杜鹃花SCoT-PCR反应体系的优化及引物筛选
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摘要
本试验以马银花(R.ovatum)DNA为模板,采用L16(45)正交试验对目标起始密码子多态性-聚合酶链式反应(SCoT-PCR)体系中5因素(Taq聚合酶用量,Mg~(2+)浓度,模板DNA用量,dNTPs浓度,引物浓度)进行了优化,建立了杜鹃花植物SCoT-PCR反应体系。结果表明,在20μL的反应体系中,模板DNA 1.00 ng/μL,Mg~(2+)为3.00 mmol/L,dNTPs为0.25 mmol/L,引物为0.50μmol/L,Taq DNA聚合酶为2.00 U。比较了各因素对反应体系的影响,发现引物物浓度的差异对体系影响显著,各因素影响的先后顺序为引物>Taq DNA聚合酶>dNTPs>Mg~(2+)>模板DNA。并运用了杜鹃属的4个不同种对最优体系进行了稳定性验证,选择了10个通用性的引物进行筛选,发现10个引物均能扩增出条带,其中有6个引物能够得到清晰可辨、多态性丰富的条带。该体系的建立将为杜鹃花遗传多样性、差异基因的表达分析,分子辅助育种、遗传图谱构建等研究提供技术支持。
In this experiment, the R. ovatum DNA was used as template, and the orthogonal experiment of L16(45)was designed to optimize the five factors(Taq DNA polymerase, Mg~(2+), template DNA, dNTPs and primer concentrations) of SCoT-PCR(start codon targeted polymorphism) system in Rhododendron plants. The results showed that the total volume of 20 μL system included template DNA 1.00 ng/μL, Mg~(2+)3.00 mmol/L, dNTPs 0.25 mmol/L,primer 0.50 μmol/L and Taq DNA polymerase 2.00 U. The comparison of the effects of various factors on the reaction system indicated that different concentrations of primers had significantly different effect on reaction system, and the order of the effective degree was primer >Taq DNA polymerase >dNTPs>Mg~(2+)>template DNA.4 different species of Rhododendron were used to verify the stability of the optimal system, and 10 general-purpose primers were selected to screen. It was found that 10 primers all could amplify bands, in which 6 primers could amplify bands with clear and polymorphic strips. The establishment of the system could provide technical support for the study of Rhododendron genetic diversity, gene expression profile change, molecular assisted breeding,genetic map construction and other aspects.
In this experiment, the R. ovatum DNA was used as template, and the orthogonal experiment of L16(45)was designed to optimize the five factors(Taq DNA polymerase, Mg~(2+), template DNA, dNTPs and primer concentrations) of SCoT-PCR(start codon targeted polymorphism) system in Rhododendron plants. The results showed that the total volume of 20 μL system included template DNA 1.00 ng/μL, Mg~(2+)3.00 mmol/L, dNTPs 0.25 mmol/L,primer 0.50 μmol/L and Taq DNA polymerase 2.00 U. The comparison of the effects of various factors on the reaction system indicated that different concentrations of primers had significantly different effect on reaction system, and the order of the effective degree was primer >Taq DNA polymerase >dNTPs>Mg~(2+)>template DNA.4 different species of Rhododendron were used to verify the stability of the optimal system, and 10 general-purpose primers were selected to screen. It was found that 10 primers all could amplify bands, in which 6 primers could amplify bands with clear and polymorphic strips. The establishment of the system could provide technical support for the study of Rhododendron genetic diversity, gene expression profile change, molecular assisted breeding,genetic map construction and other aspects.
引文
Chen H.,He X.H.,Lu o C.,Gao M.P.,and Zhu J.H.,2009,The optimization of SCo T-PCR system of Longan(Dimocarpus Longan),Jiyinzuxue Yu Yingyong Shengwuxue(Genomic and Applied Biology),28(5):970-974(陈虎,何新华,罗聪,高美萍,朱建华,2009,龙眼SCo T-PCR反应体系的优化,基因组学与应用生物学,28(5):970-974)
Chen X.L.,Li Y.R.,Yang L.T.,Wu J.M.,Luo C.,Xiong F.Q.,and Yang L.,2010,c DNA-SCo T differential display of cold resistance related genes in sugarcane,Shengwu Jishu Tongbao(Biotechnology Bulletin),(8):120-124(陈香玲,李杨瑞,杨丽涛,吴建明,罗聪,熊发前,杨柳,2010,低温胁迫下甘蔗抗寒相关基因的c DNA-SCo T差异显示,生物技术通报,(8):120-124)
Collard B.C.Y.,and Mackill D.J.2009,Start codon targeted(SCo T)polymorphism:a simple,novel DNA marker technique for generating gene-targeted markers in plants,Plant Mol.Biol.Rep.,27(1):86-93
Ding X.Y.,Qiu S.,Xi M.L.,and Shi J.S.,2012,Optimizing the ISSR-PCR reaction system for oriental lily based on orthogonal design,Nangjing Linye Daxue Xuebao(Journal of Nanjing Forestry University,36(5):42-46(丁信誉,邱帅,席梦利,施季森,2012,东方百合ISSR-PCR反应体系的正交优化,南京林业大学学报,36(5):42-46)
Geng X.M.,Yang Q.Y.,Zheng F.C.,Wang L.G.,and Xie W.J.,2016,Differences in the protein expression of four Rhododendron species under high temperature,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(6):1574-1581(耿兴敏杨秋玉,郑福超,王良桂,解玮佳,2016,4种杜鹃幼苗高温胁迫下蛋白表达差异,分子植物育种,14(6):1574-1581)
Gong Y.,Liu X.H.,Li M.S.,Xu M.Z.,Zhang C.Y.,and Wu Q.2010,Optimization for rapid reaction system of Rhododendron chrysanthum Pall,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),38(12):6134-6135,6196(宫宇刘宪虎,李美善,许明子,张春影,吴琼,2010,牛皮杜鹃RAPD反应体系的优化,安徽农业科学,38(12):6134-61356196)
Gu Q.P.,Jin Z.X.,and Li J.M.,2007,Optimization of ISSR amplification condition for Rhododendron fortunei,Guangxi Zhiwu(Guihaia),27(4):560-563(顾奇萍,金则新,李钧敏2007,云锦杜鹃ISSR扩增条件的优化,广西植物,27(4):560-563)
Han G.H.,Xiang S.Q.,Wang W.X.,Jia Z.G.,Hong Q.B.,and Liang G.L.,2011,Establishment and application of scot molecular marker system for citrus,Yuanyi Xuebao(Acta Horticulturae Sinica),38(7):1243-1250(韩国辉,向素琼,汪卫星,贾志刚,洪棋斌,梁国鲁,2011,柑橘SCo T分子标记技术体系的建立及其在遗传分析中的应用,园艺学报,38(7):1243-1250)
He R.F.,Feng S.G.,Chen Z.,Gao L.,Shen X.X.,Shen Y.F.Wang Z.A.,and Wang H.Z.,2014,Optimization of start codon targeted polymorphism system in medicinal Chrysanthemum morifolium based on orthogonal design,Henongxue Bao(Acta Agriculturae Nucleatae Sinica),28(10):1781-1789(何仁锋,冯尚国,陈喆,高岭,沈晓霞,沈宇峰,王志安,王慧中,2014,药用菊花SCo T-PCR反应体系的正交优化核农学报,28(10):1781-1789)
Hou X.G.,Wang J.,Jia T.,Zhang Y.Q.,Hou J.,and Li J.J.,2011,Orthogonal optimization of SCo T-PCR system and primer screening of tree peony,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),26(5):92-96(侯小改,王娟,贾甜,张钰乾,侯娟,李嘉珏,2011,牡丹SCo T分子标记正交优化及引物筛选,华北农学报,26(5):92-96)
Jiang Q.Q.,Long G.Y.,Li W.W.,and Deng Z.N.,2011,Identification of genetic variation of sweet orange clones from hunan by using SCo T markers and sequence analysis,Zhong guo Nongxue Tongbao(Chinese Agricultural Science Bulletin),27(6):148-154(蒋巧巧,龙桂友,李武文,邓子牛,2011,SCo T结合克隆测序鉴别湖南甜橙变异类型,中国农学通报,27(6):148-154)
Jiang X.F.,Gao Y.H.,Tong Z.K.,and Huang C.H.,2013,Establishment and optimizing of SCo T-PCR system for Lycoris,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&FUniversity),30(3):444-452(姜小凤,高燕会,童再康,黄春红,2013,石蒜属植物SCo T-PCR反应体系构建及优化,浙江农林大学学报,30(3):444-452)
Li P.R.,Jiang J.F.,Chen S.M.,Guan Z.Y.,Liao Y.,Fang W.M.,and Chen F.D.,2013,Establishment and optimization of SCo T molecular marker system in chrysanthemum and its application of analysis on genetic diversity,Yuanyi Xuebao(Acta Horticulturae Sinica),40(10):2015-2025(李丕睿,蒋甲福,陈素梅,管志勇,廖园,房伟民,陈发棣,2013,菊属植物SCo T分子标记技术在遗传多样性分析中的应用,园艺学报,40(10):2015-2025)
Li X.F.,Zhang H.G.,Guan C.Y.,and Zhang Y.,2009,Establishment and optimization of RAPD-PCR reaction system for Larix gmeinii(Rupr.)using orthogonal design,Zhiwu Yanjiu(Bullet of Botanical Research),29(1):80-85(李雪峰,张含国,贯春雨,张瑶,2009,利用正交设计优化兴安落叶松RAPD-PCR反应体系,植物研究,29(1):80-85)
Liu X.Q.,Su J.L.,Li C.,He L.S.,Xiao Z.,and Jia X.P.,2016,Anew Rhododendron cultiva‘Zijin Fenyu’,Yuanyi Xuebao(Acta Horticulturae Sinica),43(10):2067-2068(刘晓青,苏家乐,李畅,何丽斯,肖政,贾新平,2016,杜鹃花新品种‘紫金粉玉’,园艺学报,43(10):2067-2068)
Long Z.J.,Fan L.Z.,Xu G.,Hu S.L.,and Han G.H.,2015,Application advance of SCo T molecular markers in plants,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),16(2):336-343(龙治坚,范理璋,徐刚,胡尚连,韩国辉,2015,SCo T分子标记在植物研究中的应用进展,植物遗传资源学报,16(2):336-343)
Long Z.J.,Wang Y.,Han G.H.,Chen Z.,Xiang S.Q.,Liang G.L.,and Lin Q.,2013,Establishment and optimization of SCo Tsystem of mustard,Xi'nan Daxue Xuebao(Journal of Southwest University(Nature Science Edition)),35(4):20-25(龙治坚,王莹,韩国辉,陈志,向素琼,梁国鲁,林清,2013,芥菜SCo T分析体系的建立与优化,西南大学学报(自然科学版),35(4):20-25)
Lu C.R.,Yu S.X.,Yu J.W.,Fan S.L.,Song M.Z.,Wang W.,and Ma S.J.,2008,Development and appraisement of functiona molecular marker:intron sequence amplified polymorphism Yichuan(Hereditas),30(9):1207-1216(陆才瑞,喻树迅,于霁雯,范术丽,宋美珍,王武,马淑娟,2008,功能型分子标记(ISAP)的开发及评价,遗传,30(9):1207-1216)
Luo C.,2012,Study on SCo T maker and analysis on genes of stress-related and important flowering time in mango,Disser tation for Ph.D.,Guangxi University,Supervisor:He XH.,pp.77(罗聪,2012,芒果SCo T分子标记与逆境和重要开花时间相关基因研究,广西大学,导师:何新华,pp.77)
Luo T.,Yang H.X.,Cen H.F.,Liu X.H.,Gao Y.J.,Duan W.X.Zhang R.H.,Liu X.H.,Song H.Z.,Huang Y.X.,and Zhang G.M.,2013,Application of scot molecular marker in construction of molecular genetic linkage map of Saccharum spontaneum L.,Zhiwu Yichuan Ziyuan Xuebao(Jounal of Plant Genetic Resources),14(4):704-710(罗霆,杨海霞,岑华飞,刘许辉,高轶静,段维兴,张荣华,刘昔辉,宋焕忠黄玉新,张革民,2013,SCo T分子标记在割手密遗传图谱构建中的应用,植物遗传资源学报,14(4):704-710)
Pang X.A.,Bai B.W.,Jiang X.,Chen J.L.,and Jiao P.P.,2015Total DNA extraction and RAPD system optimization of Tamarix plants in tarim basin desert,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(9)2015-2023(庞新安,白宝伟,姜喜,陈加利,焦培培,2015塔里木盆地荒漠区柽柳属植物总DNA提取及RAPD体系优化,基因组学与应用生物学,34(9):2015-2023)
Qin G.X.,He Q.,Liang G.L.,Lei J.J.,and Guo Q.G.,2012,Establishment and optimization of SCo T-PCR system in Fragaria,Guoshu Xuebao(Journal of Fruit Science),29(3):393-397(秦国新,何桥,梁国鲁,雷家军,郭启高,2012,草莓属植物SCo T分析体系的建立及优化,果树学报,29(3):393-397)
Qiu L.H.,Chen R.F.,Huang X.,Wu J.M.,Li Y.R.,Li Q.,and Luo H.M.,2016,Research and application of c DNA-SCo T on plants:a technology about gene differential expression,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Bio-technology),24(11):1771-1780(丘立杭,陈荣发,黄杏,吴建明,李杨瑞,李强,罗含敏,2016,基因差异表达技术c D-NA-SCo T在植物上的研究应用,农业生物技术学报,24(11):1771-1780)
Scariot V.,Keyser E.D.,Handa T.,and Riek D.,2007,Comparative study of the discriminating capacity and effectiveness of AFLR,STMS and EST markers in assessing genetic relationships among evergreen azaleas,Plant Breeding,126(10):207-212
Shi S.G.,Sun Z.H.,Li S.F.,Xie W.J.,Liu B.,and Wang Y.L.2013,Establishment of ISSR-PCR reaction system for Rhododendron lapponicum,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),41(27):10977-10979,11040(石绍高,孙正海,李世峰,解玮佳,刘邦,王永路,2013,高山杜鹃ISSR-PCR反应体系建立,安徽农业科学,41(27):10977-10979,11040)
Shi W.D.,Yang C.F.,Yang L.T.,Ju X.X.,Luo H.L.,Kang H.W.,and Zhang L.,2015,Assessment of phenotype diversity and SCo T polymorphism of 15 Chinese kales,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):615-621(史卫东,杨翠芳,杨丽婷,琚茜茜,罗海玲,康红卫,张力,2015,15个芥蓝的表型多样性和SCo T多态性评价,基因组学与应用生物学,34(3):615-621)
Wang Y.,Zhao L.Y.,Shen H.W.,Qi Y.,Shi L.H.,and Wu D.,2014,Optimization for SCo T system and primer selection of Rose rugosa,Beifang Yuanyi(Northern Horticulture),(23):91-96(王玉,赵兰勇,申宏伟,齐宇,石良红,吴迪,2014,玫瑰SCo T-PCR反应体系优化及引物筛选,北方园艺,(23):91-96)
Wu Y.Y.,Tao Q.J.,Li B.,and Xu D.Y.,2013,An optimal SRAP-PCR system of Rhododendron and its genetic diversi ty,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),30(6):844-851(吴月燕,陶巧静,李波,许丹叶,2013,西洋杜鹃SRAP体系优化及遗传多样性分析,浙江农林大学学报,30(6):844-851)
Xie Y.H.,Xia D.A.,Jiang J.,and Lin P.,2005,Optimization for ISSR-PCR system of Fraxinus mandshurica Rupr,using orthogonal design,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),3(3):445-450(谢运海,夏德安,姜静,林萍,2005,利用正交设计优化水曲柳ISSR-PCR反应体系,分子植物育种,3(3):445-450)
Yan S.H.,Zheng W.H.,and Ding B.Y.,2012,Opitimization of ISSR reaction conditions and analysis of phylogenetic relationship for Rhododendron huadingense,Guangxi Zhiwu(Guihaia),32(5):593-598(颜士辉,郑蔚虹,丁炳扬,2012,华顶杜鹃ISSR反应体系的优化及亲缘关系的初步分析,广西植物,32(5):593-598)
Yang X.X.,Zheng J.,Leng P.S.,Wang Y.,and Wang Y.T.,2014,Optimization of ISSR-PCR system and selection of primers for Syringa reticulate var.amurensis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(5):1018-1026(杨晓霞,郑健,冷平生,王羽,王宇婷,2014,暴马丁香ISSR-PCR反应体系优化及引物筛选,分子植物育种,12(5):1018-1026)
Yuan W.J.,Ma Y.F.,Dong M.F.,Han Y.J.,Zhang W.R.,and Shang F.D.,2015,Establishment and genetic diversity analysis of SCo T molecular maker system for Osmanthus fragrans,Yuanyi Xuebao(Acta Horticulturae Sinica),34(3):569-575(袁王俊,马寅峰,董美芳,韩远记,张维瑞,尚富德,2015,桂花SCo T标记体系的建立及其在遗传多样性分析中的应用,园艺学报,34(3):569-575)
Zhang B.,Lu S.J.,Shi L.,Zou D.F.,Wang D.H.,Xu S.Y.,Long Z.J.,and Han G.H.,2016,Establishment and optimization of SCo T-PCR system of an endangered and rare species Urophysa rockii Ulbr,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(9):2453-2459(张波,陆世家,石力,邹大方王德怀,徐双燕,龙治坚,韩国辉,2016,珍稀濒危植物距瓣尾囊草(Urophysa rockii Ulbr)SCo T-PCR分析体系的建立与优化,分子植物育种,14(9):2453-2459)
Zhang J.Y.,Guo D.L.,Gong Y.,Liu C.H.,Li M.,and Zhang G.H.2011,Optimization of start codon targeted polymorphism PCR(SCo T-PCR)system in Vitis vinifera,Guoshu Xuebao(Journal of Fruit Science),28(2):209-214(张君玉,郭大龙龚莹,刘崇怀,李猛,张国海,2011,葡萄目标起始密码子多态性反应体系的优化,果树学报,28(2):209-214)
Zhang L.,Zhou L.Y.,Xiao Q.W.,and Wu Z.K.,2007,Application of orthogonal design in rhododendron RAPD reaction system,Beifang Yuanyi(Northern Horticulture),(5):124-126(张丽,周兰英,肖千文,吴开志,2007,正交试验设计在建立杜鹃花RAPD-PCR反应体系中的应用,北方园艺,(5)124-126)
Zhang Y.H.,Shen X.Q.,Liu X.Y.,and Zhao F.J.,2010,Optimation for ISSR-PCR system of Rhododendron mucronulatum Hubei Nongye Kexue(Hubei Agricultural Sciences),49(11)2657-2659(张艳红,沈向群,刘旭颖,赵凤军,2010,迎红杜鹃ISSR-PCR反应体系建立,湖北农业科学,49(11):2657-2659)
Zhao R.Q.,Gao Y.H.,Zhang X.L.,and Si J.P.,2012,Establishment and optimization of SCo T-PCR reaction system for Dendrobium officinale,Henongxue Bao(Journal of Nuclear Agricultural Scinences),26(4):648-655(赵瑞强,高燕会,章晓玲,斯金平,2012,铁皮石斛SCo T-PCR反应体系构建及优化,核农学报,26(4):648-655)
Zhao X.H.,and Wang M.Y.,2005,Optimization of RAPD condition of rhododendron,Shengwu Jishu(Biotechnology),15(1):41-43(赵喜华,王曼莹,2005,杜鹃花RAPD条件的优化,生物技术,15(1):41-43)
Zhuang D.F.,Cao H.N.,Zhou L.,Xu Y.,He W.,and Huang Y.,2008,Establishmet of RAPD system and DNA extraction from rhododendron in Changbai Mountain,Hubei Nongye Kexue(Hubei Agricultural Sciences),47(3):254-257(庄得凤,曹后男,周兰,徐颖,何伟,黄岳,2008,长白山杜鹃花基因组DNA提取及RAPD体系的建立,湖北农业科学,47(3):254-257)
Chen X.L.,Li Y.R.,Yang L.T.,Wu J.M.,Luo C.,Xiong F.Q.,and Yang L.,2010,c DNA-SCo T differential display of cold resistance related genes in sugarcane,Shengwu Jishu Tongbao(Biotechnology Bulletin),(8):120-124(陈香玲,李杨瑞,杨丽涛,吴建明,罗聪,熊发前,杨柳,2010,低温胁迫下甘蔗抗寒相关基因的c DNA-SCo T差异显示,生物技术通报,(8):120-124)
Collard B.C.Y.,and Mackill D.J.2009,Start codon targeted(SCo T)polymorphism:a simple,novel DNA marker technique for generating gene-targeted markers in plants,Plant Mol.Biol.Rep.,27(1):86-93
Ding X.Y.,Qiu S.,Xi M.L.,and Shi J.S.,2012,Optimizing the ISSR-PCR reaction system for oriental lily based on orthogonal design,Nangjing Linye Daxue Xuebao(Journal of Nanjing Forestry University,36(5):42-46(丁信誉,邱帅,席梦利,施季森,2012,东方百合ISSR-PCR反应体系的正交优化,南京林业大学学报,36(5):42-46)
Geng X.M.,Yang Q.Y.,Zheng F.C.,Wang L.G.,and Xie W.J.,2016,Differences in the protein expression of four Rhododendron species under high temperature,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(6):1574-1581(耿兴敏杨秋玉,郑福超,王良桂,解玮佳,2016,4种杜鹃幼苗高温胁迫下蛋白表达差异,分子植物育种,14(6):1574-1581)
Gong Y.,Liu X.H.,Li M.S.,Xu M.Z.,Zhang C.Y.,and Wu Q.2010,Optimization for rapid reaction system of Rhododendron chrysanthum Pall,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),38(12):6134-6135,6196(宫宇刘宪虎,李美善,许明子,张春影,吴琼,2010,牛皮杜鹃RAPD反应体系的优化,安徽农业科学,38(12):6134-61356196)
Gu Q.P.,Jin Z.X.,and Li J.M.,2007,Optimization of ISSR amplification condition for Rhododendron fortunei,Guangxi Zhiwu(Guihaia),27(4):560-563(顾奇萍,金则新,李钧敏2007,云锦杜鹃ISSR扩增条件的优化,广西植物,27(4):560-563)
Han G.H.,Xiang S.Q.,Wang W.X.,Jia Z.G.,Hong Q.B.,and Liang G.L.,2011,Establishment and application of scot molecular marker system for citrus,Yuanyi Xuebao(Acta Horticulturae Sinica),38(7):1243-1250(韩国辉,向素琼,汪卫星,贾志刚,洪棋斌,梁国鲁,2011,柑橘SCo T分子标记技术体系的建立及其在遗传分析中的应用,园艺学报,38(7):1243-1250)
He R.F.,Feng S.G.,Chen Z.,Gao L.,Shen X.X.,Shen Y.F.Wang Z.A.,and Wang H.Z.,2014,Optimization of start codon targeted polymorphism system in medicinal Chrysanthemum morifolium based on orthogonal design,Henongxue Bao(Acta Agriculturae Nucleatae Sinica),28(10):1781-1789(何仁锋,冯尚国,陈喆,高岭,沈晓霞,沈宇峰,王志安,王慧中,2014,药用菊花SCo T-PCR反应体系的正交优化核农学报,28(10):1781-1789)
Hou X.G.,Wang J.,Jia T.,Zhang Y.Q.,Hou J.,and Li J.J.,2011,Orthogonal optimization of SCo T-PCR system and primer screening of tree peony,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),26(5):92-96(侯小改,王娟,贾甜,张钰乾,侯娟,李嘉珏,2011,牡丹SCo T分子标记正交优化及引物筛选,华北农学报,26(5):92-96)
Jiang Q.Q.,Long G.Y.,Li W.W.,and Deng Z.N.,2011,Identification of genetic variation of sweet orange clones from hunan by using SCo T markers and sequence analysis,Zhong guo Nongxue Tongbao(Chinese Agricultural Science Bulletin),27(6):148-154(蒋巧巧,龙桂友,李武文,邓子牛,2011,SCo T结合克隆测序鉴别湖南甜橙变异类型,中国农学通报,27(6):148-154)
Jiang X.F.,Gao Y.H.,Tong Z.K.,and Huang C.H.,2013,Establishment and optimizing of SCo T-PCR system for Lycoris,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&FUniversity),30(3):444-452(姜小凤,高燕会,童再康,黄春红,2013,石蒜属植物SCo T-PCR反应体系构建及优化,浙江农林大学学报,30(3):444-452)
Li P.R.,Jiang J.F.,Chen S.M.,Guan Z.Y.,Liao Y.,Fang W.M.,and Chen F.D.,2013,Establishment and optimization of SCo T molecular marker system in chrysanthemum and its application of analysis on genetic diversity,Yuanyi Xuebao(Acta Horticulturae Sinica),40(10):2015-2025(李丕睿,蒋甲福,陈素梅,管志勇,廖园,房伟民,陈发棣,2013,菊属植物SCo T分子标记技术在遗传多样性分析中的应用,园艺学报,40(10):2015-2025)
Li X.F.,Zhang H.G.,Guan C.Y.,and Zhang Y.,2009,Establishment and optimization of RAPD-PCR reaction system for Larix gmeinii(Rupr.)using orthogonal design,Zhiwu Yanjiu(Bullet of Botanical Research),29(1):80-85(李雪峰,张含国,贯春雨,张瑶,2009,利用正交设计优化兴安落叶松RAPD-PCR反应体系,植物研究,29(1):80-85)
Liu X.Q.,Su J.L.,Li C.,He L.S.,Xiao Z.,and Jia X.P.,2016,Anew Rhododendron cultiva‘Zijin Fenyu’,Yuanyi Xuebao(Acta Horticulturae Sinica),43(10):2067-2068(刘晓青,苏家乐,李畅,何丽斯,肖政,贾新平,2016,杜鹃花新品种‘紫金粉玉’,园艺学报,43(10):2067-2068)
Long Z.J.,Fan L.Z.,Xu G.,Hu S.L.,and Han G.H.,2015,Application advance of SCo T molecular markers in plants,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),16(2):336-343(龙治坚,范理璋,徐刚,胡尚连,韩国辉,2015,SCo T分子标记在植物研究中的应用进展,植物遗传资源学报,16(2):336-343)
Long Z.J.,Wang Y.,Han G.H.,Chen Z.,Xiang S.Q.,Liang G.L.,and Lin Q.,2013,Establishment and optimization of SCo Tsystem of mustard,Xi'nan Daxue Xuebao(Journal of Southwest University(Nature Science Edition)),35(4):20-25(龙治坚,王莹,韩国辉,陈志,向素琼,梁国鲁,林清,2013,芥菜SCo T分析体系的建立与优化,西南大学学报(自然科学版),35(4):20-25)
Lu C.R.,Yu S.X.,Yu J.W.,Fan S.L.,Song M.Z.,Wang W.,and Ma S.J.,2008,Development and appraisement of functiona molecular marker:intron sequence amplified polymorphism Yichuan(Hereditas),30(9):1207-1216(陆才瑞,喻树迅,于霁雯,范术丽,宋美珍,王武,马淑娟,2008,功能型分子标记(ISAP)的开发及评价,遗传,30(9):1207-1216)
Luo C.,2012,Study on SCo T maker and analysis on genes of stress-related and important flowering time in mango,Disser tation for Ph.D.,Guangxi University,Supervisor:He XH.,pp.77(罗聪,2012,芒果SCo T分子标记与逆境和重要开花时间相关基因研究,广西大学,导师:何新华,pp.77)
Luo T.,Yang H.X.,Cen H.F.,Liu X.H.,Gao Y.J.,Duan W.X.Zhang R.H.,Liu X.H.,Song H.Z.,Huang Y.X.,and Zhang G.M.,2013,Application of scot molecular marker in construction of molecular genetic linkage map of Saccharum spontaneum L.,Zhiwu Yichuan Ziyuan Xuebao(Jounal of Plant Genetic Resources),14(4):704-710(罗霆,杨海霞,岑华飞,刘许辉,高轶静,段维兴,张荣华,刘昔辉,宋焕忠黄玉新,张革民,2013,SCo T分子标记在割手密遗传图谱构建中的应用,植物遗传资源学报,14(4):704-710)
Pang X.A.,Bai B.W.,Jiang X.,Chen J.L.,and Jiao P.P.,2015Total DNA extraction and RAPD system optimization of Tamarix plants in tarim basin desert,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(9)2015-2023(庞新安,白宝伟,姜喜,陈加利,焦培培,2015塔里木盆地荒漠区柽柳属植物总DNA提取及RAPD体系优化,基因组学与应用生物学,34(9):2015-2023)
Qin G.X.,He Q.,Liang G.L.,Lei J.J.,and Guo Q.G.,2012,Establishment and optimization of SCo T-PCR system in Fragaria,Guoshu Xuebao(Journal of Fruit Science),29(3):393-397(秦国新,何桥,梁国鲁,雷家军,郭启高,2012,草莓属植物SCo T分析体系的建立及优化,果树学报,29(3):393-397)
Qiu L.H.,Chen R.F.,Huang X.,Wu J.M.,Li Y.R.,Li Q.,and Luo H.M.,2016,Research and application of c DNA-SCo T on plants:a technology about gene differential expression,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Bio-technology),24(11):1771-1780(丘立杭,陈荣发,黄杏,吴建明,李杨瑞,李强,罗含敏,2016,基因差异表达技术c D-NA-SCo T在植物上的研究应用,农业生物技术学报,24(11):1771-1780)
Scariot V.,Keyser E.D.,Handa T.,and Riek D.,2007,Comparative study of the discriminating capacity and effectiveness of AFLR,STMS and EST markers in assessing genetic relationships among evergreen azaleas,Plant Breeding,126(10):207-212
Shi S.G.,Sun Z.H.,Li S.F.,Xie W.J.,Liu B.,and Wang Y.L.2013,Establishment of ISSR-PCR reaction system for Rhododendron lapponicum,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),41(27):10977-10979,11040(石绍高,孙正海,李世峰,解玮佳,刘邦,王永路,2013,高山杜鹃ISSR-PCR反应体系建立,安徽农业科学,41(27):10977-10979,11040)
Shi W.D.,Yang C.F.,Yang L.T.,Ju X.X.,Luo H.L.,Kang H.W.,and Zhang L.,2015,Assessment of phenotype diversity and SCo T polymorphism of 15 Chinese kales,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):615-621(史卫东,杨翠芳,杨丽婷,琚茜茜,罗海玲,康红卫,张力,2015,15个芥蓝的表型多样性和SCo T多态性评价,基因组学与应用生物学,34(3):615-621)
Wang Y.,Zhao L.Y.,Shen H.W.,Qi Y.,Shi L.H.,and Wu D.,2014,Optimization for SCo T system and primer selection of Rose rugosa,Beifang Yuanyi(Northern Horticulture),(23):91-96(王玉,赵兰勇,申宏伟,齐宇,石良红,吴迪,2014,玫瑰SCo T-PCR反应体系优化及引物筛选,北方园艺,(23):91-96)
Wu Y.Y.,Tao Q.J.,Li B.,and Xu D.Y.,2013,An optimal SRAP-PCR system of Rhododendron and its genetic diversi ty,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),30(6):844-851(吴月燕,陶巧静,李波,许丹叶,2013,西洋杜鹃SRAP体系优化及遗传多样性分析,浙江农林大学学报,30(6):844-851)
Xie Y.H.,Xia D.A.,Jiang J.,and Lin P.,2005,Optimization for ISSR-PCR system of Fraxinus mandshurica Rupr,using orthogonal design,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),3(3):445-450(谢运海,夏德安,姜静,林萍,2005,利用正交设计优化水曲柳ISSR-PCR反应体系,分子植物育种,3(3):445-450)
Yan S.H.,Zheng W.H.,and Ding B.Y.,2012,Opitimization of ISSR reaction conditions and analysis of phylogenetic relationship for Rhododendron huadingense,Guangxi Zhiwu(Guihaia),32(5):593-598(颜士辉,郑蔚虹,丁炳扬,2012,华顶杜鹃ISSR反应体系的优化及亲缘关系的初步分析,广西植物,32(5):593-598)
Yang X.X.,Zheng J.,Leng P.S.,Wang Y.,and Wang Y.T.,2014,Optimization of ISSR-PCR system and selection of primers for Syringa reticulate var.amurensis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(5):1018-1026(杨晓霞,郑健,冷平生,王羽,王宇婷,2014,暴马丁香ISSR-PCR反应体系优化及引物筛选,分子植物育种,12(5):1018-1026)
Yuan W.J.,Ma Y.F.,Dong M.F.,Han Y.J.,Zhang W.R.,and Shang F.D.,2015,Establishment and genetic diversity analysis of SCo T molecular maker system for Osmanthus fragrans,Yuanyi Xuebao(Acta Horticulturae Sinica),34(3):569-575(袁王俊,马寅峰,董美芳,韩远记,张维瑞,尚富德,2015,桂花SCo T标记体系的建立及其在遗传多样性分析中的应用,园艺学报,34(3):569-575)
Zhang B.,Lu S.J.,Shi L.,Zou D.F.,Wang D.H.,Xu S.Y.,Long Z.J.,and Han G.H.,2016,Establishment and optimization of SCo T-PCR system of an endangered and rare species Urophysa rockii Ulbr,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(9):2453-2459(张波,陆世家,石力,邹大方王德怀,徐双燕,龙治坚,韩国辉,2016,珍稀濒危植物距瓣尾囊草(Urophysa rockii Ulbr)SCo T-PCR分析体系的建立与优化,分子植物育种,14(9):2453-2459)
Zhang J.Y.,Guo D.L.,Gong Y.,Liu C.H.,Li M.,and Zhang G.H.2011,Optimization of start codon targeted polymorphism PCR(SCo T-PCR)system in Vitis vinifera,Guoshu Xuebao(Journal of Fruit Science),28(2):209-214(张君玉,郭大龙龚莹,刘崇怀,李猛,张国海,2011,葡萄目标起始密码子多态性反应体系的优化,果树学报,28(2):209-214)
Zhang L.,Zhou L.Y.,Xiao Q.W.,and Wu Z.K.,2007,Application of orthogonal design in rhododendron RAPD reaction system,Beifang Yuanyi(Northern Horticulture),(5):124-126(张丽,周兰英,肖千文,吴开志,2007,正交试验设计在建立杜鹃花RAPD-PCR反应体系中的应用,北方园艺,(5)124-126)
Zhang Y.H.,Shen X.Q.,Liu X.Y.,and Zhao F.J.,2010,Optimation for ISSR-PCR system of Rhododendron mucronulatum Hubei Nongye Kexue(Hubei Agricultural Sciences),49(11)2657-2659(张艳红,沈向群,刘旭颖,赵凤军,2010,迎红杜鹃ISSR-PCR反应体系建立,湖北农业科学,49(11):2657-2659)
Zhao R.Q.,Gao Y.H.,Zhang X.L.,and Si J.P.,2012,Establishment and optimization of SCo T-PCR reaction system for Dendrobium officinale,Henongxue Bao(Journal of Nuclear Agricultural Scinences),26(4):648-655(赵瑞强,高燕会,章晓玲,斯金平,2012,铁皮石斛SCo T-PCR反应体系构建及优化,核农学报,26(4):648-655)
Zhao X.H.,and Wang M.Y.,2005,Optimization of RAPD condition of rhododendron,Shengwu Jishu(Biotechnology),15(1):41-43(赵喜华,王曼莹,2005,杜鹃花RAPD条件的优化,生物技术,15(1):41-43)
Zhuang D.F.,Cao H.N.,Zhou L.,Xu Y.,He W.,and Huang Y.,2008,Establishmet of RAPD system and DNA extraction from rhododendron in Changbai Mountain,Hubei Nongye Kexue(Hubei Agricultural Sciences),47(3):254-257(庄得凤,曹后男,周兰,徐颖,何伟,黄岳,2008,长白山杜鹃花基因组DNA提取及RAPD体系的建立,湖北农业科学,47(3):254-257)