三叶青种质资源遗传多样性的SSR荧光标记分析
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摘要
目的研究三叶青64个种质的遗传多样性和亲缘关系。方法利用SSR荧光标记对其进行PCR扩增,利用POPGENE32软件分析三叶青64个种质的遗传多样性和亲缘关系,利用UPGMA法构建其亲缘关系树状图,利用NTSYS软件构建其主成分分析二维图和三维散点图。结果从14对引物中筛选出8对条带清晰、重复性好的引物,对64份供试材料的基因组DNA进行扩增。8个SSR标记的观察等位基因数(Na)变化范围为3～13,均值为7.875 0;有效等位基因数(Ne)变化范围为1.424 9～6.087 4,均值3.605 2;Shannon信息指数(I)变化范围为0.689 5～2.082 4,均值1.424 0;观测杂合度(Ho)变化范围为0.206 3～0.734 4,均值0.524 7;期望杂合度(He)变化范围为0.300 6～0.842 4,均值0.658 4;Nei’s基因多样性(H)0.298 2～0.835 7,均值0.653 2;多态信息含量(PIC)变化范围为0.288 0～0.817 5,均值0.614 5,遗传相似系数变化范围为0.115 4～0.954 5,遗传距离变化范围为0.000 0～3.218 1,说明64份三叶青种质亲缘关系较远,遗传分化程度较大。在遗传距离1.018 9处,64份三叶青种质可分为5大组。结论地理差异与种质遗传差异无必然联系。三叶青种质资源具有丰富的遗传多样性,SSR荧光标记分析结果可为三叶青种质资源的利用和品种选育提供一定的参考。
Objective To understand the genetic diversity and genetic relationship of 64 samples of Tetrastigma hemsleyanum germplasm resources. Methods Fluorescently labeled SSR were used for PCR amplification, POPGENE32 software was used to analyze genetic diversity and genetic relationship of 64 samples of T. hemsleyanum germplasm resources, UPGMA method was used to construct their genetic tree map, NTSYS software was used to construct their two-dimensional principal component analysis map and three-dimensional scatter map. Result Eight pairs of primers with clear and reproducible bands were screened from 14 pairs of primers and used for genomic DNA amplification of 64 sample materials. The observed number of alleles(Na) ranged from 3 to 13, and the mean value was 7.875 0; The effective number of alleles(Ne) ranged from 1.424 9 to 6.087 4, and the mean value was 3.605 2; The Shannon information index(I) ranged from 0.689 5 to 2.082 4, and the mean value was 1.424 0; The observed heterozyghosity(Ho) ranged from 0.206 3 to 0.734 4, and the mean value was 0.524 7; The expected heterozygosity(He) ranged from 0.300 6 to 0.842 4, and the mean value was 0.658 4; The Nei's gene diversity(H) ranged from 0.298 2 to 0.835 7, and the mean value was 0.653 2; The polymorphism information content(PIC) ranged from 0.288 0 to 0.817 5, and the mean value was 0.614 5; The genetic similarity coefficient ranged from 0.115 4 to 0.954 5; The genetic distance ranged from 0.000 0 to 3.218 1. It was indicated that the genetic relationship of 64 T. hemsleyanum germplasm resources was far, and the degree of genetic differentiation was high. At the genetic distance of 1.018 9, 64 T. hemsleyanum germplasm resources could be divided into five groups. Conclusion There was no necessary connection between geographical difference and genetic difference of germplasm. T. hemsleyanum germplasm resources are rich in genetic diversity. The results of fluorescently labeled SSR analysis can provide some references for the utilization and variety breeding of T. hemsleyanum germplasm resources.
Objective To understand the genetic diversity and genetic relationship of 64 samples of Tetrastigma hemsleyanum germplasm resources. Methods Fluorescently labeled SSR were used for PCR amplification, POPGENE32 software was used to analyze genetic diversity and genetic relationship of 64 samples of T. hemsleyanum germplasm resources, UPGMA method was used to construct their genetic tree map, NTSYS software was used to construct their two-dimensional principal component analysis map and three-dimensional scatter map. Result Eight pairs of primers with clear and reproducible bands were screened from 14 pairs of primers and used for genomic DNA amplification of 64 sample materials. The observed number of alleles(Na) ranged from 3 to 13, and the mean value was 7.875 0; The effective number of alleles(Ne) ranged from 1.424 9 to 6.087 4, and the mean value was 3.605 2; The Shannon information index(I) ranged from 0.689 5 to 2.082 4, and the mean value was 1.424 0; The observed heterozyghosity(Ho) ranged from 0.206 3 to 0.734 4, and the mean value was 0.524 7; The expected heterozygosity(He) ranged from 0.300 6 to 0.842 4, and the mean value was 0.658 4; The Nei's gene diversity(H) ranged from 0.298 2 to 0.835 7, and the mean value was 0.653 2; The polymorphism information content(PIC) ranged from 0.288 0 to 0.817 5, and the mean value was 0.614 5; The genetic similarity coefficient ranged from 0.115 4 to 0.954 5; The genetic distance ranged from 0.000 0 to 3.218 1. It was indicated that the genetic relationship of 64 T. hemsleyanum germplasm resources was far, and the degree of genetic differentiation was high. At the genetic distance of 1.018 9, 64 T. hemsleyanum germplasm resources could be divided into five groups. Conclusion There was no necessary connection between geographical difference and genetic difference of germplasm. T. hemsleyanum germplasm resources are rich in genetic diversity. The results of fluorescently labeled SSR analysis can provide some references for the utilization and variety breeding of T. hemsleyanum germplasm resources.
引文
[1]刘崟艳,周以飞,李清,等.三叶青的蒸腾作用与气孔结构研究[J].中草药,2015,46(17):2610-2617.
[2]李朝銮.中国植物志[M].北京:科学出版社,1998.
[3]许文,傅志勤,林婧,等.UPLC-MS/MS法同时测定三叶青中10种黄酮类成分[J].药学学报,2014,49(12):1711-1717.
[4]许文,傅志勤,林婧,等.HPLC-Q-TOF-MS和UPLC-QqQ-MS的三叶青主要成分定性与定量研究[J].中国中药杂志,2014,39(22):4365-4372.
[5]朱波,华金渭,程文亮,等.不同种源三叶青农艺性状比较[J].浙江农业学报,2015,27(10):1752-1756.
[6]袁带秀,吕江明.不同种源地三叶青的POD同工酶比较[J].贵州农业科学,2010,38(7):50-51.
[7]袁带秀,侯娟.3个不同种源地三叶青酯酶同工酶比较研究[J].湖南农业科学,2010(21):34-36.
[8]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.
[9]Peng X,Ji Q Y,Fan S W,et al.Genetic diversity in populations of the endangered medicinal plant Tetrastigma hemsleyanum revealed by ISSR and SRAPmarkers:implications for conservation[J].Genet Resources Crop Evol,2015,62(7):1069-1078.
[10]左力辉,韩志校,梁海永,等.不同产地中国李资源遗传多样性SSR分析[J].园艺学报,2015,42(1):111-118.
[11]宪立杰,刘兴菊,李雪雁,等.葡萄种质遗传多样性的SSR分析及指纹库构建[J].北方园艺,2013(1):87-90.
[12]刘晓鑫,谢传晓,赵琦,等.基于SSR荧光标记技术的玉米群体混合样本基因频率分析方法[J].中国农业科学,2008,41(12):3991-3998.
[13]王瑞云,季煦,陆平,等.利用荧光SSR分析中国糜子遗传多样性[J].作物学报,2017,43(4):530-548.
[14]陈斐,魏臻武,李伟民,等.基于SSR标记的苜蓿种质资源遗传多样性与群体结构分析[J].草地学报,2013,21(4):759-768.
[15]Liesebach H,Schneck V,Ewald E.Clonal fingerprinting in the genus Populus L.by nuclear microsatellite loci regarding differences between sections,species and hybrids[J].Tree Genet Genom,2010,6(2):259-269.
[16]Faria D A,Mamani E M C,Jr G J P,et al.Genotyping systems for Eucalyptus based on tetra-,penta-,and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests[J].Tree Genet Genom,2011,7(1):63-77.
[17]Dra?narováA,Krak K,Vít P,et al.Cross-amplification and multiplexing of SSR markers for Alnus glutinosa and A.incana[J].Tree Genet Genom,2014,10(4):865-873.
[18]郝晨阳,王兰芬,贾继增,等.SSR荧光标记和银染技术的比较分析[J].作物学报,2005,31(2):144-149.
[19]Kong Q S,Zhang,G P,Chen W C,et al.Identification and development of polymorphic EST-SSR markers by sequence alignment in pepper,Capsicum annuum(Solanaceae)[J].Am J Bot,2012,99(2):59-61.
[20]Wayne P,Gordon C M,Jim P.Polymorphism revealed by simple sequence repeats[J].Trends Plant Sci,1996,1(7):215-222.
[21]王瑞,张福耀,程庆军,等.利用SSR荧光标记构建20个高粱品种指纹图谱[J].作物学报,2015,41(4):658-665.
[22]郑永胜,张晗,王东建,等.基于荧光检测技术的小麦品种SSR鉴定体系的建立[J].中国农业科学,2014,47(19):3725-3735.
[23]张明泽,姚玉仙,陈世军.黔南60份茶树种质资源遗传多样性的SSR分析[J].西北植物学报,2016,36(6):1117-1124.
[24]Hamrick J L,Godt M J W.Plant Population Genetics,Breeding and Genetic Resources[M].Sunderland,Massachusetts:Sinauer,1990.
[25]Nybom H.Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants[J].Mol Ecol,2004,13(5):1143-1155.
[26]Nybom H,Bartish I V.Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants[J].Persp Plant Ecol Evol Syst,2000,3(2):93-114.
[27]庞广昌,姜冬梅.群体遗传多样性和数据分析[J].林业科学,1995,31(11):543-549.
[28]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.
[29]岳文娣,魏利斌,张体德,等.芝麻种质资源SSR标记遗传多样性与群体结构分析[J].作物学报,2012,38(12):2286-2296.
[2]李朝銮.中国植物志[M].北京:科学出版社,1998.
[3]许文,傅志勤,林婧,等.UPLC-MS/MS法同时测定三叶青中10种黄酮类成分[J].药学学报,2014,49(12):1711-1717.
[4]许文,傅志勤,林婧,等.HPLC-Q-TOF-MS和UPLC-QqQ-MS的三叶青主要成分定性与定量研究[J].中国中药杂志,2014,39(22):4365-4372.
[5]朱波,华金渭,程文亮,等.不同种源三叶青农艺性状比较[J].浙江农业学报,2015,27(10):1752-1756.
[6]袁带秀,吕江明.不同种源地三叶青的POD同工酶比较[J].贵州农业科学,2010,38(7):50-51.
[7]袁带秀,侯娟.3个不同种源地三叶青酯酶同工酶比较研究[J].湖南农业科学,2010(21):34-36.
[8]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.
[9]Peng X,Ji Q Y,Fan S W,et al.Genetic diversity in populations of the endangered medicinal plant Tetrastigma hemsleyanum revealed by ISSR and SRAPmarkers:implications for conservation[J].Genet Resources Crop Evol,2015,62(7):1069-1078.
[10]左力辉,韩志校,梁海永,等.不同产地中国李资源遗传多样性SSR分析[J].园艺学报,2015,42(1):111-118.
[11]宪立杰,刘兴菊,李雪雁,等.葡萄种质遗传多样性的SSR分析及指纹库构建[J].北方园艺,2013(1):87-90.
[12]刘晓鑫,谢传晓,赵琦,等.基于SSR荧光标记技术的玉米群体混合样本基因频率分析方法[J].中国农业科学,2008,41(12):3991-3998.
[13]王瑞云,季煦,陆平,等.利用荧光SSR分析中国糜子遗传多样性[J].作物学报,2017,43(4):530-548.
[14]陈斐,魏臻武,李伟民,等.基于SSR标记的苜蓿种质资源遗传多样性与群体结构分析[J].草地学报,2013,21(4):759-768.
[15]Liesebach H,Schneck V,Ewald E.Clonal fingerprinting in the genus Populus L.by nuclear microsatellite loci regarding differences between sections,species and hybrids[J].Tree Genet Genom,2010,6(2):259-269.
[16]Faria D A,Mamani E M C,Jr G J P,et al.Genotyping systems for Eucalyptus based on tetra-,penta-,and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests[J].Tree Genet Genom,2011,7(1):63-77.
[17]Dra?narováA,Krak K,Vít P,et al.Cross-amplification and multiplexing of SSR markers for Alnus glutinosa and A.incana[J].Tree Genet Genom,2014,10(4):865-873.
[18]郝晨阳,王兰芬,贾继增,等.SSR荧光标记和银染技术的比较分析[J].作物学报,2005,31(2):144-149.
[19]Kong Q S,Zhang,G P,Chen W C,et al.Identification and development of polymorphic EST-SSR markers by sequence alignment in pepper,Capsicum annuum(Solanaceae)[J].Am J Bot,2012,99(2):59-61.
[20]Wayne P,Gordon C M,Jim P.Polymorphism revealed by simple sequence repeats[J].Trends Plant Sci,1996,1(7):215-222.
[21]王瑞,张福耀,程庆军,等.利用SSR荧光标记构建20个高粱品种指纹图谱[J].作物学报,2015,41(4):658-665.
[22]郑永胜,张晗,王东建,等.基于荧光检测技术的小麦品种SSR鉴定体系的建立[J].中国农业科学,2014,47(19):3725-3735.
[23]张明泽,姚玉仙,陈世军.黔南60份茶树种质资源遗传多样性的SSR分析[J].西北植物学报,2016,36(6):1117-1124.
[24]Hamrick J L,Godt M J W.Plant Population Genetics,Breeding and Genetic Resources[M].Sunderland,Massachusetts:Sinauer,1990.
[25]Nybom H.Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants[J].Mol Ecol,2004,13(5):1143-1155.
[26]Nybom H,Bartish I V.Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants[J].Persp Plant Ecol Evol Syst,2000,3(2):93-114.
[27]庞广昌,姜冬梅.群体遗传多样性和数据分析[J].林业科学,1995,31(11):543-549.
[28]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.
[29]岳文娣,魏利斌,张体德,等.芝麻种质资源SSR标记遗传多样性与群体结构分析[J].作物学报,2012,38(12):2286-2296.
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