运用MSAP技术测定谷子基因组DNA胞嘧啶甲基化水平
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摘要
DNA甲基化是真核生物一种重要的表观修饰形式。为了探讨谷子基因组DNA胞嘧啶甲基化的水平和模式,以谷子Setaria italica的两个品种朝谷58号和豫谷1号为实验材料,利用Eco RⅠ和HpaⅡ/MspⅠ双酶切建立适合于谷子基因组的甲基化敏感扩增多态性(MSAP)分析体系。结果表明,从100对MSAP选扩引物中,筛选出32对MSAP引物组合,在朝谷58号和豫谷1号中分别扩增产生1 615、1 482条清晰可辨且可重复的DNA条带,其中包括3种类型的甲基化条带,朝谷58号和豫谷1号的基因组中CCGG序列胞嘧啶甲基化水平分别为6.93%和8.77%。这种谷子不同品种间甲基化水平和分布位点的差异为从表观遗传学的角度培育新品种提供了初步的理论依据和参考。
DNA methylation is an important type of epigenetic modification in eukaryotes. In order to research genome-wide methylation levels and patterns in foxtail millet(Setaria italica), the Methylation Sensitive Amplified Polymorphism(MSAP) analysis(employing double digestion with Eco R I and Hpa II/Msp I) was established and applied in two foxtail millet cultivars(Chaogu 58 and Yugu 1). The results showed that 32 pairs of MSAP primers were selected from 100 MSAP primers, and 1 615 and 1 482 clearly distinguishable and reproducible bands were amplified from Chaogu 58 and Yugu 1 respectively, including 3 types of methylation patterns. Cytosine methylation levels of CCGG context in Chaogu 58 and Yugu 1 were characterized as 6.93% and 8.77% respectively. Such different genomic DNA methylation levels between two foxtail millet varieties may provide a preliminary reference for the cultivation of this crop from a novel epigenetic viewpoint.
DNA methylation is an important type of epigenetic modification in eukaryotes. In order to research genome-wide methylation levels and patterns in foxtail millet(Setaria italica), the Methylation Sensitive Amplified Polymorphism(MSAP) analysis(employing double digestion with Eco R I and Hpa II/Msp I) was established and applied in two foxtail millet cultivars(Chaogu 58 and Yugu 1). The results showed that 32 pairs of MSAP primers were selected from 100 MSAP primers, and 1 615 and 1 482 clearly distinguishable and reproducible bands were amplified from Chaogu 58 and Yugu 1 respectively, including 3 types of methylation patterns. Cytosine methylation levels of CCGG context in Chaogu 58 and Yugu 1 were characterized as 6.93% and 8.77% respectively. Such different genomic DNA methylation levels between two foxtail millet varieties may provide a preliminary reference for the cultivation of this crop from a novel epigenetic viewpoint.
引文
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[11]Xu ZG,Fu LB,Zhong FN.Analysis on the regional comparative advantage of grain production in China.Chin Agric Resour Region Plann,2001,22(1):45-48(in Chinese).徐志刚,傅龙波,钟甫宁.中国粮食生产的区域比较优势分析.中国农业资源与区划,2001,22(1):45-48.
[12]Zhang GY,Liu X,Quan ZW,et al.Genome sequence of foxtail millet(Setaria italica)provides insights into grass evolution and biofuel potential.Nat Biotechnol,2012,30(6):549-554.
[13]Jo Y,Lian S,Cho JK,et al.De novo transcriptome assembly of Setatria italica variety Taejin.Genomics Data,2016,8:121-122.
[14]Tang S,Li L,Wang YQ,et al.Genotype-specific physiological and transcriptomic responses to drought stress in Setaria italica(an emerging model for Panicoideae grasses).Sci Rep,2017,7:10009.
[15]Bennetzen JL,Schmutz J,Wang H,et al.Reference genome sequence of the model plant Setaria.Nature Biotechnol,2012,30(6):555-561.
[16]Li W,Zhi H,Wang YF,et al.Analysis on the conditions of EMS treatment on Foxtail Millet.J Hebei Agric Sci,2010,14(11):77-79(in Chinese).李伟,智慧,王永芳,等.谷子EMS诱变的处理条件分析.河北农业科学,2010,14(11):77-79.
[17]Wang SS,Zhang N,Wang KX,et al.Genetic diversity of foxtail millet cultivars in western Liaoning province revealed by SSR markers.Mol Plant Breed,2015,13(5):1091-1097(in Chinese).王姗姗,张宁,王凯玺,等.中国辽西地区谷子品种遗传多样性的SSR分析.分子植物育种,2015,13(5):1091-1097.
[18]Kidwell KK,Osborn TC.Simple plant DNA isolation procedures//Beckmann JS,Osborn TC.Plant Genomes:Methods for Genetic and Physical Mapping.Netherlands:Springer 1992:1-13.
[19]Wang QM,Wang YZ,Sun LL,et al.Direct and indirect organogenesis of Clivia miniata and assessment of DNAmethylation changes in various regenerated plantlets.Plant Cell Rep,2012,31(7):1283-1296.
[20]Dong ZY,Wang YM,Zhang ZJ,et al.Extent and pattern of DNA methylation alteration in rice lines derived from introgressive hybridization of rice and Zizania latifolia Griseb.Theoret Appl Genet,2006,113(2):196-205.
[21]Yi ZB,Sun Y,Niu TT,et al.Genomic DNA cystoine methylations of corn hybrids and their parents.Acta Bot Bor-Occidental Sin,2005,25(12):2420-2425(in Chinese).仪治本,孙毅,牛天堂,等.玉米杂交种及其亲本基因组胞嘧啶甲基化水平研究.西北植物学报,2005,25(12):2420-2425.
[22]Gehring M,Henikoff S.DNA methylation dynamics in plant genomes.Biochim Biophys Acta Gene Struct Expr,2007,1769(5/6):276-286.
[23]Sung S,He YH,Eshoo TW,et al.Epigenetic maintenance of the vernalized state in Arabidopsis thaliana requires LIKE HETEROCHROMATINPROTEIN 1.Nat Genetics,2006,38(6):706-710.
[24]Zhong SL,Fei ZJ,Chen YR,et al.Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening.Nat Biotechnol,2013,31(2):154-159.
[25]Xiong LZ,Xu CG,Maroof MAS,et al.Patterns of cytosine methylation in an elite rice hybrid and its parental lines,detected by a methylation-sensitive amplification polymorphism technique.Mol Gen Genet,1999,261(3):439-446.
[2]Feng SH,Jacobsen SE,Reik W.Epigenetic reprogramming in plant and animal development.Science,2010,330(6004):623-627.
[3]Finnegan EJ,Kovac KA.Plant DNA methyltransferases.Plant Mol Biol,2000,43(2/3):189-201.
[4]Singer T,Yordan C,Martienssen RA.Robertson’s mutator transposons in A.thaliana are regulated by the chromatin-remodeling gene decrease in DNAmethylation(DDM1).Genes Dev,2001,15(5):591-602.
[5]Wang ZC,Li ZA,Li SP.The technology of MSAP and its applications in plant.Biotechnol Bull,2006(S1):195-196(in Chinese).王子成,李忠爱,李锁平.MSAP技术及其在植物上的应用.生物技术通报,2006(S1):195-196.
[6]Han YN,Zhao XJ,Cai L.Application of methylation-sensitive amplified polymorphism technology in plant stress resistance.Biotechnol Bull,2010(6):71-74,79(in Chinese).韩雅楠,赵秀娟,蔡禄.MSAP技术在植物抗逆性方面的应用.生物技术通报,2010(6):71-74,79.
[7]Li WG,Chang TJ,Gong HM.Methylation-sensitive amplified polymorphism and its application to plant genetics.Biotechnology,2008,18(1):83-87(in Chinese).李卫国,常天俊,龚红梅.MSAP技术及其在植物遗传学研究中的应用.生物技术,2008,18(1):83-87.
[8]Wang CC,Xing SY,Li JH,et al.DNA methlation levels and patterns in different ginkgo Biloba L.var.Epiphylla Mak.by methlation-sensitive amplification polymorphism(MSAP).J Nucl Agric Sci,2013,27(4):399-407(in Chinese).王聪聪,邢世岩,李际红,等.不同单株叶籽银杏DNA甲基化水平与模式的MSAP分析.核农学报,2013,27(4):399-407.
[9]Zheng XG,Chen L,Li MS,et al.Transgenerational variations in DNA methylation induced by drought stress in two rice varieties with distinguished difference to drought resistance.PLoS ONE,2013,8(11):e80253.
[10]Li ZL,Wang HT,Chen HJ,et al.Studying genomic methylation of arabidopsis thaliana seedlings under cadmium stress using MSAP.J Agro-Environ Sci,2014,33(1):28-36(in Chinese).李照令,王鹤潼,陈瑞娟,等.运用MSAP研究镉胁迫对拟南芥幼苗基因甲基化的影响.农业环境科学学报,2014,33(1):28-36.
[11]Xu ZG,Fu LB,Zhong FN.Analysis on the regional comparative advantage of grain production in China.Chin Agric Resour Region Plann,2001,22(1):45-48(in Chinese).徐志刚,傅龙波,钟甫宁.中国粮食生产的区域比较优势分析.中国农业资源与区划,2001,22(1):45-48.
[12]Zhang GY,Liu X,Quan ZW,et al.Genome sequence of foxtail millet(Setaria italica)provides insights into grass evolution and biofuel potential.Nat Biotechnol,2012,30(6):549-554.
[13]Jo Y,Lian S,Cho JK,et al.De novo transcriptome assembly of Setatria italica variety Taejin.Genomics Data,2016,8:121-122.
[14]Tang S,Li L,Wang YQ,et al.Genotype-specific physiological and transcriptomic responses to drought stress in Setaria italica(an emerging model for Panicoideae grasses).Sci Rep,2017,7:10009.
[15]Bennetzen JL,Schmutz J,Wang H,et al.Reference genome sequence of the model plant Setaria.Nature Biotechnol,2012,30(6):555-561.
[16]Li W,Zhi H,Wang YF,et al.Analysis on the conditions of EMS treatment on Foxtail Millet.J Hebei Agric Sci,2010,14(11):77-79(in Chinese).李伟,智慧,王永芳,等.谷子EMS诱变的处理条件分析.河北农业科学,2010,14(11):77-79.
[17]Wang SS,Zhang N,Wang KX,et al.Genetic diversity of foxtail millet cultivars in western Liaoning province revealed by SSR markers.Mol Plant Breed,2015,13(5):1091-1097(in Chinese).王姗姗,张宁,王凯玺,等.中国辽西地区谷子品种遗传多样性的SSR分析.分子植物育种,2015,13(5):1091-1097.
[18]Kidwell KK,Osborn TC.Simple plant DNA isolation procedures//Beckmann JS,Osborn TC.Plant Genomes:Methods for Genetic and Physical Mapping.Netherlands:Springer 1992:1-13.
[19]Wang QM,Wang YZ,Sun LL,et al.Direct and indirect organogenesis of Clivia miniata and assessment of DNAmethylation changes in various regenerated plantlets.Plant Cell Rep,2012,31(7):1283-1296.
[20]Dong ZY,Wang YM,Zhang ZJ,et al.Extent and pattern of DNA methylation alteration in rice lines derived from introgressive hybridization of rice and Zizania latifolia Griseb.Theoret Appl Genet,2006,113(2):196-205.
[21]Yi ZB,Sun Y,Niu TT,et al.Genomic DNA cystoine methylations of corn hybrids and their parents.Acta Bot Bor-Occidental Sin,2005,25(12):2420-2425(in Chinese).仪治本,孙毅,牛天堂,等.玉米杂交种及其亲本基因组胞嘧啶甲基化水平研究.西北植物学报,2005,25(12):2420-2425.
[22]Gehring M,Henikoff S.DNA methylation dynamics in plant genomes.Biochim Biophys Acta Gene Struct Expr,2007,1769(5/6):276-286.
[23]Sung S,He YH,Eshoo TW,et al.Epigenetic maintenance of the vernalized state in Arabidopsis thaliana requires LIKE HETEROCHROMATINPROTEIN 1.Nat Genetics,2006,38(6):706-710.
[24]Zhong SL,Fei ZJ,Chen YR,et al.Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening.Nat Biotechnol,2013,31(2):154-159.
[25]Xiong LZ,Xu CG,Maroof MAS,et al.Patterns of cytosine methylation in an elite rice hybrid and its parental lines,detected by a methylation-sensitive amplification polymorphism technique.Mol Gen Genet,1999,261(3):439-446.
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