短丝木犀SSR-PCR反应体系优化及基于DNA混合池的引物快速筛选
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摘要
本研究采用L9(23)正交试验设计确定短丝木犀的SSR-PCR最佳反应体系,同时设置12个不同的温度梯度优化引物退火温度;利用优化后的体系以50对候选SSR引物为对象,对6个短丝木犀基因组DNA样品等量混合,进行PCR扩增筛选引物。结果表明,第一,短丝木犀SSR-PCR最优反应体系为:1.0μL50ng/μL模板DNA,1.0μL 100μmol/L引物,12.5μL 2×Taq PCR Master Mix,补ddH2O至25μL;第二,DNA混合池方法比常规的筛选方法具有效率高,实验资源消耗低的优点,可用于短丝木犀SSR引物的大量筛选;第三,最终筛选出10对具有高多态性、高稳定性且重复性好的SSR引物。本研究旨在建立适合短丝木犀的SSR-PCR最佳反应体系,并以DNA混合池为模板进行引物筛选,为短丝木犀引物的大量开发提供了一种新的途径。
In this study, SSR-PCR optimum reaction system of Osmanthus serrulatus was established by using L9(23) orthogonal experimental design, and 12 different temperature gradients were set to optimize the annealing temperature of primers. Using the optimized system of SSR-PCR, 50 pairs of candidate SSR primers were mixed into 6 genomic DNA samples of O. serrulatus by equal quantity, and the primers were screened by PCR amplification. The results showed that firstly, the optimal SSR-PCR reaction system was as follows: 1.0 μL 50 ng/μL genomic DNA, 1.0 μL 100 μmol/L primer, 12.5μL 2×Taq PCR Master Mix, and replenishing ddH2 O to 25 μL. Secondly, compared with conventional screening method, the DNA mixing pooling method could greatly shorten experimental period and significantly reduce the consumption of experimental resources, which would be useful for screening substantive SSR primers in O. serrulatus. Thirdly, 10 pairs of SSR primers with high polymorphism,high stability, and good repeatability were finally screened out. The purpose of this study was to establish an optimal SSR-PCR reaction system suitable for O. serrulatus, and to screen primers by using DNA mixing pool, which would provide a new way for the massive screening of SSR primers in O. serrulatus.
In this study, SSR-PCR optimum reaction system of Osmanthus serrulatus was established by using L9(23) orthogonal experimental design, and 12 different temperature gradients were set to optimize the annealing temperature of primers. Using the optimized system of SSR-PCR, 50 pairs of candidate SSR primers were mixed into 6 genomic DNA samples of O. serrulatus by equal quantity, and the primers were screened by PCR amplification. The results showed that firstly, the optimal SSR-PCR reaction system was as follows: 1.0 μL 50 ng/μL genomic DNA, 1.0 μL 100 μmol/L primer, 12.5μL 2×Taq PCR Master Mix, and replenishing ddH2 O to 25 μL. Secondly, compared with conventional screening method, the DNA mixing pooling method could greatly shorten experimental period and significantly reduce the consumption of experimental resources, which would be useful for screening substantive SSR primers in O. serrulatus. Thirdly, 10 pairs of SSR primers with high polymorphism,high stability, and good repeatability were finally screened out. The purpose of this study was to establish an optimal SSR-PCR reaction system suitable for O. serrulatus, and to screen primers by using DNA mixing pool, which would provide a new way for the massive screening of SSR primers in O. serrulatus.
引文
Ali S.,Müller C.R.,and Epplen J.T.,1986,DNA finger printing by oligonucleotide probes specific for simple repeats,Human Genetics,74(3):239-243
Chen L.,Li L.N.,Dai Y.P.,Wang X.R.,Duan Y.F.,and Yang G.D.,2015,De novo transcriptome analysis of Osmanthus serrulatus Rehd.flowers and leaves by Illumina sequencing,Biochemical Systematics and Ecology,61(10-11):531-540
Chen L.,Li L.N.,Yang G.D.,and Dai Y.P.,2016,Microsatellites characteristics of transcriptomic sequences from Osmanthus serrulatus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):959-965(陈林,李龙娜,杨国栋,戴亚平,2016,特有植物短丝木犀(Osmanthus serrulatus)转录组微卫星特征分析,分子植物育种,14(4):959-965)
Gupta P.K.,and Varshney R.K.,2000,The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat,Euphytica,113(3):163-185
He R.F.,Feng S.G.,Chen Z.,Shen X.X.,Shen Y.F.,Wang Z.A.,and Wang H.Z.,2015,Optimization and primer screening of SSR-PCR reaction system in medicinal Chrysanthemum morifolium based on orthogonal design,Fenzi Zhiwu Zyuzhong(Molecular Plant Breeding),13(2):367-378(何仁锋,冯尚国,陈喆,沈晓霞,沈宇峰,王志安,王慧中,2015,药用菊花SSR-PCR反应体系优化及引物筛选,分子植物育种,13(2):367-378)
Hu Y.,Li Y.,Huang Y.Z.,and Liu P.W.,2016,Genetic diversity evaluation of sugarcane varieties using SSR and RAPDmarkers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2494-2503(胡杨,李赟,黄有总,刘平武,2016,利用SSR与RAPD分子标记评估甘蔗品种的遗传多样性,基因组学与应用生物学,35(9):2494-2503)
Liang H.W.,Wang C.Z.,Li Z.,Luo X.Z.,and Zou G.W.,2008,Improvement of the silver-stained technique of polyacrylamide gel electrophoresis,Yichuan(Hereditas(Beijing)),30(10):1379-1382(梁宏伟,王长忠,李忠,罗相忠,邹桂伟,2008,聚丙烯酰胺凝胶快速、高效银染方法的建立,遗传,30(10):1379-1382)
Li X.L.,Lu J.H.,Xie L.B.,Zhang A.X.,Chen X.C.,and Li X.Y.,2015,Development of EST-SSR primers and genetic relationship analysis in four Glyeyrrhiza L.species,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),35(3):480-485(李晓岚,陆嘉惠,谢良碧,张爱霞,陈晓翠,李学禹,2015,4种甘草属植物EST-SSR引物开发及其亲缘关系分析,西北植物学报,35(3):480-485)
Liu W.W.,Hao Z.F.,Wen J.F.,Li X.H.,Song X.Y.,Zhang S.H.,and Xie C.X.,2012,An efficient method of qualitative screening positive transgenic samples based on PCR technique and pooling strategy,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),45(2):399-404(刘文文,郝转芳,翁建峰,李新海,宋新元,张世煌,谢传晓,2012,一种基于PCR技术混合样本高效定性筛查转基因阳性样本的方法,中国农业科学,45(2):399-404)
Wang B.,and Chen G.,2007,DNA pooling technique and its application in genetic study of schizophrenia,Jingshen Yixue Zazhi(Journal of Psychiatry),20(6):404-407(王博,陈刚,2007,DNA混合池技术及其在精神分裂症遗传研究中的应用,精神医学杂志,20(6):404-407)
Wang D.L.,2012,Construction of a SNP fingerprint databases for Lentinula edodes cultivars,Thesis for M.S.,Huazhong Agricultural University,Supervisors:Bian Y.B.,and Zhang J.X.,pp.27-51(王大莉,2012,香菇栽培品种SNP指纹图谱库的构建,硕士学位论文,华中农业大学,导师:边银丙,张金霞,pp.27-51)
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Zhang L.,Ding X.P.,Yan L.L.,Hu S.N.,and Bai C.J.,2014,Optimization of SSR-PCR system and primers screening of eight species of Stylosanthes,Caoye Kexue(Pratacultural Science),31(2):232-242(张龙,丁西朋,严琳玲,胡珊娜,白昌军,2014,8种柱花草属牧草SSR-PCR反应体系优化及引物筛选,草业科学,31(2):232-242)
Chen L.,Li L.N.,Dai Y.P.,Wang X.R.,Duan Y.F.,and Yang G.D.,2015,De novo transcriptome analysis of Osmanthus serrulatus Rehd.flowers and leaves by Illumina sequencing,Biochemical Systematics and Ecology,61(10-11):531-540
Chen L.,Li L.N.,Yang G.D.,and Dai Y.P.,2016,Microsatellites characteristics of transcriptomic sequences from Osmanthus serrulatus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):959-965(陈林,李龙娜,杨国栋,戴亚平,2016,特有植物短丝木犀(Osmanthus serrulatus)转录组微卫星特征分析,分子植物育种,14(4):959-965)
Gupta P.K.,and Varshney R.K.,2000,The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat,Euphytica,113(3):163-185
He R.F.,Feng S.G.,Chen Z.,Shen X.X.,Shen Y.F.,Wang Z.A.,and Wang H.Z.,2015,Optimization and primer screening of SSR-PCR reaction system in medicinal Chrysanthemum morifolium based on orthogonal design,Fenzi Zhiwu Zyuzhong(Molecular Plant Breeding),13(2):367-378(何仁锋,冯尚国,陈喆,沈晓霞,沈宇峰,王志安,王慧中,2015,药用菊花SSR-PCR反应体系优化及引物筛选,分子植物育种,13(2):367-378)
Hu Y.,Li Y.,Huang Y.Z.,and Liu P.W.,2016,Genetic diversity evaluation of sugarcane varieties using SSR and RAPDmarkers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2494-2503(胡杨,李赟,黄有总,刘平武,2016,利用SSR与RAPD分子标记评估甘蔗品种的遗传多样性,基因组学与应用生物学,35(9):2494-2503)
Liang H.W.,Wang C.Z.,Li Z.,Luo X.Z.,and Zou G.W.,2008,Improvement of the silver-stained technique of polyacrylamide gel electrophoresis,Yichuan(Hereditas(Beijing)),30(10):1379-1382(梁宏伟,王长忠,李忠,罗相忠,邹桂伟,2008,聚丙烯酰胺凝胶快速、高效银染方法的建立,遗传,30(10):1379-1382)
Li X.L.,Lu J.H.,Xie L.B.,Zhang A.X.,Chen X.C.,and Li X.Y.,2015,Development of EST-SSR primers and genetic relationship analysis in four Glyeyrrhiza L.species,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),35(3):480-485(李晓岚,陆嘉惠,谢良碧,张爱霞,陈晓翠,李学禹,2015,4种甘草属植物EST-SSR引物开发及其亲缘关系分析,西北植物学报,35(3):480-485)
Liu W.W.,Hao Z.F.,Wen J.F.,Li X.H.,Song X.Y.,Zhang S.H.,and Xie C.X.,2012,An efficient method of qualitative screening positive transgenic samples based on PCR technique and pooling strategy,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),45(2):399-404(刘文文,郝转芳,翁建峰,李新海,宋新元,张世煌,谢传晓,2012,一种基于PCR技术混合样本高效定性筛查转基因阳性样本的方法,中国农业科学,45(2):399-404)
Wang B.,and Chen G.,2007,DNA pooling technique and its application in genetic study of schizophrenia,Jingshen Yixue Zazhi(Journal of Psychiatry),20(6):404-407(王博,陈刚,2007,DNA混合池技术及其在精神分裂症遗传研究中的应用,精神医学杂志,20(6):404-407)
Wang D.L.,2012,Construction of a SNP fingerprint databases for Lentinula edodes cultivars,Thesis for M.S.,Huazhong Agricultural University,Supervisors:Bian Y.B.,and Zhang J.X.,pp.27-51(王大莉,2012,香菇栽培品种SNP指纹图谱库的构建,硕士学位论文,华中农业大学,导师:边银丙,张金霞,pp.27-51)
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Zhang L.,Ding X.P.,Yan L.L.,Hu S.N.,and Bai C.J.,2014,Optimization of SSR-PCR system and primers screening of eight species of Stylosanthes,Caoye Kexue(Pratacultural Science),31(2):232-242(张龙,丁西朋,严琳玲,胡珊娜,白昌军,2014,8种柱花草属牧草SSR-PCR反应体系优化及引物筛选,草业科学,31(2):232-242)