青稞AFLP体系的建立及优化
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摘要
本文以青稞为材料,对AFLP酶切连接、预扩增、选择性扩增过程进行优化。结果表明,酶切反应条件为DNA模板200 ng、限制性内切酶Eco RI/Mse I 4U、酶切时间4h;连接反应条件为连接温度16℃,T4连接酶3 U,连接时间12 h以上;预扩增反应条件为r Taq聚合酶3 U、dNTP 4 mmol/L、镁离子浓度1.00 mmol/L;选择性扩增反应条件为r Taq聚合酶4 U、d NTP 4 mmol/L、镁离子浓度1.25 mmol/L及预扩产物稀释10倍。利用优化后的AFLP体系,以藏青2000、冬青18这2个供试材料的基因组为模板,从100对引物组合中筛选出10对重复性好、稳定性强、扩增条带清晰、分布均匀、多态性高的引物组合,为青稞种质资源鉴定及遗传多样性分析提供了基础。
Taking hulless barley as the material for AFLP reaction system,the enzyme cut-link reaction,pre-amplification reaction and selective amplification reaction were optimized in this paper.The results showed that the enzyme cut-link reaction required 200 ng genomic DNA digested by 4 U Eco RI and Mse I for 4 h,then linked by 3 U T4 DNA ligase for 12 h at 16 ℃.The pre-amplification reaction required 3 U r Taq DNA polymerase,4 mmol/L dNTP and 1.00 mmol/L Mg~(2+).The selective amplification reaction required 4 U r Taq DNA polymerase,4 mmol/L d NTP,1.25 mmol/L Mg~(2+) and pre-amplification product diluted 10 times.Using the genomes of Zangqing 2000 and Dongqing 18 as templates and the optimized AFLP system,10 primers were selected from 100 primers.The selective primers could provide better repeatability,stability and polymorphism.The amplified bands were clearer and more evenly distributed.The optimization of AFLP system provides a basis for identification and genetic diversity analysis of hulless barley germplasm resource.
Taking hulless barley as the material for AFLP reaction system,the enzyme cut-link reaction,pre-amplification reaction and selective amplification reaction were optimized in this paper.The results showed that the enzyme cut-link reaction required 200 ng genomic DNA digested by 4 U Eco RI and Mse I for 4 h,then linked by 3 U T4 DNA ligase for 12 h at 16 ℃.The pre-amplification reaction required 3 U r Taq DNA polymerase,4 mmol/L dNTP and 1.00 mmol/L Mg~(2+).The selective amplification reaction required 4 U r Taq DNA polymerase,4 mmol/L d NTP,1.25 mmol/L Mg~(2+) and pre-amplification product diluted 10 times.Using the genomes of Zangqing 2000 and Dongqing 18 as templates and the optimized AFLP system,10 primers were selected from 100 primers.The selective primers could provide better repeatability,stability and polymorphism.The amplified bands were clearer and more evenly distributed.The optimization of AFLP system provides a basis for identification and genetic diversity analysis of hulless barley germplasm resource.
引文
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[3]VOS P,HOGERS R,ZABEAU M,et al.AFLP:a new technique for DNAfingerprinting[J].Nucleic Acids Res,1995,23(21):4407-4417.
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[5]LIU Y G,LIU L X.Isolation and characterization of twenty two polymorphic simple sequence repeat markers from AFLP sequences of Crassostrea gigas[J].Conservation Genetics Resources,2015,7(3):659-661.
[6]WANG X,XING S Y,SUN L M,et al.Genetic diversity of Styrax obassia Sieb.et Zucc.based on AFLP markers[J].Biochemical Systematics&Ecology,2015:6128-6134.
[7]BIAN F H,PANG Y J,WANG Z,et al.Genetic diversity of the rare plant Anemone shikokiana(Makino)Makino(Ranunculaceae)inferred from AFLP markers[J].Plant Systematics and Evolution,2015,301(2):677-684.
[8]LING Y,HUANG L K,ZHANG X Q,et al.Assessment of genetic diversity of bermudagrass germplasm from southwest China and Africa by using AFLP markers[J].Genet Mol Res,2015,14(1):1748-1756.
[9]MUELLER U G,WOLFENBARGER L L.AFLP genotyping and fingerprinting[J].Trends in Ecology and Evolution,1999,14(10):389-394.
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[11]李双铃,任艳,陶海腾,等.山东花生主栽品种AFLP指纹图谱的构建[J].花生学报,2006,35(1):18-21.
[12]张瑞萍,吴俊,李秀根,等.梨AFLP标记遗传图谱构建及果实相关性状的QTL定位[J].园艺学报,2011,38(10):1991-1998.!!!!!!!!!!!!!!!!!!!!!!!
[2]孙立军,陆炜,张京,等.中国大麦种质资源鉴定评价及其利用研究[J].中国农业科学,1999,32(2):24-31.!!!!!!!!!!!!!!!!!!!!!!!
[3]VOS P,HOGERS R,ZABEAU M,et al.AFLP:a new technique for DNAfingerprinting[J].Nucleic Acids Res,1995,23(21):4407-4417.
[4]TURPEINEN T,VANHALA T,NEVO E,et al.AFLP genetic polymorphism in wild barley(Hordeum spontaneum)populations in Israel[J].Theoretical and Applied Genetics,2003,106(7):1333-1339.
[5]LIU Y G,LIU L X.Isolation and characterization of twenty two polymorphic simple sequence repeat markers from AFLP sequences of Crassostrea gigas[J].Conservation Genetics Resources,2015,7(3):659-661.
[6]WANG X,XING S Y,SUN L M,et al.Genetic diversity of Styrax obassia Sieb.et Zucc.based on AFLP markers[J].Biochemical Systematics&Ecology,2015:6128-6134.
[7]BIAN F H,PANG Y J,WANG Z,et al.Genetic diversity of the rare plant Anemone shikokiana(Makino)Makino(Ranunculaceae)inferred from AFLP markers[J].Plant Systematics and Evolution,2015,301(2):677-684.
[8]LING Y,HUANG L K,ZHANG X Q,et al.Assessment of genetic diversity of bermudagrass germplasm from southwest China and Africa by using AFLP markers[J].Genet Mol Res,2015,14(1):1748-1756.
[9]MUELLER U G,WOLFENBARGER L L.AFLP genotyping and fingerprinting[J].Trends in Ecology and Evolution,1999,14(10):389-394.
[10]SIEGFRIED L K.Complete exclusion of nonsires in an analysis of paternity in a natural plant population using amplified fragment length polymorphism(AFLP)[J].Molecular Ecology,1999,8(2):217-226.
[11]李双铃,任艳,陶海腾,等.山东花生主栽品种AFLP指纹图谱的构建[J].花生学报,2006,35(1):18-21.
[12]张瑞萍,吴俊,李秀根,等.梨AFLP标记遗传图谱构建及果实相关性状的QTL定位[J].园艺学报,2011,38(10):1991-1998.!!!!!!!!!!!!!!!!!!!!!!!