橡胶草及其近缘种ISSR反应体系及扩增程序的优化
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摘要
本研究旨在建立和优化橡胶草及其近缘种ISSR (Inter-simple sequence repeat)反应体系和扩增程序。以橡胶草及其近缘种叶片为供试材料,采用单因素试验方法,正交试验方法 L16(44)和极差分析方法,对橡胶草ISSR-PCR反应中4个主要影响因素(dNTP浓度, DNA浓度,引物浓度和Taq DNA聚合酶浓度)进行优化,并在最优反应体系的基础上进行引物和退火温度的筛选。结果表明:dNTP和DNA对PCR扩增结果有较为显著的影响,引物和Taq酶的浓度变化对扩增结果无显著影响。最后确定橡胶草及其近缘种ISSR-PCR反应体系(20.0μL)为:双蒸水14.2μL,10×Buffer (含Mg2+) 2.0μL,DNA模板50.0 ng,10 mmol/L引物1.2μL,2.5 mmol/L dNTP 1.6μL,5 U Taq DNA聚合酶0.1μL。PCR扩增程序为:94℃5 min,94℃45 s,退火1 min,72℃70 s,45个循环,72℃10 min,4℃保存。本研究为橡胶草及其近缘种的遗传多样性和交配系统等后续研究提供理论参考。
The purpose of this study was to establish and optimize the ISSR(Inter-simple sequence repeat) reaction system and amplification procedure for Taraxacum kok-saghyz and its related species. Taraxacum kok-saghyz and its related species leaves were used as test materials, and the single factor test method, orthogonal experiment method L16(44) and range analysis method were used to optimize the four main influencing factors(dNTP concentration, DNA concentration, primer concentration and Taq DNA polymerase concentration) in the ISSR-PCR reaction of Taraxacum kok-saghyz. Also, primer and annealing temperatures were screened based on the optimal reaction system. The results showed that d NTP and DNA had significant effects on PCR amplification results, while the concentration changes of primers and Taq enzyme had no significant effect on the amplification results. Finally,the ISSR reaction system of Taraxacum kok-saghyz and its related species(20.0 μL) was determined as: double distilled water 14.2 μL, 10×Buffer(containing Mg2+) 2.0 μL, DNA template 50.0 ng, 10 mmol/L primer 1.2 μL, 2.5 mmol/L dNTP 1.6 μL, and 5 U Taq DNA polymerase 0.1 μL. The PCR amplification procedure was: 94℃ for 5 min, 94℃for 45 s, annealing 1 min, 72℃ for 70 s, 45 cycles, 72℃ for 10 min, 4℃ for storage. This study could provide a theoretical reference for the follow-up studies on genetic diversity and mating system of Taraxacum kok-saghyz and its related species.
The purpose of this study was to establish and optimize the ISSR(Inter-simple sequence repeat) reaction system and amplification procedure for Taraxacum kok-saghyz and its related species. Taraxacum kok-saghyz and its related species leaves were used as test materials, and the single factor test method, orthogonal experiment method L16(44) and range analysis method were used to optimize the four main influencing factors(dNTP concentration, DNA concentration, primer concentration and Taq DNA polymerase concentration) in the ISSR-PCR reaction of Taraxacum kok-saghyz. Also, primer and annealing temperatures were screened based on the optimal reaction system. The results showed that d NTP and DNA had significant effects on PCR amplification results, while the concentration changes of primers and Taq enzyme had no significant effect on the amplification results. Finally,the ISSR reaction system of Taraxacum kok-saghyz and its related species(20.0 μL) was determined as: double distilled water 14.2 μL, 10×Buffer(containing Mg2+) 2.0 μL, DNA template 50.0 ng, 10 mmol/L primer 1.2 μL, 2.5 mmol/L dNTP 1.6 μL, and 5 U Taq DNA polymerase 0.1 μL. The PCR amplification procedure was: 94℃ for 5 min, 94℃for 45 s, annealing 1 min, 72℃ for 70 s, 45 cycles, 72℃ for 10 min, 4℃ for storage. This study could provide a theoretical reference for the follow-up studies on genetic diversity and mating system of Taraxacum kok-saghyz and its related species.
引文
A kkaya M.S.,Bhagwat A.A.,and Cregan P.B.,1992,Length polymorphism of simple sequence repeat DNA in soybean,Genetics,132(4):1131-1139
An C.,Lee H.,Lee J.,Cheong E.J.,Li Y.H.,and Yi J.S.,2016,Analysis of genetic diversity and differentiation of artificial populations of yellowhorn(Xanthoceras sorbifolium)in China using ISSR markers,J.For.Res.,27(5):1099-1104
Bai J.J.,Wei A.Z.,Wang J.,and Zhang Y.Z.,2009,Optimization of an ISSR analysis system of apricot of Kernel-Consuming based on orthogonal design,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(6):1237-1244(白锦军,魏安智,王佳,张亚转,2009,仁用杏ISSR分析体系的正交优化,分子植物育种,7(6):1237-1244)
Cota-Sánchez J.H.,Remarchuk K.,and Ubayasena K.,2006,Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue,Plant Mol.Biol.Rep.,24(2):161-167
Fu Y.,Luo N.,Yang Q.,Wang Y.Q.,Deng Q.X.,Li J.Q.,Qin H.M.,and Ruan G.L.,2009,Establishment and optimization of ISSR reaction system for species of Eriobotrya,Guoshu Xuebao(Journal of Fruit Science),26(2):180-185(付燕,罗楠,杨芩,王永清,邓群仙,李俊强,秦红玫,阮光伦,2009,枇杷属植物ISSR反应体系的建立和优化,果树学报,26(2):180-185)
He Z.W.,Liu Y.S.,Chen L.H.,Cao M.H.,and Xia J.H.,1998,Orthogonal design-direct analysis for PCR optimization,Hunan Yike Daxue Xuebao(Journal of Social Science of Hunan Medical University),23(4):76-77(何正文,刘运生,陈立华,曹美鸿,夏家辉,1998,正交设计直观分析法优化PCR条件,湖南医科大学学报,23(4):76-77)
Ji J.H.,Han S.S.,Wang J.,He Q.,He B.,Zeng Z.M.,Li X.L.,Liang G.L.,and Guo Q.G.,2014,Genetic diversity analysis of balsam pear(Momordica charantia L.)by ISSR marker,Agricultural Science&Technology,15(7):1073-1075
Jin X.L.,Tian X.H.,and Du W.H.,2015,Orthogonal optimization of ISSR reaction system for chickpea,Caodi Xuebao(Acta Agrestia Sinica),23(6):1303-1309(靳晓丽,田新会,杜文华,2015,鹰嘴豆ISSR反应体系的正交优化,草地学报,23(6):1303-1309)
Ju X.T.,and He Z.P.,2018,Establishment and optimization of IS-SR-PCR system for Impatiens Balsamina,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(1):184-189(巨秀婷,贺宗萍,2018,凤仙花ISSR-PCR体系建立与优化,分子植物育种,16(1):184-189)
Krotkov G.,1945,A review of literature on Taraxacum KokSaghyz rod,The Botanical Review,11(8):417-461
Li R.L.,2012,Study on the genetic diversity of taraxacum Kok-saghyz rodin germplasm in Xinjiang,Thesis for M.S.,Hainan University,Supervisor:Zhang X.C.,and Wu K.X.,pp.55-56(李若霖,2012,新疆橡胶草种质资源遗传多样性研究,硕士学位论文,海南大学,导师:张秀春,吴坤鑫,pp.55-56)
Li X.F.,Qiu T.B.,Zhang H.M.,Hu C.Y.,Hu L.X.,Zheng N.N.,and Tang L.,2012,ISSR analysis on genetic diversity of Taraxaci Herba,Zhongcaoyao(Chinese Traditional and Herbal Drugs),43(10):2025-2029(李喜凤,邱天宝,张红梅,胡春月,胡利欣,郑楠楠,唐露,2012,蒲公英遗传多样性的ISSR分析,中草药,43(10):2025-2029)
Li Y.S.,Lu L.,Lu T.,and Kang J.,2017,Study on phenotypic diversity of Taraxacum kok-saghyz and its sympatric relative species,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(6):1205-1215(李英霜,陆璐,陆婷,康健,2017,橡胶草及其同域近缘种表型多样性研究,西北植物学报,37(6):1205-1215)
Liu Z.,Sha W.,and Yu B.,2012,Study on optimization for IS-SR-PCR reaction system of Grimmiaceae using orthogonal design,Shengwu Jishu(Biotechnology),22(3):61-65(刘卓,沙伟,于冰,2012,正交设计优化紫萼藓科植物的IS-SR-PCR反应体系,生物技术,22(3):61-65)
Morshedloo M.R.,Moghadam M.R.F.,Ebadi A.,and Yazdani D.,2015,Genetic relationships of Iranian Hypericum perforatum L.wild populations as evaluated by ISSR markers,Plant Syst.Evol.,301(2):657-665
Prevost A.,and Wilkinson M.J.,1999,A new system of compar ing PCR primers applied to ISSR fingerprinting of potato cultivars,Theor.Appl.Genet.,98(1):107-112
Qiu J.,Zhang J.C.,Luo S.Q.,Xiao X.Z.,Wang F.,Zhang L.Q.,and Liu S.Z.,2015,Research advances and perspectives on rubber-producing taraxacum,Zhiwu Xuebao(Chinese Bulletin of Botany),50(1):133-141(仇键,张继川,罗世巧,校现周,王锋,张立群,刘实忠,2015,橡胶草的研究进展,植物学报,50(1):133-141)
Raji M.R.,Lotfi M.,Tohidfar M.,Zahedi B.,Carra A.,Abbate L.,and Carimi F.,2018,Somatic embryogenesis of muskmelon(Cucumis melo L.)and genetic stability assessment of regenerants using flow cytometry and ISSR markers,Protoplasma,255(3):873-883
Rasam D.V.,Gokhale N.B.,Sawardekar S.V.,and Patil D.M.,2016,Molecular characterisation of coconut(Cocos nucifera L.)varieties using ISSR and SSR markers,Journal of Pomology&Horticultural Science,91(4):347-352
Shao Q.S.,Guo Q.S.,and Xie Z.C.,2009,Optimization of IS-SR-PCR reaction system for Chrysanthemum morifolium based on analysis of variance,Zhongcaoyao(Chinese Traditional and Herbal Drugs),40(2):284-288(邵清松,郭巧生,谢作成,2009,基于方差分析优化菊花ISSR-PCR反应体系,中草药,40(2):284-288)
Thimmappaiah,Santhosh W.G.,Shobha D.,and Melwyn G.S.,2008,Assessment of genetic diversity in cashew germplasm using RAPD and ISSR markers,Sci.Hortic.,120(3):411-417
Tian Y.L.,Li Z.H.,Yang M.H.,and Zhang B.,2015,Establishment and optimization of Castanopsis tibetana hance IS-SR-PCR reaction system,Zhongnan Linye Keji Daxue Xuebao(Journal of Central South University of Forestry&Technology),35(2):32-37(田艳伶,李志辉,杨模华,张斌,2015,钩栗ISSR-PCR反应体系的建立与优化,中南林业科技大学学报,35(2):32-37)
Yan J.,Yang R.P.,Zhang Y.,Yang C.L.,and Yang Z.A.,2018,Establishment and optimization of ISSR system for peach germplasm resources in high altitude areas of Yunnan province,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(1):190-194(颜景,杨荣萍,张玉,杨春雷,杨正安,2018,云南高海拔地区桃种质资源ISSR体系的建立和优化,分子植物育种,16(1):190-194)
Zhang D.,Ren M.Y.,Zhang Y.D.,and Guan X.,2018,Genetic diversity research on Anisodus tanguticus based on ISSR molecular markers,Zhongcaoyao(Chinese Traditional and Her bal Drugs),49(1):219-226(张盾,任梦云,张银东,关潇,2018,基于ISSR分子标记的野生山莨菪遗传多样性研究,中草药,49(1):219-226)
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification,Genomics,20(2):176-183
An C.,Lee H.,Lee J.,Cheong E.J.,Li Y.H.,and Yi J.S.,2016,Analysis of genetic diversity and differentiation of artificial populations of yellowhorn(Xanthoceras sorbifolium)in China using ISSR markers,J.For.Res.,27(5):1099-1104
Bai J.J.,Wei A.Z.,Wang J.,and Zhang Y.Z.,2009,Optimization of an ISSR analysis system of apricot of Kernel-Consuming based on orthogonal design,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(6):1237-1244(白锦军,魏安智,王佳,张亚转,2009,仁用杏ISSR分析体系的正交优化,分子植物育种,7(6):1237-1244)
Cota-Sánchez J.H.,Remarchuk K.,and Ubayasena K.,2006,Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue,Plant Mol.Biol.Rep.,24(2):161-167
Fu Y.,Luo N.,Yang Q.,Wang Y.Q.,Deng Q.X.,Li J.Q.,Qin H.M.,and Ruan G.L.,2009,Establishment and optimization of ISSR reaction system for species of Eriobotrya,Guoshu Xuebao(Journal of Fruit Science),26(2):180-185(付燕,罗楠,杨芩,王永清,邓群仙,李俊强,秦红玫,阮光伦,2009,枇杷属植物ISSR反应体系的建立和优化,果树学报,26(2):180-185)
He Z.W.,Liu Y.S.,Chen L.H.,Cao M.H.,and Xia J.H.,1998,Orthogonal design-direct analysis for PCR optimization,Hunan Yike Daxue Xuebao(Journal of Social Science of Hunan Medical University),23(4):76-77(何正文,刘运生,陈立华,曹美鸿,夏家辉,1998,正交设计直观分析法优化PCR条件,湖南医科大学学报,23(4):76-77)
Ji J.H.,Han S.S.,Wang J.,He Q.,He B.,Zeng Z.M.,Li X.L.,Liang G.L.,and Guo Q.G.,2014,Genetic diversity analysis of balsam pear(Momordica charantia L.)by ISSR marker,Agricultural Science&Technology,15(7):1073-1075
Jin X.L.,Tian X.H.,and Du W.H.,2015,Orthogonal optimization of ISSR reaction system for chickpea,Caodi Xuebao(Acta Agrestia Sinica),23(6):1303-1309(靳晓丽,田新会,杜文华,2015,鹰嘴豆ISSR反应体系的正交优化,草地学报,23(6):1303-1309)
Ju X.T.,and He Z.P.,2018,Establishment and optimization of IS-SR-PCR system for Impatiens Balsamina,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(1):184-189(巨秀婷,贺宗萍,2018,凤仙花ISSR-PCR体系建立与优化,分子植物育种,16(1):184-189)
Krotkov G.,1945,A review of literature on Taraxacum KokSaghyz rod,The Botanical Review,11(8):417-461
Li R.L.,2012,Study on the genetic diversity of taraxacum Kok-saghyz rodin germplasm in Xinjiang,Thesis for M.S.,Hainan University,Supervisor:Zhang X.C.,and Wu K.X.,pp.55-56(李若霖,2012,新疆橡胶草种质资源遗传多样性研究,硕士学位论文,海南大学,导师:张秀春,吴坤鑫,pp.55-56)
Li X.F.,Qiu T.B.,Zhang H.M.,Hu C.Y.,Hu L.X.,Zheng N.N.,and Tang L.,2012,ISSR analysis on genetic diversity of Taraxaci Herba,Zhongcaoyao(Chinese Traditional and Herbal Drugs),43(10):2025-2029(李喜凤,邱天宝,张红梅,胡春月,胡利欣,郑楠楠,唐露,2012,蒲公英遗传多样性的ISSR分析,中草药,43(10):2025-2029)
Li Y.S.,Lu L.,Lu T.,and Kang J.,2017,Study on phenotypic diversity of Taraxacum kok-saghyz and its sympatric relative species,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(6):1205-1215(李英霜,陆璐,陆婷,康健,2017,橡胶草及其同域近缘种表型多样性研究,西北植物学报,37(6):1205-1215)
Liu Z.,Sha W.,and Yu B.,2012,Study on optimization for IS-SR-PCR reaction system of Grimmiaceae using orthogonal design,Shengwu Jishu(Biotechnology),22(3):61-65(刘卓,沙伟,于冰,2012,正交设计优化紫萼藓科植物的IS-SR-PCR反应体系,生物技术,22(3):61-65)
Morshedloo M.R.,Moghadam M.R.F.,Ebadi A.,and Yazdani D.,2015,Genetic relationships of Iranian Hypericum perforatum L.wild populations as evaluated by ISSR markers,Plant Syst.Evol.,301(2):657-665
Prevost A.,and Wilkinson M.J.,1999,A new system of compar ing PCR primers applied to ISSR fingerprinting of potato cultivars,Theor.Appl.Genet.,98(1):107-112
Qiu J.,Zhang J.C.,Luo S.Q.,Xiao X.Z.,Wang F.,Zhang L.Q.,and Liu S.Z.,2015,Research advances and perspectives on rubber-producing taraxacum,Zhiwu Xuebao(Chinese Bulletin of Botany),50(1):133-141(仇键,张继川,罗世巧,校现周,王锋,张立群,刘实忠,2015,橡胶草的研究进展,植物学报,50(1):133-141)
Raji M.R.,Lotfi M.,Tohidfar M.,Zahedi B.,Carra A.,Abbate L.,and Carimi F.,2018,Somatic embryogenesis of muskmelon(Cucumis melo L.)and genetic stability assessment of regenerants using flow cytometry and ISSR markers,Protoplasma,255(3):873-883
Rasam D.V.,Gokhale N.B.,Sawardekar S.V.,and Patil D.M.,2016,Molecular characterisation of coconut(Cocos nucifera L.)varieties using ISSR and SSR markers,Journal of Pomology&Horticultural Science,91(4):347-352
Shao Q.S.,Guo Q.S.,and Xie Z.C.,2009,Optimization of IS-SR-PCR reaction system for Chrysanthemum morifolium based on analysis of variance,Zhongcaoyao(Chinese Traditional and Herbal Drugs),40(2):284-288(邵清松,郭巧生,谢作成,2009,基于方差分析优化菊花ISSR-PCR反应体系,中草药,40(2):284-288)
Thimmappaiah,Santhosh W.G.,Shobha D.,and Melwyn G.S.,2008,Assessment of genetic diversity in cashew germplasm using RAPD and ISSR markers,Sci.Hortic.,120(3):411-417
Tian Y.L.,Li Z.H.,Yang M.H.,and Zhang B.,2015,Establishment and optimization of Castanopsis tibetana hance IS-SR-PCR reaction system,Zhongnan Linye Keji Daxue Xuebao(Journal of Central South University of Forestry&Technology),35(2):32-37(田艳伶,李志辉,杨模华,张斌,2015,钩栗ISSR-PCR反应体系的建立与优化,中南林业科技大学学报,35(2):32-37)
Yan J.,Yang R.P.,Zhang Y.,Yang C.L.,and Yang Z.A.,2018,Establishment and optimization of ISSR system for peach germplasm resources in high altitude areas of Yunnan province,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(1):190-194(颜景,杨荣萍,张玉,杨春雷,杨正安,2018,云南高海拔地区桃种质资源ISSR体系的建立和优化,分子植物育种,16(1):190-194)
Zhang D.,Ren M.Y.,Zhang Y.D.,and Guan X.,2018,Genetic diversity research on Anisodus tanguticus based on ISSR molecular markers,Zhongcaoyao(Chinese Traditional and Her bal Drugs),49(1):219-226(张盾,任梦云,张银东,关潇,2018,基于ISSR分子标记的野生山莨菪遗传多样性研究,中草药,49(1):219-226)
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification,Genomics,20(2):176-183
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