草莓组培苗遗传稳定性的ISSR检测及DNA甲基化变异
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摘要
以贵州主栽草莓品种-‘红颜’和‘章姬’,继代次数为第0、5、7、9、10、12、14、18次以及移栽时长为1月、2月、3月的试管苗为材料,利用ISSR标记和MSAP检测,对其遗传变异情况作了探讨。红颜采用49个ISSR引物扩增出207个标记,章姬采用49个ISSR引物扩增出214个标记。用MS+0.5 mg/L BA+0.1 mg/L IBA+30 g/L蔗糖+7 g/L琼脂(pH 6.0)培养基继代红颜至12代及章姬至18代DNA水平突变率为0,仍然保持其遗传稳定性,可采用组培快繁;利用MSAP技术得出随继代次数的增加,组培苗的甲基化敏感多态性呈现出下降趋势。但在田间无性繁殖过程中,组培苗DNA甲基化水平又逐渐升高,且两个品种的DNA甲基化变异程度大致相同。推测在组培环境下诱导草莓基因组DNA发生的去甲基化并不稳定,可能与适应组培环境有关。
Based on ISSR markers and MSAP detection, we explored effects of the times of subculture(0, 5, 7,9, 10, 12, 14, 18) and transplantation(1, 2, 3 months) on the genetic diversity of strawberry(Fragariag ananassa 'Benihoppe' and 'Akihime') which is widely cultivated in Guizhou Province. The results showed that 207 bands were amplified from 49 ISSR primers of 'Benihoppe', 214 bands were amplified from 49 ISSR primers of 'Akihime'.After subculture with MS+0.5 mg/L BA+0.1 mg/L IBA+30 g/L sucrose+7 g/L agar(pH 6.0) culture medium, the mutation rate of DNA level of 'Benihoppe' from 0 to 12 generation and 'Akihime' from 0 to 18 generation were 0,and their genetic stability still could be maintained, so tissue culture could be used for rapid propagation.Methylation-sensitive amplified polymorphism of tissue culture seedlings showed a downward trend with the increase of subculture times by MSAP. However DNA methylation levels of tissue culture seedlings during the asexual propagation in the field increased gradually,and the DNA methylation variation was similar between the two strawberry cultivars. We speculated that the demethylation of strawberry genomic DNA induced by tissue culture might be unstable, which might be related to the adaptation to tissue culture environment.
Based on ISSR markers and MSAP detection, we explored effects of the times of subculture(0, 5, 7,9, 10, 12, 14, 18) and transplantation(1, 2, 3 months) on the genetic diversity of strawberry(Fragariag ananassa 'Benihoppe' and 'Akihime') which is widely cultivated in Guizhou Province. The results showed that 207 bands were amplified from 49 ISSR primers of 'Benihoppe', 214 bands were amplified from 49 ISSR primers of 'Akihime'.After subculture with MS+0.5 mg/L BA+0.1 mg/L IBA+30 g/L sucrose+7 g/L agar(pH 6.0) culture medium, the mutation rate of DNA level of 'Benihoppe' from 0 to 12 generation and 'Akihime' from 0 to 18 generation were 0,and their genetic stability still could be maintained, so tissue culture could be used for rapid propagation.Methylation-sensitive amplified polymorphism of tissue culture seedlings showed a downward trend with the increase of subculture times by MSAP. However DNA methylation levels of tissue culture seedlings during the asexual propagation in the field increased gradually,and the DNA methylation variation was similar between the two strawberry cultivars. We speculated that the demethylation of strawberry genomic DNA induced by tissue culture might be unstable, which might be related to the adaptation to tissue culture environment.
引文
Fan Q.J.,Zheng S.C.,Yan F.X.,Zhang B.X.,Qiao G.,and Wen X.P.,2013,Efficient regeneration of dragon fruit(Hylocereus undatus)and an assessment of the genetic fidelity of in vitro-derived plants using ISSR markers,The Journal of Pomology Horticultural Science,88(5):631-637
Finnegan E.J.,Peacock W.J.,and Dennis E.S.,2000,DNAmethylation,a key regulator of plant development and other processes,Curr.Opin.Genet.Dev.,10(2):217-223
Gao G.Z.,Ying F.,Chen B.Y.,Li H.,Lv X.D.,Yan G.X.,Xu K.,and Wu X.M.,2011,DNA methylation of seed in response to heat stress in Brassica rapa L.,Zuowu Xuebao(Journal of Crop),37(9):1597-1604(高桂珍,应菲,陈碧云,李浩,吕晓丹,闫贵欣,许鲲,伍晓明,2011,热胁迫过程中白菜型油菜种子DNA的甲基化,作物学报,37(9):1597-1604)
Giménez C.,García E.D.,Enrech N.X.D.,and Blanca I.,2001,Somaclonal variation in banana:cytogenetic and molecular characterization of the somaclonal variant cien BTA-03,In Vitro Cell.Dev-Pl.,37(2):217-222
Gu D.Z.,Zhu J.Y.,Feng Y.,Jiang Y.T.,Xie Y.J.,Zhang L.N.,and Ba C.Y.,2010,Technology for stolon seedling in vitro of strawberry and virus eradication by combing the high temperature treatment with shoot tip culture,Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest A&F University),38(11):89-94(顾地周,朱俊义,冯颖,姜云天,谢艳君,张丽娜,巴春影,2010,草莓试管内诱导匍匐茎和高温处理结合茎尖培养脱毒技术研究,西北农林科技大学学报,38(11):89-94)
Han B.M.,Zhao K.,Li H.,Gao X.Y.,and Zhang Z.H.,2010,DNA methylation variation induced by tissue culture in Fragaria ananassa Duch,Zhiwu Shengli Xuebao(Journal of Plant Physiology),46(8):797-802(韩柏明,赵恺,李贺,高秀岩,张志宏,2010,组织培养导致的草莓DNA甲基化变异,植物生理学报,46(8):797-802)
Haque M.S.,Nath U.K.,Iqbal M.S.,and Rowshon A.K.,2015,Assessment of field performance and genetic diversity analysis of tissue culture variants of strawberry,Journal of Agricultural Technology,11(1):107-125
Huang Y.Y.,Zhang H.J.,Xing Y.X.,Qi Y.,Sun Q.Q.,Zhou C.L.,Zhao B.,and Guo Y.D.,2013,Effects of NaCl stress on seed germination and DNA methylation status detected by MSAP analysis in cucumber,Zhongguo Nongye Kexue(S-cientia Agricultura Sinica),46(8):1646-1656(黄韫宇,张海军,邢燕霞,齐艳,孙倩倩,周春蕾,赵冰,郭仰东,2013,NaCl胁迫对黄瓜种子萌发的影响及DNA甲基化的MSAP分析,中国农业科学,46(8):1646-1656)
Li H.L.,Wu C.T.,and Li W.G.,2011,Analysis on DNA methylation of rubber by mehylation sensitive amplification polymorphism,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),9(1):69-73(李海林,吴春太,李维国,2011,巴西橡胶树DNA甲基化的MSAP分析,分子植物育种,9(1):69-73)
Li W.D.,2006,The optimization of produce system and the establishment of quality monitor system of Carica papaya L.propagating plantlets,Thesis for M.S.,South China University of Tropical Agriculture,Supervisor:Zhou P.,pp.53(李卫东,2006,番木瓜组培苗生产体系的优化及质量监测体系的建立,硕士学位论文,华南热带农业大学,导师:周鹏,pp.53)
Li X.,Yu X.,Wang N.,and Liu B.,2007,Genetic and epigenetic instabilities induced by tissue culture in wild barley(Hordeum brevisubulatum(Trin.)Link),Plant Cell Tiss.Org.,90(2):153-168
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),30(10):1379-1382(梁弘伟,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)
Liu F.P.,2010,The genetic bases of plant somaclonal variation and the main influence factors,Jiyinzuxue Yu Yingyong S-hengwuxue(Genomics and Applied Biology),29(6):1142-1151(刘福平,2010,植物体细胞无性系变异的遗传基础及主要影响因素,基因组学与应用生物学,29(6):1142-1151)
Liu P.F.,Qiao G.,and Wen X.P.,2016,DNA methylation variation of in vitro pitaya shoots and its response to exogenous GA application,Huazhong Nongye Daxue Xuebao(Journal of Huazhong Agricultural University),35(5):18-26(刘鹏飞,乔光,文晓鹏,2016,火龙果组培苗DNA甲基化变化及应答赤霉素效应,华中农业大学学报,35(5):18-26)
Liu Y.,Peng Y.,He R.,Li L.S.,Tang G.H.,Wang C.Y.,Xu M.,Zeng C.B.,and Tang H.,2016,Genetic diversity of Camellia oleifera in Hainan by ISSR,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(2):517-523(刘扬,彭赟,贺瑞,李连胜,唐光华,王辰尧,徐敏,曾灿彬,汤华,2016,基于ISSR标记的海南油茶种质资源遗传多样性分析,分子植物育种,14(2):517-523)
Martienssen R.A.,and Colot V.,2001,DNA methylation and epigenetic inheritance in plants and filamentous fungi,Science,293(5532):1070
Nie Q.,and Wen X.P.,2017,Somaclonal variation of pitaya(Hylocereus undatus)in vitro plants and identification by molecular markers,Guoshu Xuebao(Journal of Fruit Science),34(12):1527-1536(聂琼,文晓鹏,2017,火龙果组培苗体细胞无性系变异及其分子检测,果树学报,34(12):1527-1536)
Tang X.M.,Tao X.,Wang Y.,and Ma X.R.,2014,Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism(MSAP)technique,Mol.Genet.Genomics,289(6):1075-1084
Tian T.,Liu P.F.,Qiao G.,and Wen X.P.,2015,Micropropagation of Prunus pseudocerasu'Manaohong'and assessments of its genetic variation,Xinan Daxue Xuebao(Journal of Southwest University),37(10):30-37(田田,刘鹏飞,乔光,文晓鹏,2015,玛瑙红樱桃离体快繁及组培苗的变异检测,西南大学学报,37(10):30-37)
Wang Z.M.,Niu Y.,Tang Q.L.,and Song M.,2013,RAPD and ISSR analyses of genomic DNA of diploid and autotetraploid Zingiber of ficinale rosco,Xinan Daxue Xuebao(Journal of Southwest University),35(2):15-19(王志敏,牛义,汤青林,宋明,2013,生姜二倍体与四倍体基因组DNA的RAPD和ISSR分析,西南大学学报,35(2):15-19)
Zeng L.,Yuan Q.H.,Wang F.,and Wang Y.,2013,Genetic diversity analysis of Agropyon germplasm resources by ISSR,Caoye Xuebao(Acta Prataculturae Sinica),22(1):260-267(曾亮,袁庆华,王方,王瑜,2013,冰草属植物种质资源遗传多样性的ISSR分析,草业学报,22(1):260-267)
Zhang X.Y.,Zhang Z.H.,Gao X.Y.,Li H.,and Du G.D.,2006,Epigenetic variation in characteristics of the micro-propagated strawberry plants and their offsprings,Guoshu Xuebao(Journal of Fruit Science),23(4):542-546(张馨宇,张志宏,高秀岩,李贺,杜国栋,2006,草莓微繁殖苗及其后代性状表观遗传变异研究,果树学报,23(4):542-546)
Finnegan E.J.,Peacock W.J.,and Dennis E.S.,2000,DNAmethylation,a key regulator of plant development and other processes,Curr.Opin.Genet.Dev.,10(2):217-223
Gao G.Z.,Ying F.,Chen B.Y.,Li H.,Lv X.D.,Yan G.X.,Xu K.,and Wu X.M.,2011,DNA methylation of seed in response to heat stress in Brassica rapa L.,Zuowu Xuebao(Journal of Crop),37(9):1597-1604(高桂珍,应菲,陈碧云,李浩,吕晓丹,闫贵欣,许鲲,伍晓明,2011,热胁迫过程中白菜型油菜种子DNA的甲基化,作物学报,37(9):1597-1604)
Giménez C.,García E.D.,Enrech N.X.D.,and Blanca I.,2001,Somaclonal variation in banana:cytogenetic and molecular characterization of the somaclonal variant cien BTA-03,In Vitro Cell.Dev-Pl.,37(2):217-222
Gu D.Z.,Zhu J.Y.,Feng Y.,Jiang Y.T.,Xie Y.J.,Zhang L.N.,and Ba C.Y.,2010,Technology for stolon seedling in vitro of strawberry and virus eradication by combing the high temperature treatment with shoot tip culture,Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest A&F University),38(11):89-94(顾地周,朱俊义,冯颖,姜云天,谢艳君,张丽娜,巴春影,2010,草莓试管内诱导匍匐茎和高温处理结合茎尖培养脱毒技术研究,西北农林科技大学学报,38(11):89-94)
Han B.M.,Zhao K.,Li H.,Gao X.Y.,and Zhang Z.H.,2010,DNA methylation variation induced by tissue culture in Fragaria ananassa Duch,Zhiwu Shengli Xuebao(Journal of Plant Physiology),46(8):797-802(韩柏明,赵恺,李贺,高秀岩,张志宏,2010,组织培养导致的草莓DNA甲基化变异,植物生理学报,46(8):797-802)
Haque M.S.,Nath U.K.,Iqbal M.S.,and Rowshon A.K.,2015,Assessment of field performance and genetic diversity analysis of tissue culture variants of strawberry,Journal of Agricultural Technology,11(1):107-125
Huang Y.Y.,Zhang H.J.,Xing Y.X.,Qi Y.,Sun Q.Q.,Zhou C.L.,Zhao B.,and Guo Y.D.,2013,Effects of NaCl stress on seed germination and DNA methylation status detected by MSAP analysis in cucumber,Zhongguo Nongye Kexue(S-cientia Agricultura Sinica),46(8):1646-1656(黄韫宇,张海军,邢燕霞,齐艳,孙倩倩,周春蕾,赵冰,郭仰东,2013,NaCl胁迫对黄瓜种子萌发的影响及DNA甲基化的MSAP分析,中国农业科学,46(8):1646-1656)
Li H.L.,Wu C.T.,and Li W.G.,2011,Analysis on DNA methylation of rubber by mehylation sensitive amplification polymorphism,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),9(1):69-73(李海林,吴春太,李维国,2011,巴西橡胶树DNA甲基化的MSAP分析,分子植物育种,9(1):69-73)
Li W.D.,2006,The optimization of produce system and the establishment of quality monitor system of Carica papaya L.propagating plantlets,Thesis for M.S.,South China University of Tropical Agriculture,Supervisor:Zhou P.,pp.53(李卫东,2006,番木瓜组培苗生产体系的优化及质量监测体系的建立,硕士学位论文,华南热带农业大学,导师:周鹏,pp.53)
Li X.,Yu X.,Wang N.,and Liu B.,2007,Genetic and epigenetic instabilities induced by tissue culture in wild barley(Hordeum brevisubulatum(Trin.)Link),Plant Cell Tiss.Org.,90(2):153-168
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),30(10):1379-1382(梁弘伟,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)
Liu F.P.,2010,The genetic bases of plant somaclonal variation and the main influence factors,Jiyinzuxue Yu Yingyong S-hengwuxue(Genomics and Applied Biology),29(6):1142-1151(刘福平,2010,植物体细胞无性系变异的遗传基础及主要影响因素,基因组学与应用生物学,29(6):1142-1151)
Liu P.F.,Qiao G.,and Wen X.P.,2016,DNA methylation variation of in vitro pitaya shoots and its response to exogenous GA application,Huazhong Nongye Daxue Xuebao(Journal of Huazhong Agricultural University),35(5):18-26(刘鹏飞,乔光,文晓鹏,2016,火龙果组培苗DNA甲基化变化及应答赤霉素效应,华中农业大学学报,35(5):18-26)
Liu Y.,Peng Y.,He R.,Li L.S.,Tang G.H.,Wang C.Y.,Xu M.,Zeng C.B.,and Tang H.,2016,Genetic diversity of Camellia oleifera in Hainan by ISSR,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(2):517-523(刘扬,彭赟,贺瑞,李连胜,唐光华,王辰尧,徐敏,曾灿彬,汤华,2016,基于ISSR标记的海南油茶种质资源遗传多样性分析,分子植物育种,14(2):517-523)
Martienssen R.A.,and Colot V.,2001,DNA methylation and epigenetic inheritance in plants and filamentous fungi,Science,293(5532):1070
Nie Q.,and Wen X.P.,2017,Somaclonal variation of pitaya(Hylocereus undatus)in vitro plants and identification by molecular markers,Guoshu Xuebao(Journal of Fruit Science),34(12):1527-1536(聂琼,文晓鹏,2017,火龙果组培苗体细胞无性系变异及其分子检测,果树学报,34(12):1527-1536)
Tang X.M.,Tao X.,Wang Y.,and Ma X.R.,2014,Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism(MSAP)technique,Mol.Genet.Genomics,289(6):1075-1084
Tian T.,Liu P.F.,Qiao G.,and Wen X.P.,2015,Micropropagation of Prunus pseudocerasu'Manaohong'and assessments of its genetic variation,Xinan Daxue Xuebao(Journal of Southwest University),37(10):30-37(田田,刘鹏飞,乔光,文晓鹏,2015,玛瑙红樱桃离体快繁及组培苗的变异检测,西南大学学报,37(10):30-37)
Wang Z.M.,Niu Y.,Tang Q.L.,and Song M.,2013,RAPD and ISSR analyses of genomic DNA of diploid and autotetraploid Zingiber of ficinale rosco,Xinan Daxue Xuebao(Journal of Southwest University),35(2):15-19(王志敏,牛义,汤青林,宋明,2013,生姜二倍体与四倍体基因组DNA的RAPD和ISSR分析,西南大学学报,35(2):15-19)
Zeng L.,Yuan Q.H.,Wang F.,and Wang Y.,2013,Genetic diversity analysis of Agropyon germplasm resources by ISSR,Caoye Xuebao(Acta Prataculturae Sinica),22(1):260-267(曾亮,袁庆华,王方,王瑜,2013,冰草属植物种质资源遗传多样性的ISSR分析,草业学报,22(1):260-267)
Zhang X.Y.,Zhang Z.H.,Gao X.Y.,Li H.,and Du G.D.,2006,Epigenetic variation in characteristics of the micro-propagated strawberry plants and their offsprings,Guoshu Xuebao(Journal of Fruit Science),23(4):542-546(张馨宇,张志宏,高秀岩,李贺,杜国栋,2006,草莓微繁殖苗及其后代性状表观遗传变异研究,果树学报,23(4):542-546)