基于RNA-Seq SSR标记的沙棘种质遗传多样性和群体结构分析
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摘要
沙棘是中国重要的生态经济植物,遗传多样性分析对于其种质资源的评价与保存十分重要。现以80个沙棘品种/品系为试材,利用17对多态性的RNA-Seq SSR引物,研究其遗传多样性和群体遗传结构。17对SSR引物共扩增出50条谱带,其中多态性谱带41条,每个位点条带数在2～5之间;多样性指数(PIC)介于0.150 7～0.588 6,平均0.271 0。利用PowerMarker 3.25和UPGMA方法进行聚类分析。结果显示,供试材料可分为两大类群,蒙古沙棘(ssp. mongolica)和杂交品种/品系聚为一个类群,中国沙棘(ssp. sinensis)构成另外一个类群。群体遗传结构分析中所有种质也被分成两个类群,杂交品种/品系介于中国沙棘和蒙古沙棘之间,与中国沙棘聚为一个类群。这些结果表明,沙棘的遗传多样性与它们的来源和遗传背景相符,可为沙棘种质资源保存和分子标记辅助育种提供理论依据。
Sea buckt horn is an ecologically and economically valuable species in China. The genetic diversity analysis of sea buckthorn is very important for the evaluation and conservation of its germplasm resources. In this study, 80 sea buckthorn cultivars/lines were used as materials and 17 pairs of polymorphic RNA-Seq SSR primers were used to analyze their genetic diversity and structure. Among 50 alleles generated by the 17 SSR loci, 41 were polymorphic alleles, and the number of bands ranged from 2 to 5 per locus. The value of polymorphism information content(PIC) varied from 0.150 7 to 0.588 6, with an average of 0.271 0. Cluster analysis based on unweighted pair group method with arithmetic average(UPGMA) using PowerMarker 3.25 divided all the genotypes into two main groups, with all of the ssp. mongolica cultivars and hybrids in one group and ssp. sinensis cultivars in the other group. 80 cultivars/lines were also divided into two populations by the population genetic structure analysis.Hybrids were between ssp. mongolica and ssp. sinensis cultivars and close to the latter. These results indicated the genetic diversity of sea buckthorn samples was in good agreement with their source and genetic background, which could provide theoretical reference for the conservation and molecular marker assisted breeding of sea buckthorn germplasm.
Sea buckt horn is an ecologically and economically valuable species in China. The genetic diversity analysis of sea buckthorn is very important for the evaluation and conservation of its germplasm resources. In this study, 80 sea buckthorn cultivars/lines were used as materials and 17 pairs of polymorphic RNA-Seq SSR primers were used to analyze their genetic diversity and structure. Among 50 alleles generated by the 17 SSR loci, 41 were polymorphic alleles, and the number of bands ranged from 2 to 5 per locus. The value of polymorphism information content(PIC) varied from 0.150 7 to 0.588 6, with an average of 0.271 0. Cluster analysis based on unweighted pair group method with arithmetic average(UPGMA) using PowerMarker 3.25 divided all the genotypes into two main groups, with all of the ssp. mongolica cultivars and hybrids in one group and ssp. sinensis cultivars in the other group. 80 cultivars/lines were also divided into two populations by the population genetic structure analysis.Hybrids were between ssp. mongolica and ssp. sinensis cultivars and close to the latter. These results indicated the genetic diversity of sea buckthorn samples was in good agreement with their source and genetic background, which could provide theoretical reference for the conservation and molecular marker assisted breeding of sea buckthorn germplasm.
引文
Bal L.M.,Me d a V.,N ai k N.S.,and Satya S.,2011,Sea buckthorn:A potential source of valuable nutrients for nutraceuticals and cosmoceuticals,Food Res.Intl.,44(7):1718-1727
Bartish G.I.,Jeppsson N.,Bartish I.V.,and Nybom H.,2000,Assessment of genetic diversity using RAPD analysis in a germplasm collection of sea buckthorn,Agr.Food Sci.Finland,9(9):279-288
Bartish I.V.,Jeppsson N.,Swenson U.,and Nybom H.,2002,Phylogeny of Hippophae(Elaeagnaceae)inferred from parsimony analysis of chloroplast DNA and morphology,Syst.Bot.,27(1):41-54
Chen C.,Xu X.M.,Chen Y.,Yu M.Y.,Wen F.Y.,and Zhang H.,2013,Identification,quantification and antioxidant activity of acylated flavonol glycosides from sea buckthorn(Hippophae rhamnoides ssp.sinensis),Food Chem.,141(3):1573-1579
Chen X.L.,Ma R.J.,Sun K.,and Lian Y.S.,2003,Germplasm resource and habitat types of seabuckthorn in China,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),23(3):451-455(陈学林,马瑞君,孙坤,廉永善,2003,中国沙棘属种质资源及其生境类型的研究,西北植物学报,23(3):451-455)
Earl D.A.,and Vonholdt B.M.,2012,STRUCTURE HARVEST-ER:a website and program for visualizing STRUCTURE output and implementing the Evanno method,Cons.Genet.Res.,4(2):359-361
Evanno G.,Regnaut S.,and Goudet J.,2005,Detecting the number of clusters of individuals using the software STRUC-TURE:a simulation study,Mol.Ecol.,14(8):2611-2620
Huang Q.,2006,The research progress on seabuckthorn breeding in China,Guoji Shaji Yanjiu Yu Kaifa(Global Seabuckthorn Research and Development),4(4):25-29(黄铨,2006,中国沙棘育种研究进展,国际沙棘研究与开发,4(4):25-29)
Jain A.,Chaudhary S.,and Sharma P.C.,2014,Mining of microsatellites using next generation sequencing of seabuckthorn(Hippophae rhamnoides L.)transcriptome,Physiol.Mol.Biol.Plants,20(1):115-123
Jain A.,Ghangal R.,Grover A.,Raghuvanshi S.,and Sharma P.C.,2010,Development of EST-based new SSR markers in seabuckthorn,Physiol.Mol.Biol.Plants,16(4):375-378
Jakobsson M.,and Rosenberg N.A.,2007,CLUMPP:a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure,Bioinformatics,23(14):1801-1806
Kallio H.,Lassila M.,J覿rvenp覿覿E.,Haraldsson G.G.,Jonsdottir S.,and Yang B.R.,2009,Inositols and methylinositol in sea buckthorn(Hippoph覿e rhamnoides)berries,J.Chromatogr.BAnalyt.Technol.,Biomed.Life Sci.,877(14-15):1426-1432
Li H.,Ruan C.J.,and Teixeira da Silva J.A.,2009,Identification and genetic relationship based on ISSR analysis in a germplasm collection of sea buckthorn(Hippophae L.)from China and other countries,Sci.Hortic.,123:263-271
Li H.,Ruan C.J.,Teixeira da Silva J.A.,and Liu B.Q.,2010,Associations of SRAP markers with dried-shrink disease resistance in a germplasm collection of sea buckthorn(Hippophae L.),Genome,53(6):447-457
Li H.,Ruan C.J.,Wang L.,Ding J.,and Tian X.J.,2017,Development of RNA-Seq SSR markers and application to genetic relationship analysis among sea buckthorn germplasm,J.Am.Soc.Hortic.Sci.,142(3):200-208
Li S.S.,Zeng Y.F.,He C.Y.,and Zhang J.G.,2017,Development of EST-SSR markers based on seabuckthorn transcriptomic sequences,Linye Kexue Yanjiu(Forest Research),30(1):69-74(李珊珊,曾艳飞,何彩云,张建国,2017,基于沙棘转录组序列开发EST-SSR分子标记,林业科学研究,30(1):69-74)
Liu K.,and Muse S.V.,2005,PowerMarker:an integrated analysis environment for genetic marker analysis,Bioinformatics,21(9):2128-2129
Liu Y.L.,Zhang P.F.,Song M.L.,Hou J.L.,Qing M.,Wang W.Q.,and Liu C.S.,2015,Transcriptome analysis and development of SSR molecular markers in Glycyrrhiza uralensis fisch,PLoS One,10(11):e0143017
Peakall R.,and Smouse P.E.,2012,GenAlEx 6.5:genetic analysis in Excel.population genetic software for teaching and research-an update,Bioinformatics,28(19):2537-2539
Pritchard J.K.,Stephens M.,and Donnelly P.,2000,Inference of population structure using multilocus genotype data,Genetics,155(2):945-959
Raffo A.,Paoletti F.,and Antonelli M.,2004,Changes in sugar,organic acid,flavonol and carotenoid composition during ripening of berries of three seabuckthorn(Hippophae rhamnoides L.)cultivars,Eur.Food Res.Technol.,219(4):360-368
Ramchiary N.,Nguyen V.D.,Li X.,Hong C.P.,Dhandapani V.,Choi S.R.,Yu G.,Piao Z.Y.,and Lim Z.P.,2011,Genic microsatellite markers in Brassica rapa:development,characterization,mapping,and their utility in other cultivated and wild Brassica relatives,DNA Res.,18(5):305-320
Ruan C.J.,2006,Genetic relationships among some sea buckthorn cultivars from China,Russia and Mongolia using AFLP markers,J.Hortic.Sci.Biotechnol.,81(3):409-414
Ruan C.J.,Qin P.,Zheng J.W.,and He Z.X.,2004,Genetic relationships among some cultivars of sea buckthorn from China,Russia and Mongolia based on RAPD analysis,Sci.Hortic.,101(4):417-426
Ruan C.J.,Teixeira da Silva J.A.,Jin H.,Li H.,and Li D.Q.,2007,Research and biotechnology in sea buckthorn(Hippophae spp.),Med.Aromat.Plant Sci.Biotechnol.,1(1):47-60
Swenson U.,and Bartish I.V.,2003,Taxonomic synopsis of Hippophae(Elaeagnaceae),Nord.J.Bot.,22:369-374
Tamura K.,Stecher G.,Peterson D.,Filipski A.,and Kumar S.,2013,MEGA6:molecular evolutionary genetics analysis version 6.0,Mol.Biol.Evol.,30(12):2725-2729
Wang A.,Zhang Q.,Wan D.S.,Yang Y.Z.,and Liu J.Q.,2008,Nine microsatellite DNA primers for Hippophae rhamnoides ssp.sinensis(Elaeagnaceae),Conserv.Genet.,9:969-971
Wang Y.H.,Jing H.,Peng S.M.,and Korpelainen H.,2011,Genetic structure in fragmented populations of Hippophae rhamnoides ssp.sinensis in China investigated by ISSR and cpSSRmarkers,Plant System.Evol.,295(1-4):97-107
Wang Z.F.,Wang F.,Hao D.H.,Duan X.Y.,Su Y.,Feng Z.W.,and Li C.H.,2010,Simple preparation of potato SSR markers degeneration polyacrylamide gel silver stain dry PAG,Zhongzi(Seed),29(6):60-63(王兆富,王芳,郝大海,段晓艳,苏跃,冯泽蔚,李灿辉,2010,马铃薯SSR标记变性聚丙烯酰胺凝胶干胶的简易制备,种子,29(6):60-63)
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Zhang L.W.,Li Y.R.,Tao A.F.,Fang P.P.,and Qi J.M.,2015,Development and characterization of 1,906 EST-SSR markers from unigenes in jute(Corchorus spp.),PLoS One,10(10):e0140861
Bartish G.I.,Jeppsson N.,Bartish I.V.,and Nybom H.,2000,Assessment of genetic diversity using RAPD analysis in a germplasm collection of sea buckthorn,Agr.Food Sci.Finland,9(9):279-288
Bartish I.V.,Jeppsson N.,Swenson U.,and Nybom H.,2002,Phylogeny of Hippophae(Elaeagnaceae)inferred from parsimony analysis of chloroplast DNA and morphology,Syst.Bot.,27(1):41-54
Chen C.,Xu X.M.,Chen Y.,Yu M.Y.,Wen F.Y.,and Zhang H.,2013,Identification,quantification and antioxidant activity of acylated flavonol glycosides from sea buckthorn(Hippophae rhamnoides ssp.sinensis),Food Chem.,141(3):1573-1579
Chen X.L.,Ma R.J.,Sun K.,and Lian Y.S.,2003,Germplasm resource and habitat types of seabuckthorn in China,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),23(3):451-455(陈学林,马瑞君,孙坤,廉永善,2003,中国沙棘属种质资源及其生境类型的研究,西北植物学报,23(3):451-455)
Earl D.A.,and Vonholdt B.M.,2012,STRUCTURE HARVEST-ER:a website and program for visualizing STRUCTURE output and implementing the Evanno method,Cons.Genet.Res.,4(2):359-361
Evanno G.,Regnaut S.,and Goudet J.,2005,Detecting the number of clusters of individuals using the software STRUC-TURE:a simulation study,Mol.Ecol.,14(8):2611-2620
Huang Q.,2006,The research progress on seabuckthorn breeding in China,Guoji Shaji Yanjiu Yu Kaifa(Global Seabuckthorn Research and Development),4(4):25-29(黄铨,2006,中国沙棘育种研究进展,国际沙棘研究与开发,4(4):25-29)
Jain A.,Chaudhary S.,and Sharma P.C.,2014,Mining of microsatellites using next generation sequencing of seabuckthorn(Hippophae rhamnoides L.)transcriptome,Physiol.Mol.Biol.Plants,20(1):115-123
Jain A.,Ghangal R.,Grover A.,Raghuvanshi S.,and Sharma P.C.,2010,Development of EST-based new SSR markers in seabuckthorn,Physiol.Mol.Biol.Plants,16(4):375-378
Jakobsson M.,and Rosenberg N.A.,2007,CLUMPP:a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure,Bioinformatics,23(14):1801-1806
Kallio H.,Lassila M.,J覿rvenp覿覿E.,Haraldsson G.G.,Jonsdottir S.,and Yang B.R.,2009,Inositols and methylinositol in sea buckthorn(Hippoph覿e rhamnoides)berries,J.Chromatogr.BAnalyt.Technol.,Biomed.Life Sci.,877(14-15):1426-1432
Li H.,Ruan C.J.,and Teixeira da Silva J.A.,2009,Identification and genetic relationship based on ISSR analysis in a germplasm collection of sea buckthorn(Hippophae L.)from China and other countries,Sci.Hortic.,123:263-271
Li H.,Ruan C.J.,Teixeira da Silva J.A.,and Liu B.Q.,2010,Associations of SRAP markers with dried-shrink disease resistance in a germplasm collection of sea buckthorn(Hippophae L.),Genome,53(6):447-457
Li H.,Ruan C.J.,Wang L.,Ding J.,and Tian X.J.,2017,Development of RNA-Seq SSR markers and application to genetic relationship analysis among sea buckthorn germplasm,J.Am.Soc.Hortic.Sci.,142(3):200-208
Li S.S.,Zeng Y.F.,He C.Y.,and Zhang J.G.,2017,Development of EST-SSR markers based on seabuckthorn transcriptomic sequences,Linye Kexue Yanjiu(Forest Research),30(1):69-74(李珊珊,曾艳飞,何彩云,张建国,2017,基于沙棘转录组序列开发EST-SSR分子标记,林业科学研究,30(1):69-74)
Liu K.,and Muse S.V.,2005,PowerMarker:an integrated analysis environment for genetic marker analysis,Bioinformatics,21(9):2128-2129
Liu Y.L.,Zhang P.F.,Song M.L.,Hou J.L.,Qing M.,Wang W.Q.,and Liu C.S.,2015,Transcriptome analysis and development of SSR molecular markers in Glycyrrhiza uralensis fisch,PLoS One,10(11):e0143017
Peakall R.,and Smouse P.E.,2012,GenAlEx 6.5:genetic analysis in Excel.population genetic software for teaching and research-an update,Bioinformatics,28(19):2537-2539
Pritchard J.K.,Stephens M.,and Donnelly P.,2000,Inference of population structure using multilocus genotype data,Genetics,155(2):945-959
Raffo A.,Paoletti F.,and Antonelli M.,2004,Changes in sugar,organic acid,flavonol and carotenoid composition during ripening of berries of three seabuckthorn(Hippophae rhamnoides L.)cultivars,Eur.Food Res.Technol.,219(4):360-368
Ramchiary N.,Nguyen V.D.,Li X.,Hong C.P.,Dhandapani V.,Choi S.R.,Yu G.,Piao Z.Y.,and Lim Z.P.,2011,Genic microsatellite markers in Brassica rapa:development,characterization,mapping,and their utility in other cultivated and wild Brassica relatives,DNA Res.,18(5):305-320
Ruan C.J.,2006,Genetic relationships among some sea buckthorn cultivars from China,Russia and Mongolia using AFLP markers,J.Hortic.Sci.Biotechnol.,81(3):409-414
Ruan C.J.,Qin P.,Zheng J.W.,and He Z.X.,2004,Genetic relationships among some cultivars of sea buckthorn from China,Russia and Mongolia based on RAPD analysis,Sci.Hortic.,101(4):417-426
Ruan C.J.,Teixeira da Silva J.A.,Jin H.,Li H.,and Li D.Q.,2007,Research and biotechnology in sea buckthorn(Hippophae spp.),Med.Aromat.Plant Sci.Biotechnol.,1(1):47-60
Swenson U.,and Bartish I.V.,2003,Taxonomic synopsis of Hippophae(Elaeagnaceae),Nord.J.Bot.,22:369-374
Tamura K.,Stecher G.,Peterson D.,Filipski A.,and Kumar S.,2013,MEGA6:molecular evolutionary genetics analysis version 6.0,Mol.Biol.Evol.,30(12):2725-2729
Wang A.,Zhang Q.,Wan D.S.,Yang Y.Z.,and Liu J.Q.,2008,Nine microsatellite DNA primers for Hippophae rhamnoides ssp.sinensis(Elaeagnaceae),Conserv.Genet.,9:969-971
Wang Y.H.,Jing H.,Peng S.M.,and Korpelainen H.,2011,Genetic structure in fragmented populations of Hippophae rhamnoides ssp.sinensis in China investigated by ISSR and cpSSRmarkers,Plant System.Evol.,295(1-4):97-107
Wang Z.F.,Wang F.,Hao D.H.,Duan X.Y.,Su Y.,Feng Z.W.,and Li C.H.,2010,Simple preparation of potato SSR markers degeneration polyacrylamide gel silver stain dry PAG,Zhongzi(Seed),29(6):60-63(王兆富,王芳,郝大海,段晓艳,苏跃,冯泽蔚,李灿辉,2010,马铃薯SSR标记变性聚丙烯酰胺凝胶干胶的简易制备,种子,29(6):60-63)
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Zhang L.W.,Li Y.R.,Tao A.F.,Fang P.P.,and Qi J.M.,2015,Development and characterization of 1,906 EST-SSR markers from unigenes in jute(Corchorus spp.),PLoS One,10(10):e0140861
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