利用SSR标记和SNP芯片对小麦EMS突变体进行真实性鉴定
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摘要
为鉴定EMS突变的真实性,本研究利用SSR标记和90 K SNP芯片对小麦品系H261及其EMS突变体进行检测。SSR检测结果表明,H261与LF2010和LF2099的差异SSR标记为0个,但与LF2100的差异SSR标记为10个,多态性比例为47.62%。SNP芯片分析结果表明,H261与LF2010和LF2099之间的差异位点分别为66和12个,分别占总数的0.080 9%和0.014 7%,2个突变体与H261的纯合差异SNP数目均为0;而H261与LF2100之间的差异位点为2 846个,占总数的3.487 9%,二者之间纯合差异SNP为784,占总数的0.960 8%。综上所述,LF2010和LF2099突变体与亲本H261的遗传背景高度一致,是H261经过EMS诱变的后代,而LF2100是天然异交或机械混杂产生的假突变体。本研究结果为更好地发挥小麦突变体在遗传改良和功能基因组研究奠定了一定的理论基础。
In order to identify the authenticity of EMS mutation, SSR markers and 90 K SNP chip were used to detect Wheat Strain H261 and its EMS mutants. SSR analysis showed that the difference of SSR markers between H261 and LF2010 or LF2099 was 0, but the difference of SSR markers between H261 and LF2100 was 10, and the polymorphism rate was 47.62%. SNP chip analysis showed that the differences between H261 and LF2010 and LF2099 were 66 and 12, accounting for 0.0809% and 0.0147% of the total SNPS, respectively. The homozygous differences between the two mutants and H261 were 0, while the differences between H261 and LF2100 were 2 846, accounting for 3.487 9% of the total SNPS, and the homozygous differences between them were 784, accounting for the 0.960 8%. The results showed the genetic background of LF2010 and LF2099 were highly consistent with that of genetic background of parents H261, and were the mutated offspring of H261 mutated by EMS, while LF2100 was a "pseudomutant" produced by natural outcrossing or mechanical hybridization. This study laid a theoretical foundation for the better use of wheat mutants in genetic improvement and functional genome research.
In order to identify the authenticity of EMS mutation, SSR markers and 90 K SNP chip were used to detect Wheat Strain H261 and its EMS mutants. SSR analysis showed that the difference of SSR markers between H261 and LF2010 or LF2099 was 0, but the difference of SSR markers between H261 and LF2100 was 10, and the polymorphism rate was 47.62%. SNP chip analysis showed that the differences between H261 and LF2010 and LF2099 were 66 and 12, accounting for 0.0809% and 0.0147% of the total SNPS, respectively. The homozygous differences between the two mutants and H261 were 0, while the differences between H261 and LF2100 were 2 846, accounting for 3.487 9% of the total SNPS, and the homozygous differences between them were 784, accounting for the 0.960 8%. The results showed the genetic background of LF2010 and LF2099 were highly consistent with that of genetic background of parents H261, and were the mutated offspring of H261 mutated by EMS, while LF2100 was a "pseudomutant" produced by natural outcrossing or mechanical hybridization. This study laid a theoretical foundation for the better use of wheat mutants in genetic improvement and functional genome research.
引文
[1] Stadler L J. Mutations in barley induced by X-rays and radium[J]. Science,1928,68(1765): 186-187
[2] 刘录祥,郭会君,赵林姝,李军辉,古佳玉,赵世荣,王晶. 植物诱发突变技术育种研究现状与展望[J]. 核农学报,2009,23(6):1001-1007
[3] Caldecott R S, Stevens H, Robert B J. Stem rust resistant variants in irradiated populations—mutations or field hybrids[J]. Agronomy Journal,1959,51(7):401-403
[4] Knott D R. What determines the success of mutation breeding[R].Austria: Vienna,1991: 111-118
[5] Fu H W, Li Y F, Shu Q Y. A revisit of mutation induction by gamma rays in rice (Oryza sativa L.):implications of microsatellite markers for quality control[J]. Molecular Breeding,2008,22(2): 281-288
[6] 李瑞清,武立权,舒庆尧,赵海军,吴殿星,王荣富. 一个新的水稻白化转绿突变体G9的特性研究[J]. 核农学报,2010,24(5):881-886
[7] 吕兴娜,杜久元,苏萍萍,王琪琳,吴建辉,黄丽丽,康振生,韩德俊. 小麦航天诱变抗条锈病突变体的筛选与鉴定[J].麦类作物学报,2016,36(12):1599-1604
[8] Nasu S,Suzuki J,Ohta R,Hasegawa K,Yui R,Kitazawa N,Monna L,Minobe Y.Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers[J]. DNA Research,2002,9:163-171
[9] 邹喻苹,葛颂. 新一代分子标记-SNPs及其应用[J]. 生物多样性,2003,11(5):370-382
[10] 陈竹锋,严维,王娜,张文辉,谢刚,卢嘉威,简智华,刘东风,唐晓燕. 利用改进的MutMap方法克隆水稻雄性不育基因[J]. 遗传,2014,36(1):85-93
[11] Uauy C. Wheat genomics comes of age[J]. Current Opinion in Plant Biology, 2017, 36:142-148
[12] Zimin A Ⅴ, Puiu D, Hall R, Kingan S, Clavijo B J, Salzberg S L. The first near-complete assembly of the hexaploid bread wheat genome,Triticum aestivum[J]. Gigascience,2017, 6(11):1-7
[13] Avni R, Nave M, Barad O, Baruch K, Twardziok S O, Gundlach H, Hale Ⅰ, Mascher M, Spannagl M, Wiebe K, Jordan K W, Golan G, Deek J, Ben-Zvi B, Ben-Zvi G, Himmelbach A, MacLachlan R P, Sharpe A G, Fritz A, Ben-David R, Budak H, Fahima T, Korol A, Faris J D, Hernandez A, Mikel M A, Levy A A, Steffenson B, Maccaferri M, Tuberosa R, Cattivelli L, Faccioli P, Ceriotti A, Kashkush K, Pourkheirandish M, Komatsuda T, Eilam T, Sela H, Sharon A, Ohad N, Chamovitz D A, Mayer K F X, Stein N, Ronen G, Peleg Z, Pozniak C J, Akhunov E D, Distelfeld A. Wild emmer genome architecture and diversity elucidate wheat evolution and domestication[J]. Science,2017, 357(7):93-97
[14] Uauy C,Wulff B,Dubcovsky J.Combining traditional mutagenesis with new high-throughput sequencing and genome editing to reveal hidden variation in polyploid wheat[J]. Annual Review of Genetics,2017, 51(1): 435-454
[15] 闫智慧,郭会君,徐荣旗,刘录祥. TILLING技术的发展及其在不同植物中的应用[J]. 核农学报,2014,28(2): 224-233
[16] Krasileva K Ⅴ,Vasquez-Gross H A,Howell T,Bailey P,Paraiso F,Clissold L,Simmonds J,Ramirez-Gonzalez R H,Wang X,Borrill P.Uncovering hidden variation in polyploid wheat[J]. Proceeding of the National Academy of Science,2017,114(6):913-921
[17] 李明飞,谢彦周,刘录祥,王超杰,徐喜堂,邹淑芳,黄树伟,贺小弟,王成社. 叠氮化钠诱变普通小麦陕农33突变体库的构建和初步评估[J]. 麦类作物学报,2015,35(1):22-29
[18] Slade A J,Knauf V C.TILLING moves beyond functional genomics into crop improvement[J]. Transgenic Research, 2005, 14(2):109-115
[19] Uauy C,Paraiso F,Colasuonno P,Tran R K,Tsai H,Berardi S,Comai L,Dubcovsky J.A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat[J]. BMC Plant Biology,2009,9(1):115-129
[20] Cavanagh C R, Chao S, Wang S, Huang B E, Stephen S, Kiani S, Forrest K, Saintenac C, Brown-Guedira G L, Akhunova A, See D, Bai G, Pumphrey M, Tomar L, Wong D, Kong S, Reynolds M, da Silva M L, Bockelman H, Talbert L, Anderson J A, Dreisigacker S, Baenziger S, Carter A, Korzun Ⅴ, Morrell P L, Dubcovsky J, Morell M K, Sorrells M E, Hayden M J, Akhunov E. Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(5):8057-8062
[21] Wang S, Wong D, Forrest K, Allen A, Chao S, Huang B E, Maccaferri M, Salvi S, Milner S G, Cattivelli L, Mastrangelo A M, Whan A, Stephen S, Barker G, Wieseke R, Plieske J, Lillemo M, Mather D, Appels R, Dolferus R, Brown-Guedira G, Korol A, Akhunova A R, Feuillet C, Salse J, Morgante M, Pozniak C, Luo M C, Dvorak J, Morell M, Dubcovsky J, Ganal M, Tuberosa R, Lawley C, Mikoulitch Ⅰ, Cavanagh C, Edwards K J, Hayden M, Akhunov E. Characterization of polyploid wheat genomic diversity using a high-density 90000 single nucleotide polymorphism array[J]. Plant Biotechnology Journal, 2014, 12(6):787-796
[22] Cui F,Zhang N,Fan X L,Zhang W,Zhao C H,Yang L J,Pan R Q,Chen M,Han J, Zhao X Q, Ji J, Tong Y P, Zhang H X, Jia J Z, Zhao G Y, Li J M. Utilization of a Wheat 660K SNP array-derived high-density genetic map for high-resolution mapping of a major QTL for kernel number[J]. Scientific Report, 2017,7(1):3788
[23] 杜丽芬,李明飞,刘录祥,王超杰,刘洋,许喜堂,邹淑芳,谢彦周,王成社. 一个化学诱变的小麦斑点叶突变体的生理和遗传分析[J]. 作物学报,2014,46(6): 1020-1026
[24] 季伟,王立新,孙辉,王茅雁,赵昌平. 小麦 SSR 分析体系的简化[J]. 农业生物技术学报, 2007,15(5):907-908
[25] 王立新,常利芳,李宏博,季伟,刘丽华,赵昌平. 小麦区试品系DUS测试的分子标记[J]. 作物学报,2010,36(7):1114-1125
[26] 许绍斌,陶玉芬,杨昭庆,褚嘉佑. 简单快速的DNA银染和胶保存方法[J]. 遗传,2002,24(3): 335-336
[27] 王立新,常利芳,李宏博,葛玲玲,信爱华,高世庆,季伟,孙辉,赵昌平. 小麦种子纯度的分子标记检测方法[J]. 麦类作物学报,2009,29(1):1-8
[28] 许云峰,蒋方山,郭营,李瑞军,李斯深. EMS诱导小麦品种烟农15突变体的鉴定和EST-SSR分析[J]. 核农学报,2008,22(4):410-414
[29] 王瑾. 小麦抗旱突变体的离体诱发鉴定及SSR分析[D]. 保定: 河北农业大学,2005:6-20
[30] 来德娥,赵平,王敏,张从宇. 60Co-γ射线诱变小麦品质突变体的筛选及分子标记检测[J]. 种子,2012,31(5):1001-4705
[2] 刘录祥,郭会君,赵林姝,李军辉,古佳玉,赵世荣,王晶. 植物诱发突变技术育种研究现状与展望[J]. 核农学报,2009,23(6):1001-1007
[3] Caldecott R S, Stevens H, Robert B J. Stem rust resistant variants in irradiated populations—mutations or field hybrids[J]. Agronomy Journal,1959,51(7):401-403
[4] Knott D R. What determines the success of mutation breeding[R].Austria: Vienna,1991: 111-118
[5] Fu H W, Li Y F, Shu Q Y. A revisit of mutation induction by gamma rays in rice (Oryza sativa L.):implications of microsatellite markers for quality control[J]. Molecular Breeding,2008,22(2): 281-288
[6] 李瑞清,武立权,舒庆尧,赵海军,吴殿星,王荣富. 一个新的水稻白化转绿突变体G9的特性研究[J]. 核农学报,2010,24(5):881-886
[7] 吕兴娜,杜久元,苏萍萍,王琪琳,吴建辉,黄丽丽,康振生,韩德俊. 小麦航天诱变抗条锈病突变体的筛选与鉴定[J].麦类作物学报,2016,36(12):1599-1604
[8] Nasu S,Suzuki J,Ohta R,Hasegawa K,Yui R,Kitazawa N,Monna L,Minobe Y.Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers[J]. DNA Research,2002,9:163-171
[9] 邹喻苹,葛颂. 新一代分子标记-SNPs及其应用[J]. 生物多样性,2003,11(5):370-382
[10] 陈竹锋,严维,王娜,张文辉,谢刚,卢嘉威,简智华,刘东风,唐晓燕. 利用改进的MutMap方法克隆水稻雄性不育基因[J]. 遗传,2014,36(1):85-93
[11] Uauy C. Wheat genomics comes of age[J]. Current Opinion in Plant Biology, 2017, 36:142-148
[12] Zimin A Ⅴ, Puiu D, Hall R, Kingan S, Clavijo B J, Salzberg S L. The first near-complete assembly of the hexaploid bread wheat genome,Triticum aestivum[J]. Gigascience,2017, 6(11):1-7
[13] Avni R, Nave M, Barad O, Baruch K, Twardziok S O, Gundlach H, Hale Ⅰ, Mascher M, Spannagl M, Wiebe K, Jordan K W, Golan G, Deek J, Ben-Zvi B, Ben-Zvi G, Himmelbach A, MacLachlan R P, Sharpe A G, Fritz A, Ben-David R, Budak H, Fahima T, Korol A, Faris J D, Hernandez A, Mikel M A, Levy A A, Steffenson B, Maccaferri M, Tuberosa R, Cattivelli L, Faccioli P, Ceriotti A, Kashkush K, Pourkheirandish M, Komatsuda T, Eilam T, Sela H, Sharon A, Ohad N, Chamovitz D A, Mayer K F X, Stein N, Ronen G, Peleg Z, Pozniak C J, Akhunov E D, Distelfeld A. Wild emmer genome architecture and diversity elucidate wheat evolution and domestication[J]. Science,2017, 357(7):93-97
[14] Uauy C,Wulff B,Dubcovsky J.Combining traditional mutagenesis with new high-throughput sequencing and genome editing to reveal hidden variation in polyploid wheat[J]. Annual Review of Genetics,2017, 51(1): 435-454
[15] 闫智慧,郭会君,徐荣旗,刘录祥. TILLING技术的发展及其在不同植物中的应用[J]. 核农学报,2014,28(2): 224-233
[16] Krasileva K Ⅴ,Vasquez-Gross H A,Howell T,Bailey P,Paraiso F,Clissold L,Simmonds J,Ramirez-Gonzalez R H,Wang X,Borrill P.Uncovering hidden variation in polyploid wheat[J]. Proceeding of the National Academy of Science,2017,114(6):913-921
[17] 李明飞,谢彦周,刘录祥,王超杰,徐喜堂,邹淑芳,黄树伟,贺小弟,王成社. 叠氮化钠诱变普通小麦陕农33突变体库的构建和初步评估[J]. 麦类作物学报,2015,35(1):22-29
[18] Slade A J,Knauf V C.TILLING moves beyond functional genomics into crop improvement[J]. Transgenic Research, 2005, 14(2):109-115
[19] Uauy C,Paraiso F,Colasuonno P,Tran R K,Tsai H,Berardi S,Comai L,Dubcovsky J.A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat[J]. BMC Plant Biology,2009,9(1):115-129
[20] Cavanagh C R, Chao S, Wang S, Huang B E, Stephen S, Kiani S, Forrest K, Saintenac C, Brown-Guedira G L, Akhunova A, See D, Bai G, Pumphrey M, Tomar L, Wong D, Kong S, Reynolds M, da Silva M L, Bockelman H, Talbert L, Anderson J A, Dreisigacker S, Baenziger S, Carter A, Korzun Ⅴ, Morrell P L, Dubcovsky J, Morell M K, Sorrells M E, Hayden M J, Akhunov E. Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(5):8057-8062
[21] Wang S, Wong D, Forrest K, Allen A, Chao S, Huang B E, Maccaferri M, Salvi S, Milner S G, Cattivelli L, Mastrangelo A M, Whan A, Stephen S, Barker G, Wieseke R, Plieske J, Lillemo M, Mather D, Appels R, Dolferus R, Brown-Guedira G, Korol A, Akhunova A R, Feuillet C, Salse J, Morgante M, Pozniak C, Luo M C, Dvorak J, Morell M, Dubcovsky J, Ganal M, Tuberosa R, Lawley C, Mikoulitch Ⅰ, Cavanagh C, Edwards K J, Hayden M, Akhunov E. Characterization of polyploid wheat genomic diversity using a high-density 90000 single nucleotide polymorphism array[J]. Plant Biotechnology Journal, 2014, 12(6):787-796
[22] Cui F,Zhang N,Fan X L,Zhang W,Zhao C H,Yang L J,Pan R Q,Chen M,Han J, Zhao X Q, Ji J, Tong Y P, Zhang H X, Jia J Z, Zhao G Y, Li J M. Utilization of a Wheat 660K SNP array-derived high-density genetic map for high-resolution mapping of a major QTL for kernel number[J]. Scientific Report, 2017,7(1):3788
[23] 杜丽芬,李明飞,刘录祥,王超杰,刘洋,许喜堂,邹淑芳,谢彦周,王成社. 一个化学诱变的小麦斑点叶突变体的生理和遗传分析[J]. 作物学报,2014,46(6): 1020-1026
[24] 季伟,王立新,孙辉,王茅雁,赵昌平. 小麦 SSR 分析体系的简化[J]. 农业生物技术学报, 2007,15(5):907-908
[25] 王立新,常利芳,李宏博,季伟,刘丽华,赵昌平. 小麦区试品系DUS测试的分子标记[J]. 作物学报,2010,36(7):1114-1125
[26] 许绍斌,陶玉芬,杨昭庆,褚嘉佑. 简单快速的DNA银染和胶保存方法[J]. 遗传,2002,24(3): 335-336
[27] 王立新,常利芳,李宏博,葛玲玲,信爱华,高世庆,季伟,孙辉,赵昌平. 小麦种子纯度的分子标记检测方法[J]. 麦类作物学报,2009,29(1):1-8
[28] 许云峰,蒋方山,郭营,李瑞军,李斯深. EMS诱导小麦品种烟农15突变体的鉴定和EST-SSR分析[J]. 核农学报,2008,22(4):410-414
[29] 王瑾. 小麦抗旱突变体的离体诱发鉴定及SSR分析[D]. 保定: 河北农业大学,2005:6-20
[30] 来德娥,赵平,王敏,张从宇. 60Co-γ射线诱变小麦品质突变体的筛选及分子标记检测[J]. 种子,2012,31(5):1001-4705