水稻耐盐基因SKC1特异性分子标记开发及应用
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摘要
SKC1是世界上第一个被克隆的水稻耐盐性QTL,为了使SKC1在水稻分子标记辅助选择(MAS)育种中得到高效和快速利用,本研究通过将SKC1不同等位基因测序及序列比对鉴定到一个SKC1基因内特异SNP位点,使用AS-PCR (allele-specific PCR)的方法加以改进开发了一套鉴定该SNP的功能性分子标记,利用该标记及待检测水稻品种基因组DNA进行PCR扩增,通过不同PCR产物条带大小将SKC1与其它等位基因区分开,可快速判断待测水稻SKC1的等位基因类型。本方法成本费用低廉、简单高效,可应用于大规模水稻种质资源SKC1等位基因筛选和MAS育种。本研究对创制耐盐水稻新种质、培育耐盐水稻新品种、推动盐碱地水稻种植推广具有一定的意义。
SKC1 is the first salt tolerant QTL cloned in rice in the world. In order to utilize SKC1 efficiently and rapidly in rice molecular marker-assisted selection(MAS) breeding, this study identified a specific SNP locus within SKC1 gene by sequencing and sequence alignment of different alleles of SKC1, and developed a set of functional molecular markers for identifying the SNP by using AS-PCR(allele-specific PCR). Using this marker and the genomic DNA of rice varieties to be tested for PCR amplification, SKC1 and other alleles could be distinguished by different band sizes of PCR products, and the allele type of SKC1 in rice could be quickly judged. This method was cheap, simple and efficient and could be used for screening SKC1 alleles and MAS breeding of large-scale rice germplasm resources. This study could be of certain significance for the development of new salt-tolerant rice germplasm, the cultivation of new salt-tolerant rice varieties and the promotion of rice planting in saline-alkali land.
SKC1 is the first salt tolerant QTL cloned in rice in the world. In order to utilize SKC1 efficiently and rapidly in rice molecular marker-assisted selection(MAS) breeding, this study identified a specific SNP locus within SKC1 gene by sequencing and sequence alignment of different alleles of SKC1, and developed a set of functional molecular markers for identifying the SNP by using AS-PCR(allele-specific PCR). Using this marker and the genomic DNA of rice varieties to be tested for PCR amplification, SKC1 and other alleles could be distinguished by different band sizes of PCR products, and the allele type of SKC1 in rice could be quickly judged. This method was cheap, simple and efficient and could be used for screening SKC1 alleles and MAS breeding of large-scale rice germplasm resources. This study could be of certain significance for the development of new salt-tolerant rice germplasm, the cultivation of new salt-tolerant rice varieties and the promotion of rice planting in saline-alkali land.
引文
Andersen J.R.,and Lübberstedt T.,2003,Functional markers in plants,Trends Plant Sci.,8(11):554-560
Bottema C.,Sarkar G.,Cassady J.,Dutton C.,and Sommer S.,1993,Polymerase chain reaction amplification of specific alleles:a general method of detection of mutations,polymorphisms,and haplotypes,methods in enzymology,Methods Enzymol,218:388-402
Hamajima N.,Saito T.,Matsuo K.,and Tajima K.,2002,Competitive amplification and unspecific amplification in polymerase chain reaction with confronting two-pair primers,J.Mol.Diagn.,4(2):103-107
Hu S.K.,Tao H.J.,Qian Q.,and Guo L.B.,2010,Progresses on genetics and molecular breeding for salt-tolerance in rice,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(4):629-640(胡时开,陶红剑,钱前,郭龙彪,2010,水稻耐盐性的遗传和分子育种的研究进展,分子植物育种,8(4):629-640)
Jing W.,and Zhang W.H.,2017,Research progress on gene mapping and cloning for salt tolerance and variety improvement for salt tolerance by molecular marker-assisted selection in rice,Zhongguo Shuidao Kexue(Chinese Journal of Rice Science),31(2):111-123(井文,章文华,2017,水稻耐盐基因定位与克隆及品种耐盐性分子标记辅助选择改良研究进展,中国水稻科学,31(2):111-123)
Lin H.X.,Zhu M.Z.,Yano M.,Gao J.P.,Liang Z.W.,Su W.A.,Hu X.H.,Ren Z.H.,and Chao D.Y.,2004,QTLs for Na+and K+uptake of the shoots and roots controlling rice salt tolerance,Theor.Appl.Genet.,108(2):253-260
Qi D.L.,Han L.Z.,and Zhang S.Y.,2005,Methods of characterization and evaluation of salt or alkaline tolerance in rice,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),6(2):226-231(祁栋灵,韩龙植,张三元,2005,水稻耐盐/碱性鉴定评价方法,植物遗传资源学报,6(2):226-231)
Ren Z.H.,Gao J.P.,Li L.G.,Cai X.L.,Huang W.,Chao D.Y.,Zhu M.Z.,Wang Z.Y.,Luan S.,and Lin H.X.,2005,A rice quantitative trait locus for salt tolerance encodes a sodium transporter,Nat.Genet.,37(10):1141-1146
Vinod K.K.,Krishnan S.G.,Babu N.N.,Nagarajan M.,and Singh A.K.,2013,Improving salt tolerance in rice:looking beyond the conventional,In:Ahmad P.,Azooz M.M.,and Prasad M.N.V.(eds.),Salt stress in plants,Springer,New York,USA,pp.219-260
Wang C.F.,Liu D.C.,Ma X.L.,An Y.P.,Zhang W.Y.,and Ma J.,2015,Development and validation of the specific CAPSmarkers of rice salt tolerant gene SKC1,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(11):2437-2440(王彩芬,刘冬成,马晓玲,安永平,张文银,马静,2015,水稻耐盐基因SKC1特异性CAPS标记的开发与验证,分子植物育种,13(11):2437-2440)
Wang K.,Zhang J.T.,Yun Y.X.,Wu X.B.,Chen A.Q.,Wang P.,Wang K.J.,Zhang J.Y.,and Dai L.P.,2011,Improvement on PCR-CTPP:a SNP genotyping approach based on mismatch technique,Yichuan(Hereditas),33(2):182-188(王柯,章金涛,运玉霞,吴晓冰,陈阿群,王鹏,王凯娟,张建营,代丽萍,2011,PCR-CTPP:一种基于错配技术的SNP分型方法的改良,遗传,33(2):182-188)
Wang Y.Y.,Sun Y.D.,Ma J.Q.,Yang Y.,and Xu M.H.,2016,Preliminary study on the characteristics SNP of microsatellite sequence(CT)nand intron 1 in Wx gene in wild rices,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3458-3464(王炎炎,孙一丁,马继琼,杨奕,许明辉,2016,野生稻Wx基因(CT)n卫星序列和第一内含子SNP特征标签初探,基因组学与应用生物学,35(12):3458-3464)
Williams J.G.,Kubelik A.R.,Livak K.J.,Rafalski J.A.,and Tingey S.V.,1990,DNA polymorphisms amplified by arbitrary primers are useful as genetic markers,Nucleic Acids Res.,18(22):6531-6535
Yao L.S.,He C.X.,Sun M.F.,Yan G.H.,Tang H.S.,Wang A.M.,and Zhu G.Y.,2012,High efficiency screening of salt tolerant rice in coastal tidal flat,Dalei Yu Guwu Kexue(Barley and Cereal Sciences),(4):10-12(姚立生,何冲霄,孙明法,严国红,唐红生,王爱民,朱国永,2012,沿海滩涂耐盐水稻高效筛选,大麦与谷类科学,(4):10-12)
Zhang H.L.,Zhang D.L.,Wang M.X.,Sun J.L.,Qi Y.W.,Li J.J.,Wei X.H.,Han L.Z.,Qiu Z.E.,Tang S.X.,and Li Z.C.,2011,A core collection and mini core collection of Oryza sativa L.in China,Theor.Appl.Genet.,122(1):49-61
Zhang X.,Li M.J.,Zhang B.,Yin X.M.,Wang M.L.,and Xia X.J.,2016,Possibility study on improving salt tolerance of rice by overexpressing PHD-finger transcription factor gene OsM-sr16,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(7):1820-1827(张新,李明娟,张斌,殷绪明,王曼玲,夏新界,2016,水稻PHD-finger转录因子基因OsMsr16增强耐盐性的可能性研究,基因组学与应用生物学,35(7):1820-1827)
Zhou L.,Chen Z.J.,Lang X.Y.,Du B.,Liu K.,Yang G.C.,Hu G.,Li S.H.,He G.C.,and You A.Q.,2013,Development and validation of a PCR-based functional marker system for the brown planthopper resistance gene Bph14 in rice,Breed.Sci.,63(3):347-352
Bottema C.,Sarkar G.,Cassady J.,Dutton C.,and Sommer S.,1993,Polymerase chain reaction amplification of specific alleles:a general method of detection of mutations,polymorphisms,and haplotypes,methods in enzymology,Methods Enzymol,218:388-402
Hamajima N.,Saito T.,Matsuo K.,and Tajima K.,2002,Competitive amplification and unspecific amplification in polymerase chain reaction with confronting two-pair primers,J.Mol.Diagn.,4(2):103-107
Hu S.K.,Tao H.J.,Qian Q.,and Guo L.B.,2010,Progresses on genetics and molecular breeding for salt-tolerance in rice,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(4):629-640(胡时开,陶红剑,钱前,郭龙彪,2010,水稻耐盐性的遗传和分子育种的研究进展,分子植物育种,8(4):629-640)
Jing W.,and Zhang W.H.,2017,Research progress on gene mapping and cloning for salt tolerance and variety improvement for salt tolerance by molecular marker-assisted selection in rice,Zhongguo Shuidao Kexue(Chinese Journal of Rice Science),31(2):111-123(井文,章文华,2017,水稻耐盐基因定位与克隆及品种耐盐性分子标记辅助选择改良研究进展,中国水稻科学,31(2):111-123)
Lin H.X.,Zhu M.Z.,Yano M.,Gao J.P.,Liang Z.W.,Su W.A.,Hu X.H.,Ren Z.H.,and Chao D.Y.,2004,QTLs for Na+and K+uptake of the shoots and roots controlling rice salt tolerance,Theor.Appl.Genet.,108(2):253-260
Qi D.L.,Han L.Z.,and Zhang S.Y.,2005,Methods of characterization and evaluation of salt or alkaline tolerance in rice,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),6(2):226-231(祁栋灵,韩龙植,张三元,2005,水稻耐盐/碱性鉴定评价方法,植物遗传资源学报,6(2):226-231)
Ren Z.H.,Gao J.P.,Li L.G.,Cai X.L.,Huang W.,Chao D.Y.,Zhu M.Z.,Wang Z.Y.,Luan S.,and Lin H.X.,2005,A rice quantitative trait locus for salt tolerance encodes a sodium transporter,Nat.Genet.,37(10):1141-1146
Vinod K.K.,Krishnan S.G.,Babu N.N.,Nagarajan M.,and Singh A.K.,2013,Improving salt tolerance in rice:looking beyond the conventional,In:Ahmad P.,Azooz M.M.,and Prasad M.N.V.(eds.),Salt stress in plants,Springer,New York,USA,pp.219-260
Wang C.F.,Liu D.C.,Ma X.L.,An Y.P.,Zhang W.Y.,and Ma J.,2015,Development and validation of the specific CAPSmarkers of rice salt tolerant gene SKC1,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(11):2437-2440(王彩芬,刘冬成,马晓玲,安永平,张文银,马静,2015,水稻耐盐基因SKC1特异性CAPS标记的开发与验证,分子植物育种,13(11):2437-2440)
Wang K.,Zhang J.T.,Yun Y.X.,Wu X.B.,Chen A.Q.,Wang P.,Wang K.J.,Zhang J.Y.,and Dai L.P.,2011,Improvement on PCR-CTPP:a SNP genotyping approach based on mismatch technique,Yichuan(Hereditas),33(2):182-188(王柯,章金涛,运玉霞,吴晓冰,陈阿群,王鹏,王凯娟,张建营,代丽萍,2011,PCR-CTPP:一种基于错配技术的SNP分型方法的改良,遗传,33(2):182-188)
Wang Y.Y.,Sun Y.D.,Ma J.Q.,Yang Y.,and Xu M.H.,2016,Preliminary study on the characteristics SNP of microsatellite sequence(CT)nand intron 1 in Wx gene in wild rices,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3458-3464(王炎炎,孙一丁,马继琼,杨奕,许明辉,2016,野生稻Wx基因(CT)n卫星序列和第一内含子SNP特征标签初探,基因组学与应用生物学,35(12):3458-3464)
Williams J.G.,Kubelik A.R.,Livak K.J.,Rafalski J.A.,and Tingey S.V.,1990,DNA polymorphisms amplified by arbitrary primers are useful as genetic markers,Nucleic Acids Res.,18(22):6531-6535
Yao L.S.,He C.X.,Sun M.F.,Yan G.H.,Tang H.S.,Wang A.M.,and Zhu G.Y.,2012,High efficiency screening of salt tolerant rice in coastal tidal flat,Dalei Yu Guwu Kexue(Barley and Cereal Sciences),(4):10-12(姚立生,何冲霄,孙明法,严国红,唐红生,王爱民,朱国永,2012,沿海滩涂耐盐水稻高效筛选,大麦与谷类科学,(4):10-12)
Zhang H.L.,Zhang D.L.,Wang M.X.,Sun J.L.,Qi Y.W.,Li J.J.,Wei X.H.,Han L.Z.,Qiu Z.E.,Tang S.X.,and Li Z.C.,2011,A core collection and mini core collection of Oryza sativa L.in China,Theor.Appl.Genet.,122(1):49-61
Zhang X.,Li M.J.,Zhang B.,Yin X.M.,Wang M.L.,and Xia X.J.,2016,Possibility study on improving salt tolerance of rice by overexpressing PHD-finger transcription factor gene OsM-sr16,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(7):1820-1827(张新,李明娟,张斌,殷绪明,王曼玲,夏新界,2016,水稻PHD-finger转录因子基因OsMsr16增强耐盐性的可能性研究,基因组学与应用生物学,35(7):1820-1827)
Zhou L.,Chen Z.J.,Lang X.Y.,Du B.,Liu K.,Yang G.C.,Hu G.,Li S.H.,He G.C.,and You A.Q.,2013,Development and validation of a PCR-based functional marker system for the brown planthopper resistance gene Bph14 in rice,Breed.Sci.,63(3):347-352