紫花苜蓿细胞质雄性不育系和保持系SCAR标记的鉴定研究
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摘要
本研究通过已获得细胞质雄性不育系材料MS-GN-1A和保持系材料MS-GN-1B筛选与紫花苜蓿细胞质不育恢复基因高度连锁的分子标记并转化为SCAR特异标记,经鉴定分析发现该标记的重复性好,而且稳定,对紫花苜蓿"三系"配套中不育系种子生产鉴定提供研究基础。实验应用100条ISSR引物在紫花苜蓿线粒体基因组水平进行筛选,UBC842、UBS864引物在紫花苜蓿不育系及其同型保持系间显示出特异性片段,经反复验证,其差异条带均稳定出现,条带大小分别为457bp和645bp。对已获得的差异条带进行胶回收连接克隆载体后测序并转化为SCAR特异鉴定标记,以两对特异性引物对新培育的4组紫花苜蓿杂交种的不育系母本和同型保持系父本进行鉴定分析,发现该SCAR标记的重复性稳定。本研究通过ISSR标记对紫花苜蓿的不育系与保持系进行分析,将得到的差异片段克隆测序后转化为SCAR特异标记,该标记可以作为紫花苜蓿不育系种子纯度分析的候选标记,实际应用于不育系种子纯度的鉴定。
The aim of this study was to screen molecular markers highly linked to the restoring gene of cytoplasmic male sterility in alfalfa and transform them into SCAR-specific markers.The identification and analysis showed that the marker was reproducible and stable,which provided a research basis for the production and identification of sterile line seeds in "three lines" of alfalfa.The method was to screen at the mitochondrial genome level of alfalfa with 100 ISSR primers.The UBC842 and UBS864 primers showed specific fragments between the alfalfa sterile line and its homologous maintainer line.After repeated verification,the differential bands were stable,and the band sizes were 457 bp and 645 bp,respectively.At the same time,the obtained differential bands were recovered by gel recovery method,linked to the cloning vector,sequenced and transformed into SCAR-specific identification markers.Then,two pairs of specific primers were used to identify the male sterile line and the homologous maintainer line of the newly cultured four groups of alfalfa hybrids,and the results showed that the repeatability of the SCAR marker was stable.In this study,ISSR markers were used to analyze the sterile and maintainer lines of alfalfa,and the obtained differential fragments were cloned,sequenced and transformed into SCAR-specific markers.This marker can be used as a candidate marker for seed purity analysis of alfalfa sterile lines,and is actually applied to the identification of seed purity of sterile lines.
The aim of this study was to screen molecular markers highly linked to the restoring gene of cytoplasmic male sterility in alfalfa and transform them into SCAR-specific markers.The identification and analysis showed that the marker was reproducible and stable,which provided a research basis for the production and identification of sterile line seeds in "three lines" of alfalfa.The method was to screen at the mitochondrial genome level of alfalfa with 100 ISSR primers.The UBC842 and UBS864 primers showed specific fragments between the alfalfa sterile line and its homologous maintainer line.After repeated verification,the differential bands were stable,and the band sizes were 457 bp and 645 bp,respectively.At the same time,the obtained differential bands were recovered by gel recovery method,linked to the cloning vector,sequenced and transformed into SCAR-specific identification markers.Then,two pairs of specific primers were used to identify the male sterile line and the homologous maintainer line of the newly cultured four groups of alfalfa hybrids,and the results showed that the repeatability of the SCAR marker was stable.In this study,ISSR markers were used to analyze the sterile and maintainer lines of alfalfa,and the obtained differential fragments were cloned,sequenced and transformed into SCAR-specific markers.This marker can be used as a candidate marker for seed purity analysis of alfalfa sterile lines,and is actually applied to the identification of seed purity of sterile lines.
引文
[1]高翠萍,石凤翎,李红.苜蓿雄性不育系MS-4雄蕊发育的细胞形态学研究[J].内蒙古大学学报(自然科学版),2005,(03):288-293
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[4]乌云塔娜,石凤翎,薛晓兰,等.苜蓿雄性不育系杂交组配组合光合生理特性及杂种遗传力研究[J].草地学报,2018,26(03):741-747
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[11]Tanya P,Tacprayoom P,Hadkam Y,et al.Genetic diversity among jatropha and jatropha-related species based on ISSRmarkers[J].Plant Molecular Biology Reporter,2011,29(1):252~264
[12]Shao Y C,Xu L,Chen F S.Genetic diversity analysis of Monascus strains using SRAP and ISSR markers[J].Mycoscience,2011,52(4):224~233
[13]杨本超,肖炳光,陈学军,等.基于ISSR标记的烤烟种质遗传多样性研究[J].遗传,2005,27(5):753~758
[14]Liu L,Ma X,Wei J,et al.The first genetic linkage map of Luohanguo(Siraitia grosvenorii)based on ISSR and SRAPmarkers.Genome,2011,54(1):19-25
[15]任伟,徐安凯,徐博,等.朝鲜碱茅ISSR-PCR反应体系的建立与优化[J].生物技术通报,2012,(6):59~65
[16]吴安迪.芒草遗传多样性研究[M].海南:海南大学,2011
[17]Grzebelus D,Senalik D,Jagosz B,et al.The use of AFLPmarkers for the identification of carrot breeding lines and F1hybrids[J].Plant Breeding,2001,120:526-528
[18]Livneh O,Vardi E,Stram Y,et al.The conversion of a RFLPassay into PCR for the determination of purity in a hybrid pepper cultivar[J].Euphytica,1992,62:97-102
[19]IIbi H.RAPD markers assisted varietal identification and genetic purity test in pepper,Capsicum annuum[J].Scientia Horticulturae,2003,97:211-218
[20]Daniel I O,Adetumbi J A,Oyelakin O O,et al.Application of SSR markers for genetic purity analysis of parental inbred lines and some commercial hybrid maize(Zea mays L)[J].American Journal of Experimental Agriculture,2012,2:597-606
[21]Jang I,Moon J H,Yoon J B,et al.Application of RAPD and SCAR markers for purity testing of F1hybrid seed in chili pepper(Capsicum annuum)[J].Molecules and Cells,2004,18:295-299
[22]Yashitol J,Thirumurugan T,Sundaram R M,et al.Assessment of purity of rice hybrids using microsatellite and STSmarkers[J].Crop Science,2002,42:1369-1373
[23]Paran I,Michelmore R W.Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce[J].Theor.Appl.Genet,1993,85:985-993
[24]王晓武,方智远,孙培田,等.一个用于甘蓝显性雄性不育基因转育辅助选择的SCAR标记[J].园艺学报,2000,27(2):143-144
[25]陈沁滨,侯喜林,陈晓峰,等.洋葱细胞质雄性不育基因RAPD及SCAR分子标记研究[J].南京农业大学学报,2007,30(4):16-19
[26]傅俊生,刘新锐,谢宝贵,等.草菇SCAR遗传标记建立及其杂种鉴定应用[J].中国农学通报,2010,26(17):41-46
[27]周生茂,尚小红,梁任繁,等.苦瓜抗白粉病SCAR分子标记的开发[J].南方农业学报,2013,44(10):1595-1601
[28]张春宝,李玉秋,彭宝,等.线粒体ISSR与SCAR标记鉴定大豆细胞质雄性不育系与保持系[J].大豆科学,2013,32(01):19-22+27
[29]王仁汉,相元萍,王辉.与洋葱雄性不育基因紧密连锁的SCAR标记及其应用[J].北方园艺,2017(02):99-101
[30]Li S F,Wang L J,Deng C L,et al.Identification of male-specific AFLP and SCAR markers in the dioecious plant,Humulus scandens[J].Molecular and Cellular Probes,2017:S0890850817300543
[31]亚秀秀,陈翠萍,肖麓.青海芥菜型油菜多室基因Bjln2连锁标记的SCAR转化[J].青海大学学报,2017,35(02):7-12
[32]毕波,王瑜,袁庆华,等.苜蓿褐斑病抗性相关分子标记验证[J].草地学报,2011,19(04):663-667
[33]马继鹏.辣椒细胞质雄性不育相关基因的克隆及表达研究[D].杨凌:2007,西北农林科技大学,2007:39-43
[2]李晓霞,金梁,王晓娟.几个紫花苜蓿亚(变)种划分和胚珠遗传变异分析[J].中国农业科学,2009,42,1911-1917
[3]伊风艳,龙瑞才,王璐,等.苜蓿雄性不育植株与可育植株现蕾初期花蕾蛋白质组比较[J].西北植物学报,2013,33(10),1964-1971
[4]乌云塔娜,石凤翎,薛晓兰,等.苜蓿雄性不育系杂交组配组合光合生理特性及杂种遗传力研究[J].草地学报,2018,26(03):741-747
[5]Kaul M L H.Male sterility in higher plants,Berlin Heidelberg[J].Springer-Verlag,1988,3-239
[6]张世超,王英哲,王志锋,等.紫花苜蓿雄性不育机理及其应用[J].安徽农业科学,2017,45(11):96-98
[7]Robins J G,Diane L,Austin C T.Genetic Mapping of Biomass Production in Tetraploid Alfalfa[J].Crop Science,2007,47(1):1
[8]Zietkiewicz E,Rafalski A,Labuda D.Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification[J].Genomics,1994,20(2):176~183
[9]Awasthi A K,Nagaraja G M,Naik G V,et al.Genetic diversity and relationships in mulberry(genus Morus)as revealed by RAPD and ISSR marker assays[J].BMC Genetics,2004,5(1):1~9
[10]Han Y,Wang H Y.Genetic diversity and phylogenetic relationships of two closely related northeast China Vicia species revealed with RAPD and ISSR markers[J].Biochemical Genetics,2010,48(5/6):385~401
[11]Tanya P,Tacprayoom P,Hadkam Y,et al.Genetic diversity among jatropha and jatropha-related species based on ISSRmarkers[J].Plant Molecular Biology Reporter,2011,29(1):252~264
[12]Shao Y C,Xu L,Chen F S.Genetic diversity analysis of Monascus strains using SRAP and ISSR markers[J].Mycoscience,2011,52(4):224~233
[13]杨本超,肖炳光,陈学军,等.基于ISSR标记的烤烟种质遗传多样性研究[J].遗传,2005,27(5):753~758
[14]Liu L,Ma X,Wei J,et al.The first genetic linkage map of Luohanguo(Siraitia grosvenorii)based on ISSR and SRAPmarkers.Genome,2011,54(1):19-25
[15]任伟,徐安凯,徐博,等.朝鲜碱茅ISSR-PCR反应体系的建立与优化[J].生物技术通报,2012,(6):59~65
[16]吴安迪.芒草遗传多样性研究[M].海南:海南大学,2011
[17]Grzebelus D,Senalik D,Jagosz B,et al.The use of AFLPmarkers for the identification of carrot breeding lines and F1hybrids[J].Plant Breeding,2001,120:526-528
[18]Livneh O,Vardi E,Stram Y,et al.The conversion of a RFLPassay into PCR for the determination of purity in a hybrid pepper cultivar[J].Euphytica,1992,62:97-102
[19]IIbi H.RAPD markers assisted varietal identification and genetic purity test in pepper,Capsicum annuum[J].Scientia Horticulturae,2003,97:211-218
[20]Daniel I O,Adetumbi J A,Oyelakin O O,et al.Application of SSR markers for genetic purity analysis of parental inbred lines and some commercial hybrid maize(Zea mays L)[J].American Journal of Experimental Agriculture,2012,2:597-606
[21]Jang I,Moon J H,Yoon J B,et al.Application of RAPD and SCAR markers for purity testing of F1hybrid seed in chili pepper(Capsicum annuum)[J].Molecules and Cells,2004,18:295-299
[22]Yashitol J,Thirumurugan T,Sundaram R M,et al.Assessment of purity of rice hybrids using microsatellite and STSmarkers[J].Crop Science,2002,42:1369-1373
[23]Paran I,Michelmore R W.Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce[J].Theor.Appl.Genet,1993,85:985-993
[24]王晓武,方智远,孙培田,等.一个用于甘蓝显性雄性不育基因转育辅助选择的SCAR标记[J].园艺学报,2000,27(2):143-144
[25]陈沁滨,侯喜林,陈晓峰,等.洋葱细胞质雄性不育基因RAPD及SCAR分子标记研究[J].南京农业大学学报,2007,30(4):16-19
[26]傅俊生,刘新锐,谢宝贵,等.草菇SCAR遗传标记建立及其杂种鉴定应用[J].中国农学通报,2010,26(17):41-46
[27]周生茂,尚小红,梁任繁,等.苦瓜抗白粉病SCAR分子标记的开发[J].南方农业学报,2013,44(10):1595-1601
[28]张春宝,李玉秋,彭宝,等.线粒体ISSR与SCAR标记鉴定大豆细胞质雄性不育系与保持系[J].大豆科学,2013,32(01):19-22+27
[29]王仁汉,相元萍,王辉.与洋葱雄性不育基因紧密连锁的SCAR标记及其应用[J].北方园艺,2017(02):99-101
[30]Li S F,Wang L J,Deng C L,et al.Identification of male-specific AFLP and SCAR markers in the dioecious plant,Humulus scandens[J].Molecular and Cellular Probes,2017:S0890850817300543
[31]亚秀秀,陈翠萍,肖麓.青海芥菜型油菜多室基因Bjln2连锁标记的SCAR转化[J].青海大学学报,2017,35(02):7-12
[32]毕波,王瑜,袁庆华,等.苜蓿褐斑病抗性相关分子标记验证[J].草地学报,2011,19(04):663-667
[33]马继鹏.辣椒细胞质雄性不育相关基因的克隆及表达研究[D].杨凌:2007,西北农林科技大学,2007:39-43