烟草糖酯SSR分子标记筛选及辅助育种应用
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摘要
为明确与糖酯紧密连锁的SSR分子标记在烟草实际育种工作中的可用性,加快烤烟糖酯遗传改良进程,本研究利用聚丙烯酰胺凝胶电泳技术和GC-MS检测技术,以不同遗传背景的5个烟草种质以及192份重组自交系遗传群体为试验材料,对5个SSR分子标记开展了筛选和验证研究。结果表明,5个分子标记中,PT20315、PT30354两个分子标记在不同遗传背景中多态性稳定,并且其多态性条带与糖酯表型差异的对应性较好;在重组自交系群体中,两个分子标记鉴定的基因型与糖酯表型的一致率分别为93.75%和90.58%,可用于高糖酯烟草分子辅助育种中。目前,利用两个分子标记辅助选择,已经定向改良获得高糖酯K326等新品系材料。
In order to clarify the usability of SSR molecular markers closely linked to sugar esters in the actual breeding of tobacco,and to accelerate the genetic improvement of flue-cured tobacco sugar esters, in this study, using polyacrylamide gel electrophoresis and GC-MS detection techniques, 5 tobacco germplasms with different genetic backgrounds and a genetic population with 192 recombinant inbred lines were used as experimental materials for screening and testing of 5 SSR molecular markers. The results showed that, among the 5 molecular markers, the polymorphisms of 2 molecular markers PT20315 and PT30354 were stable in different genetic backgrounds, and the correspondence between their polymorphic bands and the phenotypic difference of sugar esters were better. In the recombinant inbred line population, the consistency rate between the genotypes identified by the 2 molecular markers and phenotypes of sugar esters reached 93.75% and 90.58% respectively. The 2 molecular markers could be used in the molecular-assisted breeding of high-sugar ester tobacco. Currently, using of the 2 molecular markers in assisted selection has directionally improved new lineage materials such as high sugar ester K326 etc.
In order to clarify the usability of SSR molecular markers closely linked to sugar esters in the actual breeding of tobacco,and to accelerate the genetic improvement of flue-cured tobacco sugar esters, in this study, using polyacrylamide gel electrophoresis and GC-MS detection techniques, 5 tobacco germplasms with different genetic backgrounds and a genetic population with 192 recombinant inbred lines were used as experimental materials for screening and testing of 5 SSR molecular markers. The results showed that, among the 5 molecular markers, the polymorphisms of 2 molecular markers PT20315 and PT30354 were stable in different genetic backgrounds, and the correspondence between their polymorphic bands and the phenotypic difference of sugar esters were better. In the recombinant inbred line population, the consistency rate between the genotypes identified by the 2 molecular markers and phenotypes of sugar esters reached 93.75% and 90.58% respectively. The 2 molecular markers could be used in the molecular-assisted breeding of high-sugar ester tobacco. Currently, using of the 2 molecular markers in assisted selection has directionally improved new lineage materials such as high sugar ester K326 etc.
引文
[1]邸慧慧,史宏志,张大纯.烟草叶片腺毛及其分泌物研究进展[J].贵州农业科学,2009,37(8):61-64.DI H H,SHI H Z,ZHANG D C.Research progress on glandular hair and glandular hair secretion of tobacco leaves[J].Guizhou Agricultural Sciences,2009,37(8):61-64.
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[3]JOHNSON A W,SEVERSON R F.Leaf surface chemistry of tobacco budworm resistant tobacco[J].Journal of Agricultural Entomology,1984,1(1):23-32.
[4]CUTLER H G,SEVERSON R F,COLE P D,et al.Secondary metabolites from higher plants:their possible role as biological control agents[M]//Natural Resistance of Plants to Pests:Roles of Allelochemicals,1986:178-196.
[5]CHORTYK O T,KAYS S J,TENG Q.Characterization of insecticidal sugar esters of petunia[J].Journal of Agricultural and Food Chemistry,1997,45(1):270-275.
[6]JACKSON D M,DANEHOWER D A.Integrated case study:nicotiana leaf surface components and their effects on insect pests and disease[M].Plant Cuticles:an Integrated Functional Approach,1996:231-254.
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[11]王海燕,王鸿旻,刘百战,等.烟草糖酯的分类、检测及其应用研究进展[J].现代食品科技,2010,26(8):866-870.WANG H Y,WANG H M,LIU B Z,et al.Advances in classification,determination and application of sucrose esters from tobacco leaves[J].Modern Food Science and Technology,2010,26(8):866-870.
[12]向章敏,周淑平,耿召良.烟叶表面化学成分的分析研究进展[J].西南农业学报,2015,28(3):1395-1398.XIANG Z M,ZHOU S P,GENG Z L.Research progress in analysis of cuticular constituents of tobacco leaves[J].Southwest China Journal of Agricultural Sciences,2015,28(3):1395-1398.
[13]余金恒,许明忠,黄锋林,等.烟用香精香料物质研究进展[J].河南农业科学,2011,40(2):16-18.YU J H,XU M Z,HUANG F L,et al.Research progress of tobacco flavors and fragrances[J].Journal of Henan Agricultural Sciences,2011,40(2):16-18.
[14]王伟,孔光辉,李佛琳,等.烤烟烟叶腺毛及其分泌物研究进展[J].中国农学通报,2007,23(2):251-254.WANG W,KONG G H,LI F L,et al.Research advances on tobacco leaf glandular trichome and leaf surface exudate[J].Chinese Agricultural Science Bulletin,2007,23(2):251-254.
[15]SCHILMILLER A L,CHARBONNEAU A L,LAST R L.Identification of a BAHD acetyltransferase that produces protective acyl sugars in tomato trichomes[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(40):16377-16382.
[16]SCHILMILLER A L,MOGHE G D,FAN P,et al.Functionally divergent alleles and duplicated loci encoding an acyltransferase contribute to acylsugar metabolite diversity in solanum trichomes[J].Plant Cell,2015,27(4):1002-1017.
[17]VONTIMITTA V,DANEHOWER D A,STEEDE T,et al.Analysis of a Nicotiana tabacum L.genomic region controlling two leaf surface chemistry traits[J].Journal of Agricultural&Food Chemistry,2010,58(1):294-300.
[18]屈亚芳,许明录,曹建敏,等.不同基因型烟草腺毛主要分泌物差异分析[J].中国烟草学报,2018,24(1):45-52.QU Y F,XU M L,CAO J M,et al.Differential analysis of major glandular trichome secretion between different tobacco genotypes[J].Acta Tabacaria Sinica,2018,24(1):45-52.
[19]WANG E,WANG R,DEPARASIS J,et al.Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural product-based aphid resistance[J].Nat Biotechnology,2001,19(4):371-374.
[20]JIA C,WANG Y,ZHU Y,et al.Preparative isolation and structural characterization of sucrose ester isomers from oriental tobacco[J].Carbohydrate Research,2013,372(9):73-77.
[21]GWYNN G R,SEVERSON R F,JACKSON D M,et al.Inheritance of sucrose esters containing beta-methylvaleric acid in tobacco[J].Tob.Sci.,1985,29:79-81.
[22]ASHRAFKHORASSANI M,NAZEM N,TAYLOR L T,et al.Isolation,fractionation,and identification of sucrose esters from various oriental tobaccos employing supercritical fluids[J].Beitr?ge Zur Tabakforschung,2014,23(1):32-45.
[23]蔡莉莉,谢复炜,刘克建,等.香料烟中蔗糖酯的气相色谱/质谱分析[J].烟草科技,2009(3):40-44.CAI L L,XIE F W,LIU K J,et al.Analysis of sucrose esters in oriental tobacco by GC/MS[J].Tobacco Science and Technology,2009(3):40-44.
[24]王瑞玲,王莹莹,毛多斌,等.烟草中蔗糖四酯类化合物的GC-MS分析[J].化学研究与应用,2011,23(8):1030-1035.WANG R L,WANG Y Y,MAO D B,et al.Analysis of sucrose tetra-esters from tobaccos by GC-MS[J].Chemical Research and Application,2011,23(8):1030-1035.
[25]CHEN F,XU H X,ZHANG F Y,et al.Physical mapping of puroindoline b-2 genes and molecular characterization of a novel variant in durum wheat(Triticum turgidum L.)[J].Molecular Breeding,2011,28(2):153-161.
[26]任民,贾兴华,蒋彩虹,等.Bassam和Sanguinetti银染方法在SRAP和TRAP标记中的比较研究[J].生物技术通报,2008(1):113-116.REN M,JIA X H,JIANG C H,et al.Comparison study of bassam and sanguinetti silver staining in the detecting of SRAP and TRAP[J].Biotechnology Bulletin,2008(1):113-116.
[27]NADAKUDUTI S S,UEBLER J B,LIU X X,et al.Characterization of trichome-expressed BAHDacyltransferases in petunia axillaris reveals distinct acylsugar assembly mechanisms within the solanaceae[J].Plant Physiology,2017,175(1):36-50.
[28]KROUMOVA A B,ZAITLIN D,WAGNER G J.Natural variability in acyl moieties of sugar esters produced by certain tobacco and other solanaceae species[J].Phytochemistry,2016,130:218-227.
[29]LIU X,ENRIGHT M,BARRY C S,et al.Profiling,isolation and structure elucidation of specialized acylsucrose metabolites accumulating in trichomes of petunia,species[J].Metabolomics,2017,13(7):85.
[30]LAWSON D M,LUNDE C F,MUTSCHLER M A.Marker-assisted transfer of acylsugar-mediated pest resistance from the wild tomato,lycopersicon pennellii,to the cultivated tomato,lycopersicon esculentum[J].Molecular Breeding,1997,3(4):307-317.
[31]BONIERBALE M W,PLAISTED R L,PINEDA O,et al.QTL analysis of trichome-mediated insect resistance in potato[J].Theoretical and Applied Genetics,1994,87(8):973-987.
[32]LECKIE B M,JONG D M D,MUTSCHLER M A.Quantitative trait loci increasing acylsugars in tomato breeding lines and their impacts on silverleaf whiteflies[J].Molecular Breeding,2012,30(4):1621-1634.
[33]FAN P,MILLER A M,SCHILMILLER A L,et al.In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network[J].Proc Natl Acad Sci U S A,2016,113(2):E239-E248.
[34]FAN P,MILLER A M,LIU X,et al.Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity[J].Nature Communications,2017,8(1):2080.
[35]李传江,庞宏.中式烟叶是发展中式卷烟的原料基础[J].中国烟草科学,2008,29(6):68-70.LI C J,PANG H.Development of chinese-style cigarettes with specific chinese tobacco leaf production[J].Chinese Tobacco Science,2008,29(6):68-70.
[36]李凯,姜涛,才源,等.玉米花期性状的主效SSR标记筛选[J].玉米科学,2015,23(1):33-38.LI K,JIANG T,CAI Y,et al.Screening of the main effect SSR markers of maize flowering[J].Journal of Maize Sciences,2015,23(1):33-38.
[37]常爱霞,屈亚芳,王国平,等.烤烟品种大白筋599特征香气性状的物质及分子基础研究[J].中国烟草科学,2018,39(3):1-9.CHANG A X,QU Y F,WANG G P,et al.Study on key components and molecular basis responsible for the specific aroma traits of flue-cured tobacco dabaijin599[J].Chinese Tobacco Science,2018,39(3):1-9.
[38]王国平.烟草冷杉醇合成基因功能分析及功能标记开发[D].北京:中国农业科学院,2016.WANG G P.Functional analysis and marker development of cis-abnieol synthesis gene in tobacco[D].Beijing:Chinese Academy of Agricultural Sciences,2016.
[2]WEINHOLD A,BALDWIN I T.Trichome-derived o-acyl sugars are a first meal for caterpillars that tags them for predation[J].Proceedings of the National Academy of Sciences of The United States of America,2011,108(19):7855-7859.
[3]JOHNSON A W,SEVERSON R F.Leaf surface chemistry of tobacco budworm resistant tobacco[J].Journal of Agricultural Entomology,1984,1(1):23-32.
[4]CUTLER H G,SEVERSON R F,COLE P D,et al.Secondary metabolites from higher plants:their possible role as biological control agents[M]//Natural Resistance of Plants to Pests:Roles of Allelochemicals,1986:178-196.
[5]CHORTYK O T,KAYS S J,TENG Q.Characterization of insecticidal sugar esters of petunia[J].Journal of Agricultural and Food Chemistry,1997,45(1):270-275.
[6]JACKSON D M,DANEHOWER D A.Integrated case study:nicotiana leaf surface components and their effects on insect pests and disease[M].Plant Cuticles:an Integrated Functional Approach,1996:231-254.
[7]FERRER M,SOLIVEN J,PLOU F J.Synthesis of sugar esters in solvent mixtures by lipases from thermomyces lanuginosusand candida antarctica b,and their antimicrobial properties[J].Enzyme and Microbial Technology,2005,36(4):391-398.
[8]SHINOZAKI Y,MATSUZAKI T,SUHARA S,et al.New types of glycolipids from the surface lipids of nicotiana umbratica[J].Agricultural and Biological Chemistry,1991,55(3):751-756.
[9]SEVERSON R F,ARRENDALE R F,CHORTYK O T,et al.Isolation and characterization of the sucrose esters of the cuticular waxes of green tobacco leaf[J].Journal of Agricultural and Food Chemistry,1985,33(5):870-875.
[10]LEFFINGWELL J C.Chemical constituents of tobacco leaf and differences among tobacco types[J].Social Science Electronic Publishing,2001,1:38-44.
[11]王海燕,王鸿旻,刘百战,等.烟草糖酯的分类、检测及其应用研究进展[J].现代食品科技,2010,26(8):866-870.WANG H Y,WANG H M,LIU B Z,et al.Advances in classification,determination and application of sucrose esters from tobacco leaves[J].Modern Food Science and Technology,2010,26(8):866-870.
[12]向章敏,周淑平,耿召良.烟叶表面化学成分的分析研究进展[J].西南农业学报,2015,28(3):1395-1398.XIANG Z M,ZHOU S P,GENG Z L.Research progress in analysis of cuticular constituents of tobacco leaves[J].Southwest China Journal of Agricultural Sciences,2015,28(3):1395-1398.
[13]余金恒,许明忠,黄锋林,等.烟用香精香料物质研究进展[J].河南农业科学,2011,40(2):16-18.YU J H,XU M Z,HUANG F L,et al.Research progress of tobacco flavors and fragrances[J].Journal of Henan Agricultural Sciences,2011,40(2):16-18.
[14]王伟,孔光辉,李佛琳,等.烤烟烟叶腺毛及其分泌物研究进展[J].中国农学通报,2007,23(2):251-254.WANG W,KONG G H,LI F L,et al.Research advances on tobacco leaf glandular trichome and leaf surface exudate[J].Chinese Agricultural Science Bulletin,2007,23(2):251-254.
[15]SCHILMILLER A L,CHARBONNEAU A L,LAST R L.Identification of a BAHD acetyltransferase that produces protective acyl sugars in tomato trichomes[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(40):16377-16382.
[16]SCHILMILLER A L,MOGHE G D,FAN P,et al.Functionally divergent alleles and duplicated loci encoding an acyltransferase contribute to acylsugar metabolite diversity in solanum trichomes[J].Plant Cell,2015,27(4):1002-1017.
[17]VONTIMITTA V,DANEHOWER D A,STEEDE T,et al.Analysis of a Nicotiana tabacum L.genomic region controlling two leaf surface chemistry traits[J].Journal of Agricultural&Food Chemistry,2010,58(1):294-300.
[18]屈亚芳,许明录,曹建敏,等.不同基因型烟草腺毛主要分泌物差异分析[J].中国烟草学报,2018,24(1):45-52.QU Y F,XU M L,CAO J M,et al.Differential analysis of major glandular trichome secretion between different tobacco genotypes[J].Acta Tabacaria Sinica,2018,24(1):45-52.
[19]WANG E,WANG R,DEPARASIS J,et al.Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural product-based aphid resistance[J].Nat Biotechnology,2001,19(4):371-374.
[20]JIA C,WANG Y,ZHU Y,et al.Preparative isolation and structural characterization of sucrose ester isomers from oriental tobacco[J].Carbohydrate Research,2013,372(9):73-77.
[21]GWYNN G R,SEVERSON R F,JACKSON D M,et al.Inheritance of sucrose esters containing beta-methylvaleric acid in tobacco[J].Tob.Sci.,1985,29:79-81.
[22]ASHRAFKHORASSANI M,NAZEM N,TAYLOR L T,et al.Isolation,fractionation,and identification of sucrose esters from various oriental tobaccos employing supercritical fluids[J].Beitr?ge Zur Tabakforschung,2014,23(1):32-45.
[23]蔡莉莉,谢复炜,刘克建,等.香料烟中蔗糖酯的气相色谱/质谱分析[J].烟草科技,2009(3):40-44.CAI L L,XIE F W,LIU K J,et al.Analysis of sucrose esters in oriental tobacco by GC/MS[J].Tobacco Science and Technology,2009(3):40-44.
[24]王瑞玲,王莹莹,毛多斌,等.烟草中蔗糖四酯类化合物的GC-MS分析[J].化学研究与应用,2011,23(8):1030-1035.WANG R L,WANG Y Y,MAO D B,et al.Analysis of sucrose tetra-esters from tobaccos by GC-MS[J].Chemical Research and Application,2011,23(8):1030-1035.
[25]CHEN F,XU H X,ZHANG F Y,et al.Physical mapping of puroindoline b-2 genes and molecular characterization of a novel variant in durum wheat(Triticum turgidum L.)[J].Molecular Breeding,2011,28(2):153-161.
[26]任民,贾兴华,蒋彩虹,等.Bassam和Sanguinetti银染方法在SRAP和TRAP标记中的比较研究[J].生物技术通报,2008(1):113-116.REN M,JIA X H,JIANG C H,et al.Comparison study of bassam and sanguinetti silver staining in the detecting of SRAP and TRAP[J].Biotechnology Bulletin,2008(1):113-116.
[27]NADAKUDUTI S S,UEBLER J B,LIU X X,et al.Characterization of trichome-expressed BAHDacyltransferases in petunia axillaris reveals distinct acylsugar assembly mechanisms within the solanaceae[J].Plant Physiology,2017,175(1):36-50.
[28]KROUMOVA A B,ZAITLIN D,WAGNER G J.Natural variability in acyl moieties of sugar esters produced by certain tobacco and other solanaceae species[J].Phytochemistry,2016,130:218-227.
[29]LIU X,ENRIGHT M,BARRY C S,et al.Profiling,isolation and structure elucidation of specialized acylsucrose metabolites accumulating in trichomes of petunia,species[J].Metabolomics,2017,13(7):85.
[30]LAWSON D M,LUNDE C F,MUTSCHLER M A.Marker-assisted transfer of acylsugar-mediated pest resistance from the wild tomato,lycopersicon pennellii,to the cultivated tomato,lycopersicon esculentum[J].Molecular Breeding,1997,3(4):307-317.
[31]BONIERBALE M W,PLAISTED R L,PINEDA O,et al.QTL analysis of trichome-mediated insect resistance in potato[J].Theoretical and Applied Genetics,1994,87(8):973-987.
[32]LECKIE B M,JONG D M D,MUTSCHLER M A.Quantitative trait loci increasing acylsugars in tomato breeding lines and their impacts on silverleaf whiteflies[J].Molecular Breeding,2012,30(4):1621-1634.
[33]FAN P,MILLER A M,SCHILMILLER A L,et al.In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network[J].Proc Natl Acad Sci U S A,2016,113(2):E239-E248.
[34]FAN P,MILLER A M,LIU X,et al.Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity[J].Nature Communications,2017,8(1):2080.
[35]李传江,庞宏.中式烟叶是发展中式卷烟的原料基础[J].中国烟草科学,2008,29(6):68-70.LI C J,PANG H.Development of chinese-style cigarettes with specific chinese tobacco leaf production[J].Chinese Tobacco Science,2008,29(6):68-70.
[36]李凯,姜涛,才源,等.玉米花期性状的主效SSR标记筛选[J].玉米科学,2015,23(1):33-38.LI K,JIANG T,CAI Y,et al.Screening of the main effect SSR markers of maize flowering[J].Journal of Maize Sciences,2015,23(1):33-38.
[37]常爱霞,屈亚芳,王国平,等.烤烟品种大白筋599特征香气性状的物质及分子基础研究[J].中国烟草科学,2018,39(3):1-9.CHANG A X,QU Y F,WANG G P,et al.Study on key components and molecular basis responsible for the specific aroma traits of flue-cured tobacco dabaijin599[J].Chinese Tobacco Science,2018,39(3):1-9.
[38]王国平.烟草冷杉醇合成基因功能分析及功能标记开发[D].北京:中国农业科学院,2016.WANG G P.Functional analysis and marker development of cis-abnieol synthesis gene in tobacco[D].Beijing:Chinese Academy of Agricultural Sciences,2016.