葡萄种质资源SRAP-PCR反应体系的建立及优化
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摘要
【目的】建立葡萄种质资源稳定的SRAP-PCR分析体系,为葡萄种质资源研究和种质创新奠定基础。【方法】45份葡萄资源叶片为材料,利用单因素法对影响sRAP-PCR反应体系中的各个因素进行优化。【结果】优化的最佳SRAP-PCR反应体系为:模板DNA 50ng,Mg~(2+)1.5 mmol/L,引物0.2μmol/L,dNTPs 0.2mmol/L,TaqDNA聚合酶0.5U,反应体系总体积为25.0μL。【结论】优化后的葡萄种质资源SRAP-PCR反应体系,利用该体系能够扩增获得清晰、稳定的条带,可以用于葡萄遗传多样性及种质资源分析。
【Objective】 To establish and optimize SRAP-PCR reaction system for grape and lay the foundation for the Xinjiang grape germplasm resource research and germplasm innovation.【Method】45 grape sampies from the Xinjiang Turpan germplasm nursery were used as the experimental material and the single-factor method was applied for optimizing factors in the SRAP-PCR reaction system.【Result】The suitable PCR reaction system contained 50 ng template DNA,1.5rnmol/L Mg~(2+),0.2μmol/L primer,0.2 mmol/L dNTP,0.5U Taq DNA polymerase,and in total 25.0μL reaction solution.【Conclusion】 It was proved that optimized SRAP reaction system was optimized,with which clear and stable hands could be amplified.This would provide the basis for analysis of the genetic diversity and germplasm resources.
【Objective】 To establish and optimize SRAP-PCR reaction system for grape and lay the foundation for the Xinjiang grape germplasm resource research and germplasm innovation.【Method】45 grape sampies from the Xinjiang Turpan germplasm nursery were used as the experimental material and the single-factor method was applied for optimizing factors in the SRAP-PCR reaction system.【Result】The suitable PCR reaction system contained 50 ng template DNA,1.5rnmol/L Mg~(2+),0.2μmol/L primer,0.2 mmol/L dNTP,0.5U Taq DNA polymerase,and in total 25.0μL reaction solution.【Conclusion】 It was proved that optimized SRAP reaction system was optimized,with which clear and stable hands could be amplified.This would provide the basis for analysis of the genetic diversity and germplasm resources.
引文
[1]Wang Y J.Studies and utilization on germplaam resource of Chinese wild vitis species[J].Chinese gild Plant Resources,1989,2:11-13.
[2]Sabir,A.,Tangolar,S.,Buyukalaca,S.,&Kalkas,S.(2009).Ampelogrsphic and molecular diversity among grapevine(Vitis spp.)cultivars.Czech J Genet Plant Breed,45(4):160-168.
[3]Ahmad,R.,'Potter,D.,&Southwick,S.M.(2004).Genotyping of peach and nectarine cultivars with SSR and SRAP molecular markers.Journal of the American Society for Horticultural Science,129(2):204-210.
[4]Liu,L.,Liu,G.,Gong,Y.,Dai,W.,Wang,Y.,Yu,F.,&Ren,Y.(2007).Evaluation of genetic purity of F1 hybrid seeds in cabbage with RAPD,ISSR,SRAP,and SSR markers.HortScience,42(3):724-727.
[5]徐宗大,赵兰勇,张玲,等.玫瑰SRAP遗传多样性分析与品种指纹图谱构建[J].中国农业科学,2011,44(8):1 662-1 669.XU Zong-da,ZHAOLan-yong,ZHANG Ling,et al.(2011).Analysis of genetic diversity and construction of fingerprint of Rosa rugosa by SRAP[J].Scientia Agricultura Sinica,44(8):1,662-1,669.(in Chinese)
[6]陈晖,陈美霞,陶爱芬,等.长果种黄麻SRAP标记遗传连锁图谱的构建及3个质量性状基因定位[J].中国农业科学,2011,44(12):2 422-2 430.CHEN Hui,CHEN Mei-xia,TAO Ai-fen,et al.(2011).Construction of a molecular linkage map using SRAP and mapping of Three Qualitative Traits in Corchoras olltorius L.[J].Scientia Agricultura Sinica,44(12):2,422-2,430.(in Chinese)
[7]李仁伟,王晨,戴思兰,等.菊花品种表型性状与SRAP分子标记的关联分析[J].中国农业科学,2012,45(7):1 355-1365.LI Ren-wei,WANG Chen,DAI Si-lan,et al.(2012).The association analysis of phenotypic traits with SRAP markers in Chrysanthemum[J].Scientia Agricultura,ra Sinica,45(7):1,355-1,365.(in Chinese)
[8]黄勇.基于SRAP分子标记的小果油茶遗传多样性分析[J].林业科学,2013,49(3):43-50.HUANG Yong.(2013).Analysis of Camellia raeioearpa genetic diversity based on SRAP markers[J].&ientia Silvae Sinicae,49(3):43-50.(in Chinese)
[9]金炳奎,宗成文,曹后男,等.山葡萄SRAP技术体系的建立及其在品种鉴定中的应用EJ].吉林农业大学学报,2013,35(2):198-205.JIN Bing-kui,ZONG Cheng-wen,CAO Hou-nan,et al.(2013).Establishment of SRAP-PCR system and variety identification on Vitis amurensis[J].Journal of Jilin Agricultural University,35(2):198-205.(in Chinese)
[10]郑轶琦,王志勇,郭海林,等正交设计优化假俭草SRAP-PCR反应体系及引物筛选[J].草业学报,2008,17(4):110-117.ZHEN Yi-qi,WANG Zhi-yong,GUO Hal-lin,et al.(2008).Optimization of SRAP-PCR system on centipedegrass(Eremechloa ophiuroides)using orthoganal design and selection of primers[J].Acts Prataculturae Sinica,17(4):110-117.(in Chinese)
[11]张利平,林伯年,沈德绪,等.葡萄属植物染色体DNA的提取、纯化及RFLP鉴定[J].果树科学,1996,13(2):71-74.ZHANG Li-ping,LIN Bo-nian,SHEN De-xun,et al.(1996).Isolation,purification of genomic DNA and identification of RFLP in Vitis[J].Journal of Fruit Science,13(2):71-74.(in Chinese)
[12]Li,G.,&Quires,C.F.(2001).Sequence-related amplified polyraorphism(SRAP),a new marker system based on a simple PCR reaction:its application to mapping and gene tagging in Brassica.Theoretical and Applied Genetics,103(2-3):455-461.
[13]李元春,沈林,曾燕如.山核桃SRAP体系的建立及与RAPD和ISSR标记的比较[J].浙江农林大学学报,2011,28(3):505-512.LI Yuan-ehun,SHEN Lin,ZENG Yan-ra.et al.(2011).Establishment of a SRAP analysis protocol in Carya cathayensis and a comparison among SRAP,RAPD,ISSR analysis protocols[J].Journal of Zhefiang Forestry College,28(3):505-512.(in Chinese)
[14]赵春娥,鲍荟宇,李贺.沙棘SRAP-PCR反应体系的优化及引物筛选[J].湖北农业科学,2012.51(8):1691-1695.ZHAO Chun-e,BAO Hui-yu,13 He.(2012).Optimization of the SRAP-PCR system for Sea Buckthor(Hippophae rhamnoides)and Primers Screening[J].Hubei Agricultural Sciences,51(8):1,691-1,695.(in Chinese)
[15]尹跃,曹有龙,陈晓静,等.枸杞SIL~P反应体系建立和优化[J].福建农林大学学报(自然科学版),2013,42(3):297-301.YI Yue,CAO You-long,CHEN Xiao-jing,et al.(2013).Establishment and optimization of SRAP-PCR reaction system in Chinese wolfberry[J].Journal of Fujian Agriculture and Forestry University(Natural Science Ed.),42(3):297-301.(in Chinese)
[2]Sabir,A.,Tangolar,S.,Buyukalaca,S.,&Kalkas,S.(2009).Ampelogrsphic and molecular diversity among grapevine(Vitis spp.)cultivars.Czech J Genet Plant Breed,45(4):160-168.
[3]Ahmad,R.,'Potter,D.,&Southwick,S.M.(2004).Genotyping of peach and nectarine cultivars with SSR and SRAP molecular markers.Journal of the American Society for Horticultural Science,129(2):204-210.
[4]Liu,L.,Liu,G.,Gong,Y.,Dai,W.,Wang,Y.,Yu,F.,&Ren,Y.(2007).Evaluation of genetic purity of F1 hybrid seeds in cabbage with RAPD,ISSR,SRAP,and SSR markers.HortScience,42(3):724-727.
[5]徐宗大,赵兰勇,张玲,等.玫瑰SRAP遗传多样性分析与品种指纹图谱构建[J].中国农业科学,2011,44(8):1 662-1 669.XU Zong-da,ZHAOLan-yong,ZHANG Ling,et al.(2011).Analysis of genetic diversity and construction of fingerprint of Rosa rugosa by SRAP[J].Scientia Agricultura Sinica,44(8):1,662-1,669.(in Chinese)
[6]陈晖,陈美霞,陶爱芬,等.长果种黄麻SRAP标记遗传连锁图谱的构建及3个质量性状基因定位[J].中国农业科学,2011,44(12):2 422-2 430.CHEN Hui,CHEN Mei-xia,TAO Ai-fen,et al.(2011).Construction of a molecular linkage map using SRAP and mapping of Three Qualitative Traits in Corchoras olltorius L.[J].Scientia Agricultura Sinica,44(12):2,422-2,430.(in Chinese)
[7]李仁伟,王晨,戴思兰,等.菊花品种表型性状与SRAP分子标记的关联分析[J].中国农业科学,2012,45(7):1 355-1365.LI Ren-wei,WANG Chen,DAI Si-lan,et al.(2012).The association analysis of phenotypic traits with SRAP markers in Chrysanthemum[J].Scientia Agricultura,ra Sinica,45(7):1,355-1,365.(in Chinese)
[8]黄勇.基于SRAP分子标记的小果油茶遗传多样性分析[J].林业科学,2013,49(3):43-50.HUANG Yong.(2013).Analysis of Camellia raeioearpa genetic diversity based on SRAP markers[J].&ientia Silvae Sinicae,49(3):43-50.(in Chinese)
[9]金炳奎,宗成文,曹后男,等.山葡萄SRAP技术体系的建立及其在品种鉴定中的应用EJ].吉林农业大学学报,2013,35(2):198-205.JIN Bing-kui,ZONG Cheng-wen,CAO Hou-nan,et al.(2013).Establishment of SRAP-PCR system and variety identification on Vitis amurensis[J].Journal of Jilin Agricultural University,35(2):198-205.(in Chinese)
[10]郑轶琦,王志勇,郭海林,等正交设计优化假俭草SRAP-PCR反应体系及引物筛选[J].草业学报,2008,17(4):110-117.ZHEN Yi-qi,WANG Zhi-yong,GUO Hal-lin,et al.(2008).Optimization of SRAP-PCR system on centipedegrass(Eremechloa ophiuroides)using orthoganal design and selection of primers[J].Acts Prataculturae Sinica,17(4):110-117.(in Chinese)
[11]张利平,林伯年,沈德绪,等.葡萄属植物染色体DNA的提取、纯化及RFLP鉴定[J].果树科学,1996,13(2):71-74.ZHANG Li-ping,LIN Bo-nian,SHEN De-xun,et al.(1996).Isolation,purification of genomic DNA and identification of RFLP in Vitis[J].Journal of Fruit Science,13(2):71-74.(in Chinese)
[12]Li,G.,&Quires,C.F.(2001).Sequence-related amplified polyraorphism(SRAP),a new marker system based on a simple PCR reaction:its application to mapping and gene tagging in Brassica.Theoretical and Applied Genetics,103(2-3):455-461.
[13]李元春,沈林,曾燕如.山核桃SRAP体系的建立及与RAPD和ISSR标记的比较[J].浙江农林大学学报,2011,28(3):505-512.LI Yuan-ehun,SHEN Lin,ZENG Yan-ra.et al.(2011).Establishment of a SRAP analysis protocol in Carya cathayensis and a comparison among SRAP,RAPD,ISSR analysis protocols[J].Journal of Zhefiang Forestry College,28(3):505-512.(in Chinese)
[14]赵春娥,鲍荟宇,李贺.沙棘SRAP-PCR反应体系的优化及引物筛选[J].湖北农业科学,2012.51(8):1691-1695.ZHAO Chun-e,BAO Hui-yu,13 He.(2012).Optimization of the SRAP-PCR system for Sea Buckthor(Hippophae rhamnoides)and Primers Screening[J].Hubei Agricultural Sciences,51(8):1,691-1,695.(in Chinese)
[15]尹跃,曹有龙,陈晓静,等.枸杞SIL~P反应体系建立和优化[J].福建农林大学学报(自然科学版),2013,42(3):297-301.YI Yue,CAO You-long,CHEN Xiao-jing,et al.(2013).Establishment and optimization of SRAP-PCR reaction system in Chinese wolfberry[J].Journal of Fujian Agriculture and Forestry University(Natural Science Ed.),42(3):297-301.(in Chinese)