主要林下参地的土壤理化性质测定及林下参的RAPD分析
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摘要
为了探讨长白山区最适宜种植林下参的土壤条件,揭示不同地点人参的遗传多样性及亲缘关系,在长白山区四个不同地点采样,进行了林下参土壤理化性质分析和林下参的RAPD分析,其结果如下:
1对长白山区不同地点林下参地的土壤分析表明,抚松东岗、和龙头道、抚松北岗土壤的PH值(5.95、5.55与5.55)在0.05水平上显著的高于通化石湖(4.95);抚松东岗、和龙头道、抚松北岗土壤全P含量(1.1、1.1与0.9g/kg)在0.05水平上显著的高于通化石湖(0.7g/kg);不同层次的有机质含量、全P含量、速效K含量在0.01水平上差异显著。抚松东岗、抚松北岗以及和龙头道等3个林下参地土壤理化性质基本符合林下参的种植要求,而通化石湖林下参地不大适合种植林下参。
2应用CTAB法提取林下参基因组总DNA,以抚松林下参为材料,对林下参PCR扩增条件进行摸索与优化,结果表明:林下参扩增反应的最佳体系为:在20μL体积中,模板DNA20ng,dNTPs200~μmol/L,引物浓度20pmol/L,MgCl_2浓度2.0mmol/L,TaqDNA聚合酶1.5Unit。
3利用随机多态性扩增(RAPD)技术,对采自四个地点的林下参样品进行亲缘关系分析。用20个引物共扩增出167条带,其中92条为多态性带,其比率为55.1%;聚类分析结果表明,在遗传距离0.44水平上可以划分两大类,即长白山西侧组(抚松东岗、抚松北岗与通化石湖,其平均遗传距离为0.39)和长白山东侧组(和龙头道),两组间的遗传距离为0.68。
In order to explore the most suitable for condition of the soil parameter conditions and reveal the different the genetic diversity of transplanted wild ginseng of four different sampling sites, which done the analysis of soil physical and chemical properties and analysis of RAPD , the results are as follows
1.The results that analysis 4 diffierent sites show: The PH value of Donggang Beigang and Helong is higher remarkable than Tonghua in 0.05 level. The Total N of Donggang Beigang and Helong is higher remarkable than Tonghua in 0.05 level. Different levels of organic matter content, total P content, available K content in the 0.01 level of significant difference.Participation in three forest soil physical and chemical properties of forest plantations in line with the requirements . Tonghua is not suitable for transplanted wild ginseng cultivation.
2.The total DNA from young leaves of the translated wild ginseng of Fusong was successfully extracted by using the CTAB method .The results showed that most optimal reaction system of RAPD is : total volume20μL, containing template DNA 20ng, primer 20pmol, dNTPs 200μmol/L, MgCI_2 2.0 mmol/L, DNA polymerase 1.5unit, 10×Reaction Buffer 2.0 mmol/L, left complement with ddH_2O.
3. Using random amplified polymorphic (RAPD) techniques, collected from four locations of forest parameters related to analysis of samples.With 20 primers amplified 167 bands of which 92 were polymorphic, and their ratio was 55.1%. Cluster analysis results show that , the genetic relationship relations of Helong is far from the other three sites.
1对长白山区不同地点林下参地的土壤分析表明,抚松东岗、和龙头道、抚松北岗土壤的PH值(5.95、5.55与5.55)在0.05水平上显著的高于通化石湖(4.95);抚松东岗、和龙头道、抚松北岗土壤全P含量(1.1、1.1与0.9g/kg)在0.05水平上显著的高于通化石湖(0.7g/kg);不同层次的有机质含量、全P含量、速效K含量在0.01水平上差异显著。抚松东岗、抚松北岗以及和龙头道等3个林下参地土壤理化性质基本符合林下参的种植要求,而通化石湖林下参地不大适合种植林下参。
2应用CTAB法提取林下参基因组总DNA,以抚松林下参为材料,对林下参PCR扩增条件进行摸索与优化,结果表明:林下参扩增反应的最佳体系为:在20μL体积中,模板DNA20ng,dNTPs200~μmol/L,引物浓度20pmol/L,MgCl_2浓度2.0mmol/L,TaqDNA聚合酶1.5Unit。
3利用随机多态性扩增(RAPD)技术,对采自四个地点的林下参样品进行亲缘关系分析。用20个引物共扩增出167条带,其中92条为多态性带,其比率为55.1%;聚类分析结果表明,在遗传距离0.44水平上可以划分两大类,即长白山西侧组(抚松东岗、抚松北岗与通化石湖,其平均遗传距离为0.39)和长白山东侧组(和龙头道),两组间的遗传距离为0.68。
In order to explore the most suitable for condition of the soil parameter conditions and reveal the different the genetic diversity of transplanted wild ginseng of four different sampling sites, which done the analysis of soil physical and chemical properties and analysis of RAPD , the results are as follows
1.The results that analysis 4 diffierent sites show: The PH value of Donggang Beigang and Helong is higher remarkable than Tonghua in 0.05 level. The Total N of Donggang Beigang and Helong is higher remarkable than Tonghua in 0.05 level. Different levels of organic matter content, total P content, available K content in the 0.01 level of significant difference.Participation in three forest soil physical and chemical properties of forest plantations in line with the requirements . Tonghua is not suitable for transplanted wild ginseng cultivation.
2.The total DNA from young leaves of the translated wild ginseng of Fusong was successfully extracted by using the CTAB method .The results showed that most optimal reaction system of RAPD is : total volume20μL, containing template DNA 20ng, primer 20pmol, dNTPs 200μmol/L, MgCI_2 2.0 mmol/L, DNA polymerase 1.5unit, 10×Reaction Buffer 2.0 mmol/L, left complement with ddH_2O.
3. Using random amplified polymorphic (RAPD) techniques, collected from four locations of forest parameters related to analysis of samples.With 20 primers amplified 167 bands of which 92 were polymorphic, and their ratio was 55.1%. Cluster analysis results show that , the genetic relationship relations of Helong is far from the other three sites.
引文
[1]仲伟同,曹志强,冯家等.移山参鉴定及分等质量[J].人参研究,2007(3):27-31.
[2]吕龙石.中国人参业发展现状与展望[M].2006锦山世界人参博览会学术会,2006.
[3]韩国人参史[M].韩国人参史编纂委员会,2002:451-452.
[4]方土福.野山参经验鉴别[M].人民卫生出版社,2003(3):152-153.
[5]方土福.现代移山参研究[J].人参研究,2006(1):21-33.
[6]侯玉兵,李刚.山参人工繁衍研究生态调研报告[J].人参研究,2006(1):24-27.
[7]吉林省土壤肥料总站.吉林土壤[M],中国农业出版社,1991:579-582.
[8]李方元.中国人参和西洋参[M].中国农业科学技术出版社,2002:227-228.
[9]庞立杰,孙涛,董宇等无公害规范化栽培林下参研究[J],人参研究2004(4):31-32.
[10]杜文升、于炳发、邵守功.林下参培育研究[J],人参研究2005(4):20-23.
[11]吴平,周开亚.用聚合酶链反应产物直接测序技术鉴定中药材鳖甲.中国药科大学学报.199829(1):28-30.
[12]黄培唐,俞炜源,陈添弥.PCR技术实验指南.科学出版社,1998.
[13]WilliamsJG,Kubelik AR,Livak KJ,etal.DNA polymorphisms amplified byarbitrary primers are useful as genetic makers[J].NuclAcidsRes,1990,25(18):6531.
[14]Welsh J,Mcclelland M.Fingerprinting genomes using PCR with arbitrary primers[J].Nucl AcidsRes,1990,25(18):7213.
[15]赵妹华,张波.RAPD标记技术的实用性及稳定性探讨国外农学-杂粮作物1999,19(4):13-15.
[16]沈法富,刘凤珍,于元杰.分子标记在植物遗传育种中的应用[J].山东农业大学学报,1997,28(1):83.
[17]Jianping R,James R McFerson et al.Identites and relationships among Chinese vegetable B rassica as deter2mined by random amplified polymorphic DNA markers.J.A mer.Soc.Hort.Sci.1995,120(3):548-555.
[18]Mazzucato A,G Barcaccia et al.Random amplified polymorphic DNA and genetic diversity in Indian Musa germplasm.Genetic Resources and Crop Evolution,1995,42(2):107-118.
[19]Link W,C Dixkens et al.Genetic diversity in European and Mediterranean faba bean germplasm revealed byRAPD markers.Theoretical and Applied Genetics,1995,90(1):27-32.
[20]王康正.ONA分子诊断技术在药用植物研究上的应用[J].中国中药杂志,1997,22(12):707-710.
[21]安贤惠,李联泰.RAPD标记在植物种质资源研究中的应用[J],西北农业学报2000,9(3):126-130.
[22]李海渤.RAPD标记及其在植物育种上的应用,益阳师绊学报,2002,19(6):46-49.
[23]周宏.现代生物技术在中药鉴定方面的应用及前景.2001,8(3):55-56.
[24]王和勇,罗恒,孙敏.RAPD在中药材鉴别中的稳定性和可靠性.中药材,2004,27(1):63-66.
[25]潘光堂,荣廷昭,李晚忱.新世纪初发展我国玉米遗传育种科学技术的思考.玉米科学,2003,11(7):42-53.
[26]戴思兰,王丽霞,吴乃虎.刺五加遗传多样性的RAPD分析[J].自然科学进展.1998,8(4),420-425.
[27]李钧敏,金则新,钟章成.不同海拔高度大血藤群体遗传多样性的RAPD分析.
[28]汪小全,邹喻苹,张大明等.银杉遗传多样性分析的RAPD分析[J].中国科学C 辑,1996,26(5),436-441.
[29]任冰如,贺善安,经虹等.用RAPD技术评估苍术属群间的亲缘关系[J].中革药.2000,31(6),458-472
[30]钟方丽,李平亚.林下参研究进展[J].人参研究,2006(2):8-10
[31]钟方丽,刘金平,卢丹,李平亚.林下参化学成分的研究[J],中成药,2006(2):241-243.
[32]秦海音,谭立平等.林下参GAP种植技术规程[J].中国林副特产.2007(6):35-38.研
[33]杜文升,于炳发,邵守功.林下参培育研究[J].人参研究,2005(4):20-23.
[34]任德波.人参林下栽培新技术[J].新种植技术.2005(9):13-14.
[35]庞立杰,孙涛,董宇,刘强,朴英淑.无公害规范化栽培林下参研究人参研究.2004(4):31-32.
[36]杜韧强,康尔艳,宋迷彬.林下种植野山参新技[J]中草药栽培.2002(3):21-22.
[37]侯玉兵,李刚.山参人工繁衍研究生态调查报告.人参研究.2006(1):2427.
[38]冯薪硕,吕龙石等.长白山林下参基因组DNA提取及RAPD体系的优化[J].延边大学农学学报,2009,31(1):16-20.
[39]许自成、王林.湖南烟区土壤pH分布特点及其与土壤养分的关系[J].中国生态农业学报.2008,16(4):830-834
[40]鲍士旦.土壤农化分析.中国农业出版社[M].2000,12:25-109.
[41]Fang G,Hammar S,Grumet R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA[J].Biofeedback and self-Regulation.1992.13:52-57.
[42]Cheng F s,Brown S K,Weeden N F.A DNA extraction protocol from various tissues in woody species[J].HortScience,1997,32(2):921-922.
[43]Couch J A.Isolation of DNA from high polyphenolic[J].Plant Molecular Biology Reproter.1990,8:8-12.
[44]何聪芬,小松田隆夫.PCR筛选BAC文库和直接BAC末端测序方法的建立[J].遗传学报,2004,31(11):1262-1267.
[45]常青,周开亚.分子进化研究中系统发生树的重建生物多样性[J].生物多样性,1998,6(1):56-62.
[46]高雅,阎春霞,金天博等.中国19个不同地区汉族亚群的分子遗传学关系.西安交通大学学报(医学版).2005,26(2):111-114.
[47]陈永久,张亚平,齐瑾等中国貉随机扩增多态DNA及其亚种分化关系,遗传学报1998,25(7):16-21.
[48]邵爱娟、李欣、黄璐琦、魏建和、林淑芳用RAPD技术对人参栽培群体的遗传分析[J],中国中药杂志2004,(11):1033-1034.
[49]曹后男、高光出.桃属植物随机扩增多态性DNA分析的最适反应条件[J].延边大学农学学报,1999,21(4):249-253
[50]檀树先.人参开花生物学特性的初步观察.特产科学试验,1980,(1):7.
[2]吕龙石.中国人参业发展现状与展望[M].2006锦山世界人参博览会学术会,2006.
[3]韩国人参史[M].韩国人参史编纂委员会,2002:451-452.
[4]方土福.野山参经验鉴别[M].人民卫生出版社,2003(3):152-153.
[5]方土福.现代移山参研究[J].人参研究,2006(1):21-33.
[6]侯玉兵,李刚.山参人工繁衍研究生态调研报告[J].人参研究,2006(1):24-27.
[7]吉林省土壤肥料总站.吉林土壤[M],中国农业出版社,1991:579-582.
[8]李方元.中国人参和西洋参[M].中国农业科学技术出版社,2002:227-228.
[9]庞立杰,孙涛,董宇等无公害规范化栽培林下参研究[J],人参研究2004(4):31-32.
[10]杜文升、于炳发、邵守功.林下参培育研究[J],人参研究2005(4):20-23.
[11]吴平,周开亚.用聚合酶链反应产物直接测序技术鉴定中药材鳖甲.中国药科大学学报.199829(1):28-30.
[12]黄培唐,俞炜源,陈添弥.PCR技术实验指南.科学出版社,1998.
[13]WilliamsJG,Kubelik AR,Livak KJ,etal.DNA polymorphisms amplified byarbitrary primers are useful as genetic makers[J].NuclAcidsRes,1990,25(18):6531.
[14]Welsh J,Mcclelland M.Fingerprinting genomes using PCR with arbitrary primers[J].Nucl AcidsRes,1990,25(18):7213.
[15]赵妹华,张波.RAPD标记技术的实用性及稳定性探讨国外农学-杂粮作物1999,19(4):13-15.
[16]沈法富,刘凤珍,于元杰.分子标记在植物遗传育种中的应用[J].山东农业大学学报,1997,28(1):83.
[17]Jianping R,James R McFerson et al.Identites and relationships among Chinese vegetable B rassica as deter2mined by random amplified polymorphic DNA markers.J.A mer.Soc.Hort.Sci.1995,120(3):548-555.
[18]Mazzucato A,G Barcaccia et al.Random amplified polymorphic DNA and genetic diversity in Indian Musa germplasm.Genetic Resources and Crop Evolution,1995,42(2):107-118.
[19]Link W,C Dixkens et al.Genetic diversity in European and Mediterranean faba bean germplasm revealed byRAPD markers.Theoretical and Applied Genetics,1995,90(1):27-32.
[20]王康正.ONA分子诊断技术在药用植物研究上的应用[J].中国中药杂志,1997,22(12):707-710.
[21]安贤惠,李联泰.RAPD标记在植物种质资源研究中的应用[J],西北农业学报2000,9(3):126-130.
[22]李海渤.RAPD标记及其在植物育种上的应用,益阳师绊学报,2002,19(6):46-49.
[23]周宏.现代生物技术在中药鉴定方面的应用及前景.2001,8(3):55-56.
[24]王和勇,罗恒,孙敏.RAPD在中药材鉴别中的稳定性和可靠性.中药材,2004,27(1):63-66.
[25]潘光堂,荣廷昭,李晚忱.新世纪初发展我国玉米遗传育种科学技术的思考.玉米科学,2003,11(7):42-53.
[26]戴思兰,王丽霞,吴乃虎.刺五加遗传多样性的RAPD分析[J].自然科学进展.1998,8(4),420-425.
[27]李钧敏,金则新,钟章成.不同海拔高度大血藤群体遗传多样性的RAPD分析.
[28]汪小全,邹喻苹,张大明等.银杉遗传多样性分析的RAPD分析[J].中国科学C 辑,1996,26(5),436-441.
[29]任冰如,贺善安,经虹等.用RAPD技术评估苍术属群间的亲缘关系[J].中革药.2000,31(6),458-472
[30]钟方丽,李平亚.林下参研究进展[J].人参研究,2006(2):8-10
[31]钟方丽,刘金平,卢丹,李平亚.林下参化学成分的研究[J],中成药,2006(2):241-243.
[32]秦海音,谭立平等.林下参GAP种植技术规程[J].中国林副特产.2007(6):35-38.研
[33]杜文升,于炳发,邵守功.林下参培育研究[J].人参研究,2005(4):20-23.
[34]任德波.人参林下栽培新技术[J].新种植技术.2005(9):13-14.
[35]庞立杰,孙涛,董宇,刘强,朴英淑.无公害规范化栽培林下参研究人参研究.2004(4):31-32.
[36]杜韧强,康尔艳,宋迷彬.林下种植野山参新技[J]中草药栽培.2002(3):21-22.
[37]侯玉兵,李刚.山参人工繁衍研究生态调查报告.人参研究.2006(1):2427.
[38]冯薪硕,吕龙石等.长白山林下参基因组DNA提取及RAPD体系的优化[J].延边大学农学学报,2009,31(1):16-20.
[39]许自成、王林.湖南烟区土壤pH分布特点及其与土壤养分的关系[J].中国生态农业学报.2008,16(4):830-834
[40]鲍士旦.土壤农化分析.中国农业出版社[M].2000,12:25-109.
[41]Fang G,Hammar S,Grumet R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA[J].Biofeedback and self-Regulation.1992.13:52-57.
[42]Cheng F s,Brown S K,Weeden N F.A DNA extraction protocol from various tissues in woody species[J].HortScience,1997,32(2):921-922.
[43]Couch J A.Isolation of DNA from high polyphenolic[J].Plant Molecular Biology Reproter.1990,8:8-12.
[44]何聪芬,小松田隆夫.PCR筛选BAC文库和直接BAC末端测序方法的建立[J].遗传学报,2004,31(11):1262-1267.
[45]常青,周开亚.分子进化研究中系统发生树的重建生物多样性[J].生物多样性,1998,6(1):56-62.
[46]高雅,阎春霞,金天博等.中国19个不同地区汉族亚群的分子遗传学关系.西安交通大学学报(医学版).2005,26(2):111-114.
[47]陈永久,张亚平,齐瑾等中国貉随机扩增多态DNA及其亚种分化关系,遗传学报1998,25(7):16-21.
[48]邵爱娟、李欣、黄璐琦、魏建和、林淑芳用RAPD技术对人参栽培群体的遗传分析[J],中国中药杂志2004,(11):1033-1034.
[49]曹后男、高光出.桃属植物随机扩增多态性DNA分析的最适反应条件[J].延边大学农学学报,1999,21(4):249-253
[50]檀树先.人参开花生物学特性的初步观察.特产科学试验,1980,(1):7.