基于FCM测定的香蕉种质倍性分析
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摘要
【目的】基于流式细胞术(FCM)分析结果,从细胞学层面上准确揭示不同基因组类型香蕉种质的倍性差异,为香蕉遗传多样性研究及选择育种提供基础支撑。【方法】以二倍体(AA)‘陵水野生蕉’(Musa AA acuminate‘Lingshui Yesheng Jiao’)为内参,利用FCM技术,对11份香蕉种质资源进行了倍性鉴定分析。【结果】依据(FCM)结果分析,参与分析样品的倍性可分为4大类:第I类有‘红河矮’蕉(M.AAA Cavendish‘Honghe Ai’)和‘陵水野生蕉’,属于2 N;第II类有‘始兴BB’(M.balbisiana‘Shixing BB’)、‘东莞中把大蕉’(M.ABB Cavendish‘Dongguan Zhongba Dajiao’)、‘Pisang Ceylan’和‘FHIA-18’4个种质,属于3 N;第III类有‘Cachaco’‘FHIA-17’‘FHIA-03’和‘TMB×5259-1’4个种质,均属于4 N;第IV类‘红香蕉’(‘Red banana’),在FCM分析中,此种质出现了2个峰值,第一个峰值为52.79,第二个峰值为103.24,测定时2个峰同时出现,且重复性好,可能是二倍体与四倍体混倍形成。【结论】较之传统方法,FCM准确揭示了DNA含量与倍性的关系,能更好地反映香蕉种质资源之间的遗传特征。
【Objective】Banana(Musa spp.), origined from the southeast Asia, is the forth food crop in the world, which are important staple food and income-generating crops, especially to the people in the tropical and subtropical regions Therefore, global banana production plays an important role in dealing with the problems derived from the global warming and the population expansion of the world. However, there are some problems exsiting in banana genetic improvement due to uncertainty of ploidy of varieties or breeds. The accurate determination of banana ploidy level is important for proper genetic manipulation and is a prerequisite for interspecific hybridization. So far, various methods, including conventional breeding, genetic transformation, morphological classification, cytological study, biochemical means and molecular markers, have been used to genetically manipulate bananas so as to improve its production. Chromosome counting is tedious and laborious to determine the ploidy of bananas. Flow cytometry(FCM) was employed to detect the ploidy of bananas in this study. 11 banana varieties, named Musa AAA Cavendish‘Honghe Ai', M. AA acuminate‘Lingshui Yesheng Jiao', M. balbisiana‘Shixing BB',‘Red banana',M. ABB Cavendish‘Dongguan Zhongba Dajiao',‘Pisang Ceylan'(AAB),‘FHIA-18'(AAAB),‘Cachaco'(ABB),‘FHIA-17'(AAAA),‘FHIA-03'(AABB) and‘TMB×5259-1'(ABBB) were chosen as materials.【Methods】Approximately 0.5 cm2 of fresh leaves were chopped with a sharp razor blade in a plastic Petri dish containing 2 m L HRA buffer for each sample. The suspension of nuclei was filtered through a 30 μm nylon mesh to remove large cellular materials. After that, the nuclear DNA of the sample was stained by adding 1 600 μL of 0.02 g·L-1propidium iodide. The sample was mixed gently and incubated briefly on ice before mixing again. A known diploid wild banana germplasm resource, M. AA acuminate‘Lingshui Yesheng Jiao', was used to test the standardisation of flow cytometry by using a Partec Cy Flow?Space Flow Cytometer with a 405 nm laser. At least 5 000-10 000 nuclei were analyzed for each sample. Three leaf samples were tested from each of four plants of each accession.【Results】The FCM was successfully used to analyze the ploidy of 11 banana germplasm resources in present study,. The ploidy of 11 banana clones were divided into four groups. The first group included M. AAA Cavendish‘Honghe Ai'and M.AA acuminate.‘Lingshui Yesheng Jiao', identified as 2 N. The second group contained M. BB balbisiana‘Shixing BB', M. ABB Cavendish‘Dongguan Zhongba Dajiao',‘Pisang Ceylan'and‘FHIA-18', identified as 3 N. The third group consisted of‘Cachaco'‘FHIA-17'‘FHIA-03'and‘TMB×5259-1', identified as 4 N. The fourth group only included‘Red banana', which showed two peak values in the analysis atlas, 52.79 and 103.24, with good repeatability. Our study had some different results from the prevous research. M. AAA Cavendish‘Honghe Ai'previously was classified as triploid, M. BB balbisiana.‘Shixing BB'as diploid,‘FHIA-18'as tetraploid, and‘Cachaco'as triploid,, they were found to be diploid,triploid, triploid and triploid, respectively,in our study.【Conclusions】The ploidy of 11 banana varieties were identified by means of the FCM method and were divide into four groups.‘Red banana'might be a mixoploid derived from two diploid and tetraploid, and this needs further investigation to confirm.
【Objective】Banana(Musa spp.), origined from the southeast Asia, is the forth food crop in the world, which are important staple food and income-generating crops, especially to the people in the tropical and subtropical regions Therefore, global banana production plays an important role in dealing with the problems derived from the global warming and the population expansion of the world. However, there are some problems exsiting in banana genetic improvement due to uncertainty of ploidy of varieties or breeds. The accurate determination of banana ploidy level is important for proper genetic manipulation and is a prerequisite for interspecific hybridization. So far, various methods, including conventional breeding, genetic transformation, morphological classification, cytological study, biochemical means and molecular markers, have been used to genetically manipulate bananas so as to improve its production. Chromosome counting is tedious and laborious to determine the ploidy of bananas. Flow cytometry(FCM) was employed to detect the ploidy of bananas in this study. 11 banana varieties, named Musa AAA Cavendish‘Honghe Ai', M. AA acuminate‘Lingshui Yesheng Jiao', M. balbisiana‘Shixing BB',‘Red banana',M. ABB Cavendish‘Dongguan Zhongba Dajiao',‘Pisang Ceylan'(AAB),‘FHIA-18'(AAAB),‘Cachaco'(ABB),‘FHIA-17'(AAAA),‘FHIA-03'(AABB) and‘TMB×5259-1'(ABBB) were chosen as materials.【Methods】Approximately 0.5 cm2 of fresh leaves were chopped with a sharp razor blade in a plastic Petri dish containing 2 m L HRA buffer for each sample. The suspension of nuclei was filtered through a 30 μm nylon mesh to remove large cellular materials. After that, the nuclear DNA of the sample was stained by adding 1 600 μL of 0.02 g·L-1propidium iodide. The sample was mixed gently and incubated briefly on ice before mixing again. A known diploid wild banana germplasm resource, M. AA acuminate‘Lingshui Yesheng Jiao', was used to test the standardisation of flow cytometry by using a Partec Cy Flow?Space Flow Cytometer with a 405 nm laser. At least 5 000-10 000 nuclei were analyzed for each sample. Three leaf samples were tested from each of four plants of each accession.【Results】The FCM was successfully used to analyze the ploidy of 11 banana germplasm resources in present study,. The ploidy of 11 banana clones were divided into four groups. The first group included M. AAA Cavendish‘Honghe Ai'and M.AA acuminate.‘Lingshui Yesheng Jiao', identified as 2 N. The second group contained M. BB balbisiana‘Shixing BB', M. ABB Cavendish‘Dongguan Zhongba Dajiao',‘Pisang Ceylan'and‘FHIA-18', identified as 3 N. The third group consisted of‘Cachaco'‘FHIA-17'‘FHIA-03'and‘TMB×5259-1', identified as 4 N. The fourth group only included‘Red banana', which showed two peak values in the analysis atlas, 52.79 and 103.24, with good repeatability. Our study had some different results from the prevous research. M. AAA Cavendish‘Honghe Ai'previously was classified as triploid, M. BB balbisiana.‘Shixing BB'as diploid,‘FHIA-18'as tetraploid, and‘Cachaco'as triploid,, they were found to be diploid,triploid, triploid and triploid, respectively,in our study.【Conclusions】The ploidy of 11 banana varieties were identified by means of the FCM method and were divide into four groups.‘Red banana'might be a mixoploid derived from two diploid and tetraploid, and this needs further investigation to confirm.
引文
[1]王卓,徐碧玉,贾彩红,李健平,刘菊华,张建斌,苗红霞,金志强.香蕉MYB基因克隆和表达分析[J].果树学报,2015,32(6):1085-1092.WANG Zhuo,XU Biyu,JIA Caihong,LI Jianping,LIU Juhua,ZAHNG Jianbin,MIAO Hongxia,JIN Zhiqiang.Molecular cloning and expression of MYB gene from banana(Musa acuminata L.AAA group,‘Cavendish’)under the various stresses[J].Journal of Fruit Science,2015,32(6):1085-1092.
[2]董涛,陈新建,凡超,金燕,易干军.我国香蕉产业面临的主要问题与对策[J].广东农业科学,2013,40(11):220-223.DONG Tao,CHEN Xinjian,FAN Chao,JIN Yan,YI Ganjun.Analysis on the development of the banana industry in China[J].Guangdong Agricultural Sciences,2013,40(11):220-223.
[3]孙勇,王丹,仝征,杨倩,常丽丽,王力敏,何丽平,王旭初.香蕉幼苗叶片响应低温胁迫的比较蛋白质组学研究[J].中国农学通报,2015,31(34):216-228.SUN Yong,WANG Dan,TONG Zheng,YANG Qian,CHANG Lili,WANG Limin,HE Liping,WANG Xuchu.Proteomic analysis of banana seedling leaf response to low temperature[J].Chinese Agricultural Science Bulletin,2015,31(34):216-228.
[4]陈源.福建栽培和野生香蕉种质资源离体保存及RAPD分析[D].福州:福建农林大学,2007.CHEN Yuan.In vitro conservation and RAPD analysis of banana(Musa spp.)germplasm in Fujian[D].Fuzhou:Fujian Agriculture&Forestry University,2007.
[5]SIMMONDS N W.The evolution of the bananas[M].London:Longman,1962:1-12.
[6]王正询,林兆平,潘坤清.蕉类的细胞遗传学研究[J].遗传学报,1994,21(6):453-462.WANG Zhengxun,LIN Zhaoping,PAN Kunqing.Cytogenetical studies in Musa(Eumusa)[J].Acta Genetica Sinica,1994,21(6):453-462.
[7]郭计华,李绍鹏,张蕾,谢子四,李新国.不同基因组类型的香蕉核型分析[J].果树学报,2013,30(4):567-572.GUO Jihua,LI Shaopeng,ZHANG Lei,XIE Zisi,LI Xinguo.Karyotype analysis of bananas with different genotypes[J].Journal of Fruit Science,2013,30(4):567-572.
[8]汪艳,肖媛,刘伟,李婷婷,胡锐,乔志仙.流式细胞仪检测高等植物细胞核DNA含量的方法[J].植物科学学报,2015,33(1):126-131.WANG Yan,XIAO Yuan,LIU Wei,LI Tingting,HU Rui,QIAO Zhixian.Operation skills of flow cytometer for detecting nuclear DNA contents in higher plant cells[J].Plant Science Journal,2015,33(1):126-131.
[9]姚丽萍,高银祥,祖元刚.流式细胞术在植物学研究中的应用[J].安徽农学通报,2009,15(9):55-56.YAO Liping,GAO Yinxiang,ZU Yuangang.Applications of flow cytometry in Plant research[J].Anhui Agricultural Science Bulletin,2009,15(9):55-56.
[10]SERGIO J,OCHATT S J.Flow cytometry in plant breeding[J].Cytometry Part A,2008,73:581-598.
[11]GALBRAITH D W,SHIELDS B S.The effects of inhibitors of cell wall synthesis on tobacco protoplast development[J].Physiologia Plantarum,1982,55(1):25-30.
[12]LI G,QUIROS C F.Sequencerelated amplified polymer-phism(SRAP),a new marker system based on a simple PCR reaction:its application to mapping and gene tagging in Brassica[J].Therretical and Applied Genetics,2001,103(2-3):455-461.
[13]LEI Xiaoyong,LIAO Xudong,ZHANG Guiyou,DAI Yaoren.Flow cytometric evidence for hydroxyl radical induced apoptosis in tobacco protoplasts[J].Journal of Integrative Plant Biology,2002,45(8):944-948.
[14]LIU Jihong,XU Xiaoyong,DENG Xiuxin.Characterization of nuclear and cytoplasmic compositions of somatic hybrid plants between sweet orange and sour orange[J].Acta Botanica Sinica,2004,46(10):1206-1211.
[15]SUDA J,KRAHULCOVA A,TRAVNIBEK P,KRAHULEC F.Ploidy level versus DNA ploidy level:an appeal for consistent terminology[J].International Association for Plant Taxonomy,2006,55(2):447-450.
[16]OZAKI Y,NARIKIYO K,FUJITA C,OKUBO H.Ploidy variation of progenies from intra-and inter-ploidy crosses with regard to trisomic production in asparagus(Asparagus officinalis L.)[J].Sexual Plant Reproduction,2004,17(4):157-164.
[17]OCHATT S J.Flow cytometry:ploidy determination,cell cycle analysis,DNA content per nucleus[J].Medicago Truncatula,2006(5):1-13.
[18]潘跃芝,张亦驰,龚洵,李富生.4种人参属植物基因组大小的测定[J].植物分类与资源学报,2014,36(2):233-236.PAN Yuezhi,ZHANG Yichi,GONG Xun,LI Fusheng.Estimation of genome size of four Panax species by Flow Cytometry[J].Plant Diversity and Resources,2014,36(2):233-236.
[19]DOLEZEL J,DOLEZELOVA M,NOVAK F J.Flow cytometric estimation of nuclear DNA amount in diploid bananas(Musa acuminata and M.balbisiana)[J].Biologia Plantarum,1994,36(3):351-357.
[20]龚庆.香蕉(Musa spp.)不对称体细胞融合再生植株的鉴定[D].广州:中山大学,2009.GONG Qing.Identification of putative asymmetric somatic hybrids from banana(Musa spp.)[D].Guangzhou:Zhongshan University,2009.
[21]KAMATE K,BROWN S C,DURAND P,BUREAU J M.Nuclear DNA content and base composition in 28 taxa of Musa[J].Genome,2001,44:622-627.
[22]ROUX N,TOLOZA A,RADECKI Z,ZAPATA-ARIAS F J,DOLEZEL J.Rapid detection of aneuploidy in Musa using flow cytometry[J].Plant Cell Reports,2003,21(5):483-490.
[23]LYSAK M A,DOLEZELOVA M,HORRY J P,DOLEZEL J.Flow cytometric analysis of nuclear DNA content in Musa[J].Theoretical and Applied Genetics,1999,98(8):1344-1350.
[24]NSABIMANA A,VAN STADEN J.Ploidy investigation of bananas(Musa spp.)from the National banana germplasm collection at Rubona-Rwanda by flow cytometry[J].South African Journal of Botany,2006,72(2):302-305.
[25]邓彪.香蕉杂交育种技术初探[D].长沙:湖南农业大学,2014.DENG Biao.A preliminary syudy of cross breeding of banana(Musa)[D].Changsha:Hunan Agricultural University,2014.
[26]陶抵辉,李小红,王利群,周杰良,陈涌,霍稳根.植物染色体倍性鉴定方法研究进展[J].生命科学研究,2009,13(5):453-457.TAO Dihui,LI Xiaohong,WANG Liqun,ZHOU Jieliang,CHEN Yong,HUO Wengen.Progresses on determination of cell chromosome ploidy level of plants[J].Life Science Research,2009,13(5):453-457.
[27]郭计华,李新国,李丽,李绍鹏,罗轩,魏守兴,谢子四.基于ISSR分析28份香蕉种质的基因组DNA多样性[J].基因组学与应用生物学,2012,31(5):492-497.GUO Jihua,LI Xinguo,LI Li,LI Shaopeng,LUO Xuan,WEI shouxing,XIE Zisi.Based on the ISSR analysis of genomic DNA of 28 banana germplasms diversity[J].Genomics and Applied Biology,2012,31(5):492-497.
[28]龚玉莲,曾碧健,陈坚毅,赖海娟.红皮香蕉的核型分析初报[J].广东教育学院学报,2002,22(2):73-75.GONG Yulian,ZENG Bijian,CHEN Jianyi,LAI Haijuan.Karyotype analysis of Musa AAA Red green[J].Journal of Guangdong Education Institute,2002,22(2):73-75.
[2]董涛,陈新建,凡超,金燕,易干军.我国香蕉产业面临的主要问题与对策[J].广东农业科学,2013,40(11):220-223.DONG Tao,CHEN Xinjian,FAN Chao,JIN Yan,YI Ganjun.Analysis on the development of the banana industry in China[J].Guangdong Agricultural Sciences,2013,40(11):220-223.
[3]孙勇,王丹,仝征,杨倩,常丽丽,王力敏,何丽平,王旭初.香蕉幼苗叶片响应低温胁迫的比较蛋白质组学研究[J].中国农学通报,2015,31(34):216-228.SUN Yong,WANG Dan,TONG Zheng,YANG Qian,CHANG Lili,WANG Limin,HE Liping,WANG Xuchu.Proteomic analysis of banana seedling leaf response to low temperature[J].Chinese Agricultural Science Bulletin,2015,31(34):216-228.
[4]陈源.福建栽培和野生香蕉种质资源离体保存及RAPD分析[D].福州:福建农林大学,2007.CHEN Yuan.In vitro conservation and RAPD analysis of banana(Musa spp.)germplasm in Fujian[D].Fuzhou:Fujian Agriculture&Forestry University,2007.
[5]SIMMONDS N W.The evolution of the bananas[M].London:Longman,1962:1-12.
[6]王正询,林兆平,潘坤清.蕉类的细胞遗传学研究[J].遗传学报,1994,21(6):453-462.WANG Zhengxun,LIN Zhaoping,PAN Kunqing.Cytogenetical studies in Musa(Eumusa)[J].Acta Genetica Sinica,1994,21(6):453-462.
[7]郭计华,李绍鹏,张蕾,谢子四,李新国.不同基因组类型的香蕉核型分析[J].果树学报,2013,30(4):567-572.GUO Jihua,LI Shaopeng,ZHANG Lei,XIE Zisi,LI Xinguo.Karyotype analysis of bananas with different genotypes[J].Journal of Fruit Science,2013,30(4):567-572.
[8]汪艳,肖媛,刘伟,李婷婷,胡锐,乔志仙.流式细胞仪检测高等植物细胞核DNA含量的方法[J].植物科学学报,2015,33(1):126-131.WANG Yan,XIAO Yuan,LIU Wei,LI Tingting,HU Rui,QIAO Zhixian.Operation skills of flow cytometer for detecting nuclear DNA contents in higher plant cells[J].Plant Science Journal,2015,33(1):126-131.
[9]姚丽萍,高银祥,祖元刚.流式细胞术在植物学研究中的应用[J].安徽农学通报,2009,15(9):55-56.YAO Liping,GAO Yinxiang,ZU Yuangang.Applications of flow cytometry in Plant research[J].Anhui Agricultural Science Bulletin,2009,15(9):55-56.
[10]SERGIO J,OCHATT S J.Flow cytometry in plant breeding[J].Cytometry Part A,2008,73:581-598.
[11]GALBRAITH D W,SHIELDS B S.The effects of inhibitors of cell wall synthesis on tobacco protoplast development[J].Physiologia Plantarum,1982,55(1):25-30.
[12]LI G,QUIROS C F.Sequencerelated amplified polymer-phism(SRAP),a new marker system based on a simple PCR reaction:its application to mapping and gene tagging in Brassica[J].Therretical and Applied Genetics,2001,103(2-3):455-461.
[13]LEI Xiaoyong,LIAO Xudong,ZHANG Guiyou,DAI Yaoren.Flow cytometric evidence for hydroxyl radical induced apoptosis in tobacco protoplasts[J].Journal of Integrative Plant Biology,2002,45(8):944-948.
[14]LIU Jihong,XU Xiaoyong,DENG Xiuxin.Characterization of nuclear and cytoplasmic compositions of somatic hybrid plants between sweet orange and sour orange[J].Acta Botanica Sinica,2004,46(10):1206-1211.
[15]SUDA J,KRAHULCOVA A,TRAVNIBEK P,KRAHULEC F.Ploidy level versus DNA ploidy level:an appeal for consistent terminology[J].International Association for Plant Taxonomy,2006,55(2):447-450.
[16]OZAKI Y,NARIKIYO K,FUJITA C,OKUBO H.Ploidy variation of progenies from intra-and inter-ploidy crosses with regard to trisomic production in asparagus(Asparagus officinalis L.)[J].Sexual Plant Reproduction,2004,17(4):157-164.
[17]OCHATT S J.Flow cytometry:ploidy determination,cell cycle analysis,DNA content per nucleus[J].Medicago Truncatula,2006(5):1-13.
[18]潘跃芝,张亦驰,龚洵,李富生.4种人参属植物基因组大小的测定[J].植物分类与资源学报,2014,36(2):233-236.PAN Yuezhi,ZHANG Yichi,GONG Xun,LI Fusheng.Estimation of genome size of four Panax species by Flow Cytometry[J].Plant Diversity and Resources,2014,36(2):233-236.
[19]DOLEZEL J,DOLEZELOVA M,NOVAK F J.Flow cytometric estimation of nuclear DNA amount in diploid bananas(Musa acuminata and M.balbisiana)[J].Biologia Plantarum,1994,36(3):351-357.
[20]龚庆.香蕉(Musa spp.)不对称体细胞融合再生植株的鉴定[D].广州:中山大学,2009.GONG Qing.Identification of putative asymmetric somatic hybrids from banana(Musa spp.)[D].Guangzhou:Zhongshan University,2009.
[21]KAMATE K,BROWN S C,DURAND P,BUREAU J M.Nuclear DNA content and base composition in 28 taxa of Musa[J].Genome,2001,44:622-627.
[22]ROUX N,TOLOZA A,RADECKI Z,ZAPATA-ARIAS F J,DOLEZEL J.Rapid detection of aneuploidy in Musa using flow cytometry[J].Plant Cell Reports,2003,21(5):483-490.
[23]LYSAK M A,DOLEZELOVA M,HORRY J P,DOLEZEL J.Flow cytometric analysis of nuclear DNA content in Musa[J].Theoretical and Applied Genetics,1999,98(8):1344-1350.
[24]NSABIMANA A,VAN STADEN J.Ploidy investigation of bananas(Musa spp.)from the National banana germplasm collection at Rubona-Rwanda by flow cytometry[J].South African Journal of Botany,2006,72(2):302-305.
[25]邓彪.香蕉杂交育种技术初探[D].长沙:湖南农业大学,2014.DENG Biao.A preliminary syudy of cross breeding of banana(Musa)[D].Changsha:Hunan Agricultural University,2014.
[26]陶抵辉,李小红,王利群,周杰良,陈涌,霍稳根.植物染色体倍性鉴定方法研究进展[J].生命科学研究,2009,13(5):453-457.TAO Dihui,LI Xiaohong,WANG Liqun,ZHOU Jieliang,CHEN Yong,HUO Wengen.Progresses on determination of cell chromosome ploidy level of plants[J].Life Science Research,2009,13(5):453-457.
[27]郭计华,李新国,李丽,李绍鹏,罗轩,魏守兴,谢子四.基于ISSR分析28份香蕉种质的基因组DNA多样性[J].基因组学与应用生物学,2012,31(5):492-497.GUO Jihua,LI Xinguo,LI Li,LI Shaopeng,LUO Xuan,WEI shouxing,XIE Zisi.Based on the ISSR analysis of genomic DNA of 28 banana germplasms diversity[J].Genomics and Applied Biology,2012,31(5):492-497.
[28]龚玉莲,曾碧健,陈坚毅,赖海娟.红皮香蕉的核型分析初报[J].广东教育学院学报,2002,22(2):73-75.GONG Yulian,ZENG Bijian,CHEN Jianyi,LAI Haijuan.Karyotype analysis of Musa AAA Red green[J].Journal of Guangdong Education Institute,2002,22(2):73-75.