基于CGE技术的甘蔗SSR-PCR反应体系优化
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摘要
【目的】优化基于毛细管凝胶电泳(CGE)技术的甘蔗SSR-PCR反应体系,为甘蔗遗传多样性和亲缘关系研究、分子辅助育种及遗传图谱构建提供技术支持。【方法】基于CGE技术,采用正交试验设计,选择dNTPs浓度、DNA聚合酶量、引物浓度和DNA模板量为考察因素进行优化,确定最佳SSR-PCR反应体系,并用于8个甘蔗种质资源材料的遗传多样性分析。【结果】最佳SSR-PCR反应体系(20μL):dNTPs浓度0.15 mmol/L,DNA聚合酶1.5 U,引物浓度0.50μmol/L,DNA模板量25 ng。利用该体系对8份来自不同国家地域的甘蔗种质材料进行遗传多样性分析,发现8对SSR引物共扩增出101个片段,其中多态性片段为93个,多态性比率为92.1%。聚类分析结果表明,参试材料间的遗传相似性系数为0.521~0.871,在相似性系数为0.614处,所有参试材料可分为两大类,3份国外种质材料PINDAR、CP72-1210、CP84-1198与ROC20、GT69-156聚为一类;而ROC26、GT02-237和GT97-69聚为另一类。【结论】基于CGE技术的SSR-PCR反应体系检测结果稳定,重复性好,适用于甘蔗的遗传分析及遗传作图。
【Objective】SSR-PCR reaction system of sugarcane based on capillary gel electrophoresis(CGE) technology was optimized to provide technical support for research on genetic diversity and genetic relationship,molecular-assisted breeding and genetic map construction of sugarcane.【 Method 】Four factors affecting SSR-PCR were optimized by orthogonal exprement based on CGE technology,including dNTPs concentration,primer concentration,Tag DNA polymerase amount and DNA template amount.Then the optimal SSR-PCR reaction system was confirmed and applied to genetic analysis of8 sugarcane germplasm resources.【Result】The results showed that,the optimal reaction system(20 μL) was composed of0.15 mmoI/L dNTPs,1.5 U Taq DNA polymerase,0.50 μmol/L primers and 25 ng DNA templates.Using optimal reaction system and 8 pairs of SSR primers,a total of 101 fragments were amplified from 8 sugarcane germplasms of several countries,93 of which was polymorphic,with a polymorphism rate of 92.1%.The cluster analysis showed that,coefficient of genetic similarity among 8 germplasms ranged from 0.521 to 0.871.All tested materials could be clustered into two groups at similarity coefficient of 0.614,three foreign germplasms viz.,PINDAR,CP72-1210,CP84-1198 and ROC20,GT69-156 were clustered into the same group,but ROC26,GT02-237 and GT97-69 were clustered into another group.【Conclusion 】The established SSR-CGE system is suitable for genetic diversity analysis and genetic mapping of sugarcane due to its stable detection result and good reproducibility.
【Objective】SSR-PCR reaction system of sugarcane based on capillary gel electrophoresis(CGE) technology was optimized to provide technical support for research on genetic diversity and genetic relationship,molecular-assisted breeding and genetic map construction of sugarcane.【 Method 】Four factors affecting SSR-PCR were optimized by orthogonal exprement based on CGE technology,including dNTPs concentration,primer concentration,Tag DNA polymerase amount and DNA template amount.Then the optimal SSR-PCR reaction system was confirmed and applied to genetic analysis of8 sugarcane germplasm resources.【Result】The results showed that,the optimal reaction system(20 μL) was composed of0.15 mmoI/L dNTPs,1.5 U Taq DNA polymerase,0.50 μmol/L primers and 25 ng DNA templates.Using optimal reaction system and 8 pairs of SSR primers,a total of 101 fragments were amplified from 8 sugarcane germplasms of several countries,93 of which was polymorphic,with a polymorphism rate of 92.1%.The cluster analysis showed that,coefficient of genetic similarity among 8 germplasms ranged from 0.521 to 0.871.All tested materials could be clustered into two groups at similarity coefficient of 0.614,three foreign germplasms viz.,PINDAR,CP72-1210,CP84-1198 and ROC20,GT69-156 were clustered into the same group,but ROC26,GT02-237 and GT97-69 were clustered into another group.【Conclusion 】The established SSR-CGE system is suitable for genetic diversity analysis and genetic mapping of sugarcane due to its stable detection result and good reproducibility.
引文
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韩国辉,向素琼,汪卫星,贾志刚,洪棋斌,梁国鲁.2011.柑橘SCoT分子标记技术体系的建立及其在遗传分析中的应用[J].园艺学报,38(1):1243-1250.Han G H,Xiang S Q,Wang W X,Jia Z G,Hong Q B,Liang G L.2011.Establishment and application of SCoT molecular marker system for citms[J].Acta Horticulturae Sinica,38(1):1243-1250.
郝晨阳,王兰芬,贾继增,董玉琛,张学勇.2005.SSR荧光标记和银染技术的比较分析[J].作物学报,31(2):144-149.Hao C Y,Wang L F,Jia J Z,Dong Y C,Zhang X Y.2005.Comparison of fluorescence and silver-staining detection systems of microsatellite markers[J].Acta Agronomica Sinica,31(2):144-149.
黄东亮,覃肖良,廖青,高轶静,方锋学.2010.高质量甘蔗基因组DNA的简便快速提取方法研究[J].生物技术通报,(5):101-106.Huang D L,Qin X L,Liao Q,Gao Y J,Fang F X.2010.Simple and rapid procedure for isolation of high quality genomic DNA from sugarcane[J].Biotechnology Bulletin,(5):101-106.
李杨瑞,杨丽涛.2009.20世纪90年代以来我国甘蔗产业和科技的新发展[J].西南农业学报,22(5):1469-1475.Li Y R,Yang L T.2009.New developments in sugarcane industry and technologies in China since 1990[J].Southwest China Journal of Agricultural Sciences,22(5):1469-1475.
梁俊,PAN Yong-bao,李杨瑞,方锋学.2010.利用SSR标记与毛细管电泳技术对甘蔗属进行的遗传分析[J].广西植物,30(1):106-111.Liang J,Pan Y B,Li Y R,Fang F X.2010.Assessment of genetic diversity in Saccharum using SSR markers and capillary electrophoresis[J].Guihaia,30(1):106-111.
易红梅,王凤格,赵久然,王璐,郭景伦,原亚萍.2006.玉米品种SSR标记毛细管电泳荧光检测法与变性银染检测法的比较研究[J].华北农业学报,21(5):64-67.Yi H M,Wang F G,Zhao J R,Wang L,Guo J L,Yuan Y P.2006.Comparison of two maize SSR detection methods:capillary electrophoresis with fluorescence detection method and denaturing PAGE silver_staining detection method[J].Acta Agricuiture Boreali-Sinica,21(5):64-67.
张凤娟.2004.杂交水稻冈优527品种真实性和纯度的SSR检测方法[D].杨凌:西北农林科技大学.Zhang F G.2004.The variety identification and purity detection of the hybrid rice GANG-YOU 527 using SSR marker[D].Yangling:Northwest Sci-Tech University of Agriculture and Forestry.
邹丽梅,王凤格,赵久然,许理文,易红梅,宋伟.2011.适用于玉米特定分离群体的高通量SSR检测技术研究[J].玉米科学,19(2):26-28,33.Zou L M,Wang F G,Zhao J R,Xu L W,Yi H M,Song W.2011.Mapping population of maize by high throughput measurement ssr electrophoresis technique[J].Journal of Maize Sciences,19(2):26-28,33.
Bor P,Hindkjaer J,Kolvraa S,Ingerslev H J.2003.A new approach for screening for Y microdeletions:capillary electrophoresis combined with fluorescent multiplex PCR[J].Journal of Assisted Reproduction and Genetics,20(1):46-51.
Clark J M.1988.Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases[J].Nucleic Acids Research,16(20):9677-9686.
Cordeiro G M,Casu R,McIntyre C L,Manners J M,Henry R J.2001.Microsatellite markers from sugarcane(Saccharum spp.)ESTs cross transferable to erianthus and sorghum[J].Plant Science,160(6):1115-1123.
Huang X,Bomer A,Boder M,Ganal M.2002.Assessing genetic diversity of wheat(Triticum aestivum L.)germplasm using microsatellite markers[J].Theoretical and Applied Genetics,105(5):699-707.
Levinson G,Gutman G A.1987.Slipped-strand mispairing:a major mechanism for DNA sequence evolution[J].Molecular Biology&Evolution,4(3):203-221.
Pan Y B,Scheffler B.Richard E P.2007.High throughput genotyping of commercial sugarcane clones with microsatellite(SSR)DNA markers[J].Sugar Tech,9:176-181.
Schlotterer C,Tautz D.1992.Slippage synthesis of simple sequence DNA[J].Nucleic Acids Research,20(2):211-215.
程本义,夏俊辉,龚俊义,杨仕华.2011.SSR荧光标记毛细管电泳检测法在水稻DNA指纹鉴定中的应用[J].中国水稻科学,25(6):672-676.Cheng B Y,Xia J H,Gong J Y,Yang S H.2011.Application of capillary electrophoresis detection with fluorescent SSR marker in rice DNA fingerprint identification[J].Chinese Journal of Rice Science,25(6):672-676.
韩国辉,向素琼,汪卫星,贾志刚,洪棋斌,梁国鲁.2011.柑橘SCoT分子标记技术体系的建立及其在遗传分析中的应用[J].园艺学报,38(1):1243-1250.Han G H,Xiang S Q,Wang W X,Jia Z G,Hong Q B,Liang G L.2011.Establishment and application of SCoT molecular marker system for citms[J].Acta Horticulturae Sinica,38(1):1243-1250.
郝晨阳,王兰芬,贾继增,董玉琛,张学勇.2005.SSR荧光标记和银染技术的比较分析[J].作物学报,31(2):144-149.Hao C Y,Wang L F,Jia J Z,Dong Y C,Zhang X Y.2005.Comparison of fluorescence and silver-staining detection systems of microsatellite markers[J].Acta Agronomica Sinica,31(2):144-149.
黄东亮,覃肖良,廖青,高轶静,方锋学.2010.高质量甘蔗基因组DNA的简便快速提取方法研究[J].生物技术通报,(5):101-106.Huang D L,Qin X L,Liao Q,Gao Y J,Fang F X.2010.Simple and rapid procedure for isolation of high quality genomic DNA from sugarcane[J].Biotechnology Bulletin,(5):101-106.
李杨瑞,杨丽涛.2009.20世纪90年代以来我国甘蔗产业和科技的新发展[J].西南农业学报,22(5):1469-1475.Li Y R,Yang L T.2009.New developments in sugarcane industry and technologies in China since 1990[J].Southwest China Journal of Agricultural Sciences,22(5):1469-1475.
梁俊,PAN Yong-bao,李杨瑞,方锋学.2010.利用SSR标记与毛细管电泳技术对甘蔗属进行的遗传分析[J].广西植物,30(1):106-111.Liang J,Pan Y B,Li Y R,Fang F X.2010.Assessment of genetic diversity in Saccharum using SSR markers and capillary electrophoresis[J].Guihaia,30(1):106-111.
易红梅,王凤格,赵久然,王璐,郭景伦,原亚萍.2006.玉米品种SSR标记毛细管电泳荧光检测法与变性银染检测法的比较研究[J].华北农业学报,21(5):64-67.Yi H M,Wang F G,Zhao J R,Wang L,Guo J L,Yuan Y P.2006.Comparison of two maize SSR detection methods:capillary electrophoresis with fluorescence detection method and denaturing PAGE silver_staining detection method[J].Acta Agricuiture Boreali-Sinica,21(5):64-67.
张凤娟.2004.杂交水稻冈优527品种真实性和纯度的SSR检测方法[D].杨凌:西北农林科技大学.Zhang F G.2004.The variety identification and purity detection of the hybrid rice GANG-YOU 527 using SSR marker[D].Yangling:Northwest Sci-Tech University of Agriculture and Forestry.
邹丽梅,王凤格,赵久然,许理文,易红梅,宋伟.2011.适用于玉米特定分离群体的高通量SSR检测技术研究[J].玉米科学,19(2):26-28,33.Zou L M,Wang F G,Zhao J R,Xu L W,Yi H M,Song W.2011.Mapping population of maize by high throughput measurement ssr electrophoresis technique[J].Journal of Maize Sciences,19(2):26-28,33.
Bor P,Hindkjaer J,Kolvraa S,Ingerslev H J.2003.A new approach for screening for Y microdeletions:capillary electrophoresis combined with fluorescent multiplex PCR[J].Journal of Assisted Reproduction and Genetics,20(1):46-51.
Clark J M.1988.Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases[J].Nucleic Acids Research,16(20):9677-9686.
Cordeiro G M,Casu R,McIntyre C L,Manners J M,Henry R J.2001.Microsatellite markers from sugarcane(Saccharum spp.)ESTs cross transferable to erianthus and sorghum[J].Plant Science,160(6):1115-1123.
Huang X,Bomer A,Boder M,Ganal M.2002.Assessing genetic diversity of wheat(Triticum aestivum L.)germplasm using microsatellite markers[J].Theoretical and Applied Genetics,105(5):699-707.
Levinson G,Gutman G A.1987.Slipped-strand mispairing:a major mechanism for DNA sequence evolution[J].Molecular Biology&Evolution,4(3):203-221.
Pan Y B,Scheffler B.Richard E P.2007.High throughput genotyping of commercial sugarcane clones with microsatellite(SSR)DNA markers[J].Sugar Tech,9:176-181.
Schlotterer C,Tautz D.1992.Slippage synthesis of simple sequence DNA[J].Nucleic Acids Research,20(2):211-215.