中韩野生软枣猕猴桃种质资源遗传多样性分析
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摘要
【目的】利用RAPD分子标记技术研究国内长白山地区和韩国由来野生软枣猕猴桃种质资源的遗传多样性。【方法】以长白山地区和韩国由来的28个野生软枣猕猴桃的叶片为材料,利用RAPD分子标记技术进行了遗传多样性分析,并明确了它们之间的亲缘关系。【结果】利用131个随机引物进行PCR,从中筛选出了多态性高、重复性好且扩增条带清晰的24个引物。24个引物在28个野生软枣猕猴桃中共扩增出191条带,其中多态性条带为186条,占97.4%。平均每个引物产生7.75个多态性条带。应用NTSYSpc 2.10e软件进行遗传一致度和遗传距离分析后用UPGMA方法进行聚类分析。28个野生软枣猕猴桃种质资源之间的遗传距离为0.020 2~0.934 2。遗传距离0.58时可将28个野生种划分成2大类。在遗传距离最小的0.02处,有蛟河2号和3号2个野生种,其RAPD分子标记相似性为98%。【结论】来自不同地理区域的野生软枣猕猴桃之间存在较高的遗传多样性,而在同一地理区域内遗传多样性较低。韩国野生软枣猕猴桃之间的遗传多样性较低,且与二道白河、汪清、左家等地的野生软枣猕猴桃亲缘关系较近。即具有同一地理区域聚类趋势,且不同地理区域间存在较高的遗传多样性。
【Objective】Actinidia arguta is more and more famous for its nutritional value and medical value. China, especially the Changbai mountain area, is the main distribution area of A. arguta. Most of the wild A. arguta are dioecious plants, the genealogy was not well established during the course of cultivation, management and breeding. The origin and and pedigree of some new varieties(strains) was unclear. Distant hybridization has become an important way of germplasm innovation. Understanding the genetic background of Chinese and foreign wild A. Arguta is important for the improvement of the species via hybridization. RAPD markers were employed to study the genetic diversity of the germplasm resources of wild A. arguta from China and South Korea.【Methods】The leaf DNA of 28 wild A. arguta from Changbai Mountain region(24) and South Korea(4) was extracted by the plant genome DNA extraction kit of Solarbio company. Genomic RAPD-PCR was amplified using the following amplification systems and procedures: The system included 2 μ L, 10 × PCR buffer, 2 U Taq DNA enzyme, 0.25 mmol· L~(-1) d NTP, 2.0 mmol· L~(-1) Mg2+, 0.3 μ mol· L~(-1) primer, 40 ng DNA template, and dd H_2O supplementation to 20 μ L. The PCR reaction was carried out on the BIO-RAD T100 TMThermal Cycler PCR instrument. The running amplification reaction program was as follows: an initial denaturation 94 ℃ for 5 min, denaturation at 94 ℃ for 1 min, annealing at 36 ℃ for 1 min, extension at 72 ℃ for 2 min, reaction of 40 cycles, last extension at 72 ℃ for 5 min. The same template was repeated twice. RAPD-PCR amplification products were analyzed by 0.8% agarose gel(0.5×TAE buffer) electrophoresis.【Results】The 24 primers had high genetic polymorphisms. A total of 191 bands were amplified, of which 186 were polymorphic bands, accounting for 97.4%. Average 7.75 polymorphic bands were produced by each primer. Genetic identity and genetic distance were calculated by NTSYSpc 2.10 e software and cluster analysis was analyzed by UPGMA. The result showed that the genetic distances among the 28 wild species of A. arguta germplasm resources were 0.020 2-0.934 2. When the genetic distance was0.58, the 28 wild species could be divided into two groups. The first group included Antu coloring type,Antu No. 1-6 from Antu County and Jiaohe No. 1-5 from Jiaohe. The second group include: Erdaobaihe No. 1-4 from Erdaobaihe; Wangqing No. 1-3 from Wangqing County and Wangqing Forestry Bureau;Zuojia No. 1-3 from the Zuojia special production Institute of the Chinese Academy of Agricultural Sciences(CAAS); Liaoning Huanren and Helong Qingshan; South Korea No. 1-4 from Chungbuk, Jeonnam and Gyeongnam in South Korea. The similarity of RAPD markers of Jiaohe 2 and 3, the two wild species reached 98 % when the genetic distance was 0.0202. Almost all the wild species from the same area belonged to the same category within a very small genetic distance. For example, Antu No. 1-6 were within the 0.184 genetic distance; Jiaohe No. 1-5 were within 0.18 genetic distance; Wangqing No.1-3 and Erdaobaihe No. 1 and No. 4 were within 0.315 genetic distance; Zuojia No. 1-3 was within 0.25 genetic distance; South Korea No. 1-4 were within 0.23 genetic distance; Erdaobaihe No. 2 and No. 3 were within 0.41 genetic distance.【Conclusion】There was a high genetic diversity among wild A. arguta collected from different geographic regions, but genetic diversity among those collected from the same geographical area was low. The genetic diversity of the wild A. arguta from South Korea was relatively low and was closely related to the wild A. arguta from Erdaobaihe, Wang Qing and Zuo Jia. The species collected from the same geographical area had the same clustering trend.
【Objective】Actinidia arguta is more and more famous for its nutritional value and medical value. China, especially the Changbai mountain area, is the main distribution area of A. arguta. Most of the wild A. arguta are dioecious plants, the genealogy was not well established during the course of cultivation, management and breeding. The origin and and pedigree of some new varieties(strains) was unclear. Distant hybridization has become an important way of germplasm innovation. Understanding the genetic background of Chinese and foreign wild A. Arguta is important for the improvement of the species via hybridization. RAPD markers were employed to study the genetic diversity of the germplasm resources of wild A. arguta from China and South Korea.【Methods】The leaf DNA of 28 wild A. arguta from Changbai Mountain region(24) and South Korea(4) was extracted by the plant genome DNA extraction kit of Solarbio company. Genomic RAPD-PCR was amplified using the following amplification systems and procedures: The system included 2 μ L, 10 × PCR buffer, 2 U Taq DNA enzyme, 0.25 mmol· L~(-1) d NTP, 2.0 mmol· L~(-1) Mg2+, 0.3 μ mol· L~(-1) primer, 40 ng DNA template, and dd H_2O supplementation to 20 μ L. The PCR reaction was carried out on the BIO-RAD T100 TMThermal Cycler PCR instrument. The running amplification reaction program was as follows: an initial denaturation 94 ℃ for 5 min, denaturation at 94 ℃ for 1 min, annealing at 36 ℃ for 1 min, extension at 72 ℃ for 2 min, reaction of 40 cycles, last extension at 72 ℃ for 5 min. The same template was repeated twice. RAPD-PCR amplification products were analyzed by 0.8% agarose gel(0.5×TAE buffer) electrophoresis.【Results】The 24 primers had high genetic polymorphisms. A total of 191 bands were amplified, of which 186 were polymorphic bands, accounting for 97.4%. Average 7.75 polymorphic bands were produced by each primer. Genetic identity and genetic distance were calculated by NTSYSpc 2.10 e software and cluster analysis was analyzed by UPGMA. The result showed that the genetic distances among the 28 wild species of A. arguta germplasm resources were 0.020 2-0.934 2. When the genetic distance was0.58, the 28 wild species could be divided into two groups. The first group included Antu coloring type,Antu No. 1-6 from Antu County and Jiaohe No. 1-5 from Jiaohe. The second group include: Erdaobaihe No. 1-4 from Erdaobaihe; Wangqing No. 1-3 from Wangqing County and Wangqing Forestry Bureau;Zuojia No. 1-3 from the Zuojia special production Institute of the Chinese Academy of Agricultural Sciences(CAAS); Liaoning Huanren and Helong Qingshan; South Korea No. 1-4 from Chungbuk, Jeonnam and Gyeongnam in South Korea. The similarity of RAPD markers of Jiaohe 2 and 3, the two wild species reached 98 % when the genetic distance was 0.0202. Almost all the wild species from the same area belonged to the same category within a very small genetic distance. For example, Antu No. 1-6 were within the 0.184 genetic distance; Jiaohe No. 1-5 were within 0.18 genetic distance; Wangqing No.1-3 and Erdaobaihe No. 1 and No. 4 were within 0.315 genetic distance; Zuojia No. 1-3 was within 0.25 genetic distance; South Korea No. 1-4 were within 0.23 genetic distance; Erdaobaihe No. 2 and No. 3 were within 0.41 genetic distance.【Conclusion】There was a high genetic diversity among wild A. arguta collected from different geographic regions, but genetic diversity among those collected from the same geographical area was low. The genetic diversity of the wild A. arguta from South Korea was relatively low and was closely related to the wild A. arguta from Erdaobaihe, Wang Qing and Zuo Jia. The species collected from the same geographical area had the same clustering trend.
引文
[1]刘延吉,耿书,田晓艳.辽宁地区五种野生软枣猕猴桃RAPD遗传多样性分析[J].北方园艺,2010(18):130-132.LIU Yanji,GENG Shu,TIAN Xiaoyan.The analysis of genetic diversity on random amplified polymorphic DNA of Five Actinidia arguta species in Liaoning province[J].Northern Horticulture,2010(18):130-132.
[2]王佳卉.软枣猕猴桃EST-SSR分子标记的开发及遗传多样性分析[D].长春:吉林农业大学,2014.WANG Jiahui.Development of EST-derived SSR markers in Actinidia arguta and genetic diversity analysis[D].Changchun:Jilin Agricultural University,2014.
[3]黄宏文,龚俊杰,王圣梅,何子灿,张忠慧,李建强.猕猴桃属(Actinidia)植物的遗传多样性[J].生物多样性,2000,8(1):1-12.HUANG Hongwen,GONG Junjie,WANG Shengmei,HE Zican,ZHANG Zhonghui,LI Jianqiang.Genetic diversity in the genus Actinidia[J].Chinese Biodiversity,2000,8(1):1-12.
[4]李旭,曹万万,姜丹,孙贺,朴一龙.长白山野生软枣猕猴桃资源分布与果实和叶片性状多样性[J].北方园艺,2015(15):22-27.LI Xu,CAO Wanwan,JIANG Dan,SUN He,PIAO Yilong.Resource distribution and character diversity of fruit and leaf of wild Actinidia arguta from Changbai Mountain area[J].Northern Horticulture,2015(15):22-27.
[5]张甲生,李平亚,石毅,宋秀环,卢士香,刘景英,李滦宁,王陆黎,田莉玉.软枣猕猴桃中29种无机元素含量的测定[J].白求恩医科大学学报,1993(4):354-356.ZHANG Jiasheng,LI Pingya,SHI Yi,SONG Xiuhuan,LU Shixiang,LIU Jingying,LI Luanning,WANG Luli,TIAN Liyu.Determination of contents of inorganic elements in different parts of Actinidia arguta(Sieb.et zucc.)Planch[J].Journal of Norman Bethune University of Medical Science,1993(4):354-356.
[6]孙宁宁.长白山野生软枣猕猴桃的成分分析及保鲜研究[D].长春:吉林农业大学,2007.SUN Ningning.Component analyzes and refreshing stady on Actinidia arguta in Changbaishan[D].Changchun:Jilin Agricultural University,2007.
[7]黄祥童,刘军,金哲军,隋亚臣.软枣猕猴桃光雾扦插育苗试验[J].北华大学学报(自然科学版),2010,11(2):177-182.HUANG Xiangtong,LIU Jun,JIN Zhejun,SUI Yachen.Test on sunligh and spray cuttage seedling of Actinidia arguta[J].Jounal of Beihua University(Natural Science),2010,11(2):177-182.
[8]李红莉.东北地区野生猕猴桃生物学特性的分析与研究[J].中国林副特产,2013(2):13-15.LI Hongli.Study and analysis on biological characteristics of wild kiwifruit in northeast China region[J].Forest By-Product and Speciality in China,2013(2):13-15.
[9]戴晓锋.长春公园园林植物多样性调查和应用研究[D].长春:吉林农业大学,2016.HUANG Xiaofeng.Study on the diversity investigation and application of ornamental plants in Chengchun Park[D].Changchun:Jilin Agricultural University,2016.
[10]赵芮.软枣猕猴桃离体快繁技术的研究[D].长春:吉林农业大学,2016.ZHAO Rui.Study on rapid propagation technique in vitro of Actinidia arguta[D].Changchun:Jilin Agricultural University,2016.
[11]黄圆博,姜丹,李旭,刘香苏,朴一龙.软枣猕猴桃苗木繁育技术研究[J].延边大学农学学报,2017,39(1):29-34.HUANG Yuanbo,JIANG Dan,LI Xu,LIU Xiangsu,PIAO Yilong.A study on the propagation of nursery stock technique of Actinidia arguta fruit[J].Journal of Agricultural Science Yanbian University,2017,39(1):29-34.
[12]廖慧苹.3个软枣猕猴桃单系开花特性和果实品质调查比较及栽培技术[D].成都:四川农业大学,2012.LIAO Huiping.Comparison of the blossoms and fruits characteristic of three Actinidia Arguta strains and tts planting technique[D].Chengdu:Sichuan Agricultural University,2012.
[13]朴一龙,赵兰花.软枣猕猴桃研究进展[J].北方园艺,2008(3):76-78.PIAO Yilong,ZHAO Lanhua.Research progress of Actinidia arguta[J].Northern Horticulture,2008(3):76-78.
[14]黄宏文.猕猴桃属:分类、资源、驯化、栽培[M].北京:科学出版社,2013:50-52.HUANG Hongwen.Actinidia:taxonomy,germplasm,domestication,cultivation[M].Beijing:Science Press,2013:50-52.
[15]朴一龙,赵兰花.韩国软枣猕猴桃开发利用概况[J].中国果树,2012(4):75-76.PIAO Yilong,ZHAO Lanhua.Development and utilization of South Korean A.argute[J].China Fruits,2012(4):75-76.
[16]路文鹏,李昌禹,曲炳章,李晓红,宋润刚,赵淑兰,王军.东北原生种猕猴桃种质RAPD研究[J].特产研究,2006,28(2):24-27.LU Wenpeng,LI Changyu,QU Bingzhang,LI Xiaohong,SONG Rungang,ZHAO Shulan,WANG Jun.A study of RAPD on germplasm of northeastern Actinidia chinensis in China[J].Special Wild Economic Animal and Plant Research,2006,28(2):24-27.
[17]黄岳,朴一龙,王琳.长白山区野生软枣猕猴桃种质RAPD分析[J].延边大学农学学报,2009,31(2):119-123.HUANG Yue,PIAO Yilong,WANG Lin.Analysis of wild A.arguta resources in Changbai Mountain area by RAPD[J].Journal of Agricultural Science Yanbian University,2009,31(2):119-123.
[18]魏艳霞,豁泽春,王飞,张华敏,李金月.野生猕猴桃DNA的提取及RAPD分析体系的建立[J].西北农业学报,2008,17(2):169-173.WEI Yanxia,HOU Zechun,WANG Fei,ZHANG Huamin,LI Jinyue.Extraction of DNA and establishment of RAPD system in wild kiwifruit[J].Acta Agriculturae Boreali-occidentalis Sinica,2008,17(2):169-173.
[19]NEI M.Estimation of average heterozygosity and genetic distance from a small number of individuals[J].Genetics,1978,89(3):583-590.
[20]GRANT V.The evolutionary process:a critical study of evolutionary theory[M].New York:Columbia University Press,1991.
[21]MILLAR C I,LIBBY W J.Strategies for conserving clinal,ecotypic and disjunct population diversity in widespread species[M]//Genetics and conservation of rare plants.New York:Oxford University Press,1991:149-170.
[2]王佳卉.软枣猕猴桃EST-SSR分子标记的开发及遗传多样性分析[D].长春:吉林农业大学,2014.WANG Jiahui.Development of EST-derived SSR markers in Actinidia arguta and genetic diversity analysis[D].Changchun:Jilin Agricultural University,2014.
[3]黄宏文,龚俊杰,王圣梅,何子灿,张忠慧,李建强.猕猴桃属(Actinidia)植物的遗传多样性[J].生物多样性,2000,8(1):1-12.HUANG Hongwen,GONG Junjie,WANG Shengmei,HE Zican,ZHANG Zhonghui,LI Jianqiang.Genetic diversity in the genus Actinidia[J].Chinese Biodiversity,2000,8(1):1-12.
[4]李旭,曹万万,姜丹,孙贺,朴一龙.长白山野生软枣猕猴桃资源分布与果实和叶片性状多样性[J].北方园艺,2015(15):22-27.LI Xu,CAO Wanwan,JIANG Dan,SUN He,PIAO Yilong.Resource distribution and character diversity of fruit and leaf of wild Actinidia arguta from Changbai Mountain area[J].Northern Horticulture,2015(15):22-27.
[5]张甲生,李平亚,石毅,宋秀环,卢士香,刘景英,李滦宁,王陆黎,田莉玉.软枣猕猴桃中29种无机元素含量的测定[J].白求恩医科大学学报,1993(4):354-356.ZHANG Jiasheng,LI Pingya,SHI Yi,SONG Xiuhuan,LU Shixiang,LIU Jingying,LI Luanning,WANG Luli,TIAN Liyu.Determination of contents of inorganic elements in different parts of Actinidia arguta(Sieb.et zucc.)Planch[J].Journal of Norman Bethune University of Medical Science,1993(4):354-356.
[6]孙宁宁.长白山野生软枣猕猴桃的成分分析及保鲜研究[D].长春:吉林农业大学,2007.SUN Ningning.Component analyzes and refreshing stady on Actinidia arguta in Changbaishan[D].Changchun:Jilin Agricultural University,2007.
[7]黄祥童,刘军,金哲军,隋亚臣.软枣猕猴桃光雾扦插育苗试验[J].北华大学学报(自然科学版),2010,11(2):177-182.HUANG Xiangtong,LIU Jun,JIN Zhejun,SUI Yachen.Test on sunligh and spray cuttage seedling of Actinidia arguta[J].Jounal of Beihua University(Natural Science),2010,11(2):177-182.
[8]李红莉.东北地区野生猕猴桃生物学特性的分析与研究[J].中国林副特产,2013(2):13-15.LI Hongli.Study and analysis on biological characteristics of wild kiwifruit in northeast China region[J].Forest By-Product and Speciality in China,2013(2):13-15.
[9]戴晓锋.长春公园园林植物多样性调查和应用研究[D].长春:吉林农业大学,2016.HUANG Xiaofeng.Study on the diversity investigation and application of ornamental plants in Chengchun Park[D].Changchun:Jilin Agricultural University,2016.
[10]赵芮.软枣猕猴桃离体快繁技术的研究[D].长春:吉林农业大学,2016.ZHAO Rui.Study on rapid propagation technique in vitro of Actinidia arguta[D].Changchun:Jilin Agricultural University,2016.
[11]黄圆博,姜丹,李旭,刘香苏,朴一龙.软枣猕猴桃苗木繁育技术研究[J].延边大学农学学报,2017,39(1):29-34.HUANG Yuanbo,JIANG Dan,LI Xu,LIU Xiangsu,PIAO Yilong.A study on the propagation of nursery stock technique of Actinidia arguta fruit[J].Journal of Agricultural Science Yanbian University,2017,39(1):29-34.
[12]廖慧苹.3个软枣猕猴桃单系开花特性和果实品质调查比较及栽培技术[D].成都:四川农业大学,2012.LIAO Huiping.Comparison of the blossoms and fruits characteristic of three Actinidia Arguta strains and tts planting technique[D].Chengdu:Sichuan Agricultural University,2012.
[13]朴一龙,赵兰花.软枣猕猴桃研究进展[J].北方园艺,2008(3):76-78.PIAO Yilong,ZHAO Lanhua.Research progress of Actinidia arguta[J].Northern Horticulture,2008(3):76-78.
[14]黄宏文.猕猴桃属:分类、资源、驯化、栽培[M].北京:科学出版社,2013:50-52.HUANG Hongwen.Actinidia:taxonomy,germplasm,domestication,cultivation[M].Beijing:Science Press,2013:50-52.
[15]朴一龙,赵兰花.韩国软枣猕猴桃开发利用概况[J].中国果树,2012(4):75-76.PIAO Yilong,ZHAO Lanhua.Development and utilization of South Korean A.argute[J].China Fruits,2012(4):75-76.
[16]路文鹏,李昌禹,曲炳章,李晓红,宋润刚,赵淑兰,王军.东北原生种猕猴桃种质RAPD研究[J].特产研究,2006,28(2):24-27.LU Wenpeng,LI Changyu,QU Bingzhang,LI Xiaohong,SONG Rungang,ZHAO Shulan,WANG Jun.A study of RAPD on germplasm of northeastern Actinidia chinensis in China[J].Special Wild Economic Animal and Plant Research,2006,28(2):24-27.
[17]黄岳,朴一龙,王琳.长白山区野生软枣猕猴桃种质RAPD分析[J].延边大学农学学报,2009,31(2):119-123.HUANG Yue,PIAO Yilong,WANG Lin.Analysis of wild A.arguta resources in Changbai Mountain area by RAPD[J].Journal of Agricultural Science Yanbian University,2009,31(2):119-123.
[18]魏艳霞,豁泽春,王飞,张华敏,李金月.野生猕猴桃DNA的提取及RAPD分析体系的建立[J].西北农业学报,2008,17(2):169-173.WEI Yanxia,HOU Zechun,WANG Fei,ZHANG Huamin,LI Jinyue.Extraction of DNA and establishment of RAPD system in wild kiwifruit[J].Acta Agriculturae Boreali-occidentalis Sinica,2008,17(2):169-173.
[19]NEI M.Estimation of average heterozygosity and genetic distance from a small number of individuals[J].Genetics,1978,89(3):583-590.
[20]GRANT V.The evolutionary process:a critical study of evolutionary theory[M].New York:Columbia University Press,1991.
[21]MILLAR C I,LIBBY W J.Strategies for conserving clinal,ecotypic and disjunct population diversity in widespread species[M]//Genetics and conservation of rare plants.New York:Oxford University Press,1991:149-170.