三叶青种质资源遗传多样性的ISSR分析
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摘要
目的对我国三叶青种质资源64个样本的遗传多样性进行分析。方法利用ISSR-PCR技术进行扩增,然后利用POPGENE 32软件及NTSYS软件分析三叶青种质资源64个样本的遗传多样性及亲缘关系,并根据UPGMA法,构建亲缘关系树状图。结果从30条引物中筛选出10条条带清晰、重复性好的引物对64份供试材料的基因组DNA进行扩增。扩增出了83个多态位点,多态百分数为71.43%～100.00%,平均多态百分数为94.31%,引物S17扩增得到的多态位点最多,为11个;引物P6扩增得到的多态位点最少,为5个,10条引物扩增得到的多态位点平均为8.3个;遗传多样性分析显示,平均检测等位基因数(N_a)为1.943 1,平均有效等位基因数(N_e)为1.381 08,64个样本的平均Nei’s基因多样性指数(H)为0.242 98,平均Shannon多样性指数(I)为0.385 83。64个样本遗传相似系数的变异范围为0.431 8～0.988 6。根据相似系数矩阵按UPGMA法进行聚类,在遗传相似性系数为0.715 5处,64份供试材料可分为6组;另外,根据10个ISSR引物的扩增结果,筛选出引物ISSR 20、UBC857和S17扩增的DNA指纹图谱可用于64个三叶青样本种质的鉴定。结论我国三叶青种质资源拥有丰富的遗传多样性和基因的相对稳定性,ISSR分析可揭示我国三叶青种质资源间的亲缘关系,为评价、鉴定和新品种选育提供一定的参考依据。
Objective In this paper,the genetic diversity of 64 samples of Tetrastigma hemsleyanum germplasm resources in Chinese was analyzed.Methods ISSR-PCR was firstly used to amplify,and then POPGENE 32 software and NTSYS software was used to analyze the genetic diversity and phylogenetic relationship of 64 samples of T.hemsleyanum germplasm resources,and phylogenetic tree was constructed according to the UPGMA method.Results Ten primers with clear and reproducible bands were screened from 30 primers and used for genomic DNA amplification of 64 sample materials.A total of 83 polymorphic locis were amplified,whose polymorphic percentages were 71.43%—100% and average polymorphism percentage was 94.31%.The amplification polymorphic locis of primer S17 were the most(11) and the amplification polymorphic locis of primer P6 were the least(5),the average amplified polymorphic locis of 10 primers were 8.3.Genetic diversity analysis showed that the average number of alleles(Na) of 64 samples was 1.943 1,the average effective allele number(Ne) was 1.381 08,the average Nei's gene diversity index(H) was 0.242 98,and the average Shannon diversity index(I) was 0.385 83.The variation range of the genetic similarity coefficient of the 64 samples was 0.431 8—0.988 6.A total of 64 samples were divided to six groups by UPGMA clustering method according to the similarity coefficient matrix when the genetic similarity coefficient was 0.715 5,which showed the abundant genetic diversity and relative gene stability of T.hemsleyanum germplasm resources.In addition,amplification figures by primers ISSR20,UBC857,and S17 were screened based on amplification result of 10 ISSR primers,and DNA fingerprinting was constructed,which can be used to identify 64 samples of T.hemsleyanum tested.Conclusion There are abundant genetic diversity and relative gene stability in T.hemsleyanum germplasm esources in China.ISSR analysis can reveal the genetic relationship among T.hemsleyanum germplasm resources in China,and provide certain reference for the evaluation,identification and new variety breeding of T.hemsleyanum germplasm resources in China.
Objective In this paper,the genetic diversity of 64 samples of Tetrastigma hemsleyanum germplasm resources in Chinese was analyzed.Methods ISSR-PCR was firstly used to amplify,and then POPGENE 32 software and NTSYS software was used to analyze the genetic diversity and phylogenetic relationship of 64 samples of T.hemsleyanum germplasm resources,and phylogenetic tree was constructed according to the UPGMA method.Results Ten primers with clear and reproducible bands were screened from 30 primers and used for genomic DNA amplification of 64 sample materials.A total of 83 polymorphic locis were amplified,whose polymorphic percentages were 71.43%—100% and average polymorphism percentage was 94.31%.The amplification polymorphic locis of primer S17 were the most(11) and the amplification polymorphic locis of primer P6 were the least(5),the average amplified polymorphic locis of 10 primers were 8.3.Genetic diversity analysis showed that the average number of alleles(Na) of 64 samples was 1.943 1,the average effective allele number(Ne) was 1.381 08,the average Nei's gene diversity index(H) was 0.242 98,and the average Shannon diversity index(I) was 0.385 83.The variation range of the genetic similarity coefficient of the 64 samples was 0.431 8—0.988 6.A total of 64 samples were divided to six groups by UPGMA clustering method according to the similarity coefficient matrix when the genetic similarity coefficient was 0.715 5,which showed the abundant genetic diversity and relative gene stability of T.hemsleyanum germplasm resources.In addition,amplification figures by primers ISSR20,UBC857,and S17 were screened based on amplification result of 10 ISSR primers,and DNA fingerprinting was constructed,which can be used to identify 64 samples of T.hemsleyanum tested.Conclusion There are abundant genetic diversity and relative gene stability in T.hemsleyanum germplasm esources in China.ISSR analysis can reveal the genetic relationship among T.hemsleyanum germplasm resources in China,and provide certain reference for the evaluation,identification and new variety breeding of T.hemsleyanum germplasm resources in China.
引文
[1]汪舍平,徐建春,张亮亮,等.不同栽培生境对三叶青生长及微量元素和总黄酮含量的影响[J].浙江农业科学,2017,58(12):2177-2178.
[2]张祺箐,冯正权.三叶青黄酮对荷Lewis肺癌小鼠脾脏单个核细胞PGE2、COX-2表达的影响[J].浙江中西医结合杂志,2017,27(10):842-845.
[3]陈红,廖淑彬,陈丹,等.闽产三叶青地上部分抗炎提取物的急性毒性研究[J].福建医科大学学报,2017,51(5):287-290.
[4]吴志庄,高贵宾,欧建德,等.生物炭肥对毛竹林下三叶青叶绿素含量、光合与荧光特性的影响[J].西北林学院学报,2017,32(5):59-63.
[5]周武,张善华.不同有机肥对药用植物三叶青化感物质的影响[J].丽水学院学报,2017,39(2):66-71.
[6]王静,郭万里,孔亚泽,等.珍稀药用植物三叶青的高效微繁体系研究[J].浙江理工大学学报:自然科学版,2016,35(4):636-642.
[7]钟良瑞,陈华,景晶,等.三叶青黄酮抑制肺癌A549细胞生长与蛋白酶体活性的关系研究[J].中国临床药理学与治疗学,2017,22(10):1123-1126.
[8]于虹敏,贺文达,刘巧,等.UFLC-DAD法同时测定三叶青中原花青素B1和儿茶素含量[J].辽宁中医药大学学报,2016,18(1):71-73.
[9]范世明,谢心月,曾繁天,等.三叶青叶化学成分鉴定及其总黄酮含量测定研究[J].药物分析杂志,2017,37(8):1481-1488.
[10]于虹敏,贺文达,刘巧,等.UFLC-DAD法同时测定三叶青中原花青素B1和儿茶素含量[J].辽宁中医药大学学报,2016,18(1):71-73.
[11]郝久程,贾鑫,穆晓红,等.大连地区岛屿与大陆玉竹种群遗传多样性的ISSR分析[J].植物研究,2017,37(5):709-714.
[12]朱波,华金渭,刘昆,等.珍稀药材三叶青种质资源遗传多样性的ISSR分析[J].江西农业大学学报,2015,37(5):914-919.
[13]Peng X,Ji Q Y,Fan S W,Zhang Y J,et al.Genetic diversity in populations of the endangered medicinal plant Tetrastigma hemsleyanum revealed by ISSR and SRAP markers:Implications for conservation[J].Gene Res Crop Evol,2015,62(7):1069-1078.
[14]马丽,侯乐峰,郝兆祥,等.82个石榴品种遗传多样性的ISSR分析[J].果树学报,2015,32(5):741-750.
[15]陈永久,王文,杨跃雄,等.冬虫夏草的随机扩增多态DNA及其遗传分化[J].遗传学报,1997,24(5):410-416.
[16]汪斌,祁伟,兰涛,等.应用ISSR分子标记绘制红麻种质资源DNA指纹图谱[J].作物学报,2011,37(6):1116-1123.
[17]李钰莹,董宽虎.山西白羊草种质资源遗传多样性的ISSR分析[J].草业学报,2014,23(1):217-222.
[18]蹇京蓉.中药三叶青资源状况及其对策研究[J].中国药物经济学,2012,7(2):228-230.
[19]李朝銮.中国植物志[M].北京:科学出版社,1998.
[20]钱丽华,戴丹丽,姜慧燕,等.濒危药用植物三叶青研究进展[J].浙江农业学报,2015,27(7):1301-1308.
[21]程林,胡轶娟,浦锦宝,等.三叶青的高效液相色谱指纹图谱研究[J].浙江中医杂志,2013,48(4):296-298.
[22]宋亚玲,向太和,武盼,等.三叶青扩展蛋白家族基因全长c DNA的克隆及其生物信息学和表达分析[J].中草药,2016,47(5):810-815.
[23]张煜炯,彭昕,吉庆勇,等.聚类分析和主成分分析法研究三叶青氯仿部位HPLC指纹图谱[J].中成药,2016,38(3):607-612.
[24]陈志丹,孙威江,项丽慧,等.应用ISSR指纹图谱鉴定铁观音茶树[J].分子植物育种,2015,13(12):2803-2810.
[25]张安世,韩臣鹏,齐秀娟,等.基于ISSR标记的猕猴桃品种遗传多样性分析及指纹图谱构建[J].植物资源与环境学报,2017,26(3):19-26.
[26]查美琴,赵玉玲,李疆,等.新疆枸杞种质资源遗传多样性分析及DNA指纹图谱构建[J].植物遗传资源学报,2016,17(6):1065-1071.
[2]张祺箐,冯正权.三叶青黄酮对荷Lewis肺癌小鼠脾脏单个核细胞PGE2、COX-2表达的影响[J].浙江中西医结合杂志,2017,27(10):842-845.
[3]陈红,廖淑彬,陈丹,等.闽产三叶青地上部分抗炎提取物的急性毒性研究[J].福建医科大学学报,2017,51(5):287-290.
[4]吴志庄,高贵宾,欧建德,等.生物炭肥对毛竹林下三叶青叶绿素含量、光合与荧光特性的影响[J].西北林学院学报,2017,32(5):59-63.
[5]周武,张善华.不同有机肥对药用植物三叶青化感物质的影响[J].丽水学院学报,2017,39(2):66-71.
[6]王静,郭万里,孔亚泽,等.珍稀药用植物三叶青的高效微繁体系研究[J].浙江理工大学学报:自然科学版,2016,35(4):636-642.
[7]钟良瑞,陈华,景晶,等.三叶青黄酮抑制肺癌A549细胞生长与蛋白酶体活性的关系研究[J].中国临床药理学与治疗学,2017,22(10):1123-1126.
[8]于虹敏,贺文达,刘巧,等.UFLC-DAD法同时测定三叶青中原花青素B1和儿茶素含量[J].辽宁中医药大学学报,2016,18(1):71-73.
[9]范世明,谢心月,曾繁天,等.三叶青叶化学成分鉴定及其总黄酮含量测定研究[J].药物分析杂志,2017,37(8):1481-1488.
[10]于虹敏,贺文达,刘巧,等.UFLC-DAD法同时测定三叶青中原花青素B1和儿茶素含量[J].辽宁中医药大学学报,2016,18(1):71-73.
[11]郝久程,贾鑫,穆晓红,等.大连地区岛屿与大陆玉竹种群遗传多样性的ISSR分析[J].植物研究,2017,37(5):709-714.
[12]朱波,华金渭,刘昆,等.珍稀药材三叶青种质资源遗传多样性的ISSR分析[J].江西农业大学学报,2015,37(5):914-919.
[13]Peng X,Ji Q Y,Fan S W,Zhang Y J,et al.Genetic diversity in populations of the endangered medicinal plant Tetrastigma hemsleyanum revealed by ISSR and SRAP markers:Implications for conservation[J].Gene Res Crop Evol,2015,62(7):1069-1078.
[14]马丽,侯乐峰,郝兆祥,等.82个石榴品种遗传多样性的ISSR分析[J].果树学报,2015,32(5):741-750.
[15]陈永久,王文,杨跃雄,等.冬虫夏草的随机扩增多态DNA及其遗传分化[J].遗传学报,1997,24(5):410-416.
[16]汪斌,祁伟,兰涛,等.应用ISSR分子标记绘制红麻种质资源DNA指纹图谱[J].作物学报,2011,37(6):1116-1123.
[17]李钰莹,董宽虎.山西白羊草种质资源遗传多样性的ISSR分析[J].草业学报,2014,23(1):217-222.
[18]蹇京蓉.中药三叶青资源状况及其对策研究[J].中国药物经济学,2012,7(2):228-230.
[19]李朝銮.中国植物志[M].北京:科学出版社,1998.
[20]钱丽华,戴丹丽,姜慧燕,等.濒危药用植物三叶青研究进展[J].浙江农业学报,2015,27(7):1301-1308.
[21]程林,胡轶娟,浦锦宝,等.三叶青的高效液相色谱指纹图谱研究[J].浙江中医杂志,2013,48(4):296-298.
[22]宋亚玲,向太和,武盼,等.三叶青扩展蛋白家族基因全长c DNA的克隆及其生物信息学和表达分析[J].中草药,2016,47(5):810-815.
[23]张煜炯,彭昕,吉庆勇,等.聚类分析和主成分分析法研究三叶青氯仿部位HPLC指纹图谱[J].中成药,2016,38(3):607-612.
[24]陈志丹,孙威江,项丽慧,等.应用ISSR指纹图谱鉴定铁观音茶树[J].分子植物育种,2015,13(12):2803-2810.
[25]张安世,韩臣鹏,齐秀娟,等.基于ISSR标记的猕猴桃品种遗传多样性分析及指纹图谱构建[J].植物资源与环境学报,2017,26(3):19-26.
[26]查美琴,赵玉玲,李疆,等.新疆枸杞种质资源遗传多样性分析及DNA指纹图谱构建[J].植物遗传资源学报,2016,17(6):1065-1071.