜品种鉴定的SSR核心引物的筛选[J].中国农学通报,2015,31(15):165-169.
[24]王凤格,赵久然,郭景伦,等.一种改进的玉米SSR标记的PAGE/快速银染检测新方法[J].农业生物技术学报,2004,12(5):606-607.
[2]Grimmer M,Trybush S,Hanley S,et al.An anchored linkage map for sugar beet based on AFLP,SNP and RAPD markers and QTL mapping of a new source of resistance to Beet necrotic yellow vein virus[J].Theoretical and Applied Genetics,2007,114(7):1151-1160.
[3]Halldén C,Hjerdin A,Rading I,et al.A high density RFLP linkage map of sugar beet[J].Genome,1996,39(4):634-645.
[4]Sabir A,Newbury H,Todd G,et al.Determination of genetic stability using isozymes and RFLPs in beet plants regenerated in vitro[J].Theoretical and Applied Genetics,1992,84(1-2):113-117.
[5]Schondelmaier J,Steinrücken G,Jung C.Integration of AFLP markers into a linkage map of sugar beet(Beta vulgaris L.)[J].Plant breeding,1996,115(4):231-237.
[6]Kraft T,Hansen M,Nilsson N-O.Linkage disequilibrium and fingerprinting in sugar beet[J].Theoretical and Applied Genetics,2000,101(3):323-326.
[7]Sen A,Alikamanoglu S.Analysis of drought-tolerant sugar beet(Beta vulgaris L.)mutants induced with gamma radiation using SDS-PAGE and ISSR markers[J].Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis,2012,738:38-44.
[8]Srivastava S,Gupta PS,Saxena VK,et al.Genetic diversity analysis in sugarbeet(Beta vulgaris L.)using isozymes,RAPD and ISSR markers[J].Cytologia,2007,72(3):265-274.
[9]王茂芊,李博,王华忠.甜菜遗传连锁图谱初步构建[J].作物学报,2014,40(2):222-230.
[10]王茂芊,吴则东,王华忠.利用SRAP标记分析我国甜菜三大产区骨干材料的遗传多样性[J].作物学报,2011,37(5):811-819.
[11]Schneider K,Kulosa D,Soerensen TR,et al.Analysis of DNA polymorphisms in sugar beet(Beta vulgaris L.)and development of an SNP-based map of expressed genes[J].Theoretical and Applied Genetics,2007,115(5):601-615.
[12]M?hring S,Salamini F,Schneider K.Multiplexed,linkage groupspecific SNP marker sets for rapid genetic mapping and fingerprinting of sugar beet(Beta vulgaris L.)[J].Molecular Breeding,2004,14(4):475-488.
[13]Cureton A,Burns M,Ford-Lloyd B,et al.Development of simple sequence repeat(SSR)markers for the assessment of gene flow between sea beet(Beta vulgaris ssp.maritima)populations[J].Molecular Ecology Notes,2002,2(4):402-403.
[14]Laurent V,Devaux P,Thiel T,et al.Comparative effectiveness of sugar beet microsatellite markers isolated from genomic libraries and Gen Bank ESTs to map the sugar beet genome[J].Theoretical and Applied Genetics,2007,115(6):793-805.
[15]单志,吴宏亮,李成磊,等.改良SDS法提取多种植物基因组DNA研究[J].广东农业科学,2011,38(8):113-115.
[16]宋国立,崔荣霞.改良CTAB法快速提取棉花DNA[J].棉花学报,1998,10(5):273-275.
[17]谭君,杨俊品.玉米种子DNA快速提取及杂交种纯度的快速鉴定[J].分子植物育种,2009,7(4):811-816.
[18]郭景伦,赵久然,王凤格.适用于SSR分子标记的玉米单粒种子DNA快速提取新方法[J].玉米科学,2005,13(2):16-17,25.
[19]郭景伦,赵久然,辛景树,等.玉米单株幼芽DNA快速提取新方法[J].华北农学报,2005,20(1):38-40.
[20]李强,揭琴,刘庆昌,等.甘薯基因组DNA高效快速提取方法[J].分子植物育种,2007,5(5):743-746.
[21]黄东亮,覃肖良,廖青,等.高质量甘蔗基因组DNA的简便快速提取方法研究[J].生物技术通报,2010(5):101-106.
[22]郎需勇,刘伟霞,杨亮,等.一种快速提取棉花干种子基因组DNA的新方法[J].棉花学报,2014,26(1):87-94.
[23]吴则东,王茂芊,马龙彪,等.适于甜菜品种鉴定的SSR核心引物的筛选[J].中国农学通报,2015,31(15):165-169.
[24]王凤格,赵久然,郭景伦,等.一种改进的玉米SSR标记的PAGE/快速银染检测新方法[J].农业生物技术学报,2004,12(5):606-607.