三色堇转录组SSR分析及分子标记开发
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摘要
为解决三色堇(Viola×wittrockiana)缺乏共显性DNA标记的问题,利用MISA软件对其转录组测序获得的167 576条unigene进行筛选,共检测出23 791个SSR位点,出现频率为14.20%,平均分布距离为6.75 kb。SSR位点中主导类型为三核苷酸重复,占总SSR的28.31%;其次是二核苷酸重复,占总SSR的12.86%。AG/CT与GAA/TTC分别是二核苷酸与三核苷酸的优势重复基元,分别占总SSR重复类型的4.01%和1.85%。利用Primer 3共设计出6 863对SSR引物。随机选择30对引物进行PCR扩增,其中18对引物可扩增出清晰、可重复条带,并在42份三色堇资源中表现多态性。基于扩增的多态性SSR信息,42份三色堇种质资源的聚类结果与其遗传背景基本一致。本研究中获得的18对SSR引物可为三色堇遗传图谱构建、功能基因定位等提供有力工具。
To develop co-dominant DNA markers in pansy(Viola × wittrockiana),167 576 unigenes from pansy transcriptomic database were screened by MISA software. A total of 23 791 simple sequence repeat(SSR)loci were detected in the unigenes with the frequency of 14.20%,and the average distribution distance of SSR was 6.75 kb. The predominant type of SSR was trinucleotide repetition amounting for28.31% of total SSRs,followed by dinucleotide repetition accounting for 12.86%. AG/CT and GAA/TTC are the dominant elements in dinucleotide and trinucleotide repetitions,accounting for 4.01% and 1.85% of the total SSRs,respectively. Primer 3 was employed to design SSR primers and a total of 6 863 pairs of SSR primers were obtained. Thirty pairs of primers were randomly selected for PCR in pansies,in which 18 pairs of primers amplified clear and repeatable bands and showed polymorphism in 42 pansy germplasm accessions. Based on the amplified polymorphic SSR data,the dendrogram results were in accordance with the genetic backgrounds of 42 accessions. Therefore,a large amount of SSR markers can be developed from transcriptome sequencing and employed in genetic linkage map construction and functional gene mapping in pansy.
To develop co-dominant DNA markers in pansy(Viola × wittrockiana),167 576 unigenes from pansy transcriptomic database were screened by MISA software. A total of 23 791 simple sequence repeat(SSR)loci were detected in the unigenes with the frequency of 14.20%,and the average distribution distance of SSR was 6.75 kb. The predominant type of SSR was trinucleotide repetition amounting for28.31% of total SSRs,followed by dinucleotide repetition accounting for 12.86%. AG/CT and GAA/TTC are the dominant elements in dinucleotide and trinucleotide repetitions,accounting for 4.01% and 1.85% of the total SSRs,respectively. Primer 3 was employed to design SSR primers and a total of 6 863 pairs of SSR primers were obtained. Thirty pairs of primers were randomly selected for PCR in pansies,in which 18 pairs of primers amplified clear and repeatable bands and showed polymorphism in 42 pansy germplasm accessions. Based on the amplified polymorphic SSR data,the dendrogram results were in accordance with the genetic backgrounds of 42 accessions. Therefore,a large amount of SSR markers can be developed from transcriptome sequencing and employed in genetic linkage map construction and functional gene mapping in pansy.
引文
An M,Deng M,Zheng S,Song Y.2016.De novo transcriptome assembly and development of SSR markers of oaks Quercus austrocochinchinensis,and Q.kerrii,(Fagaceae).Tree Genetics&Genomes,12(6):103.
Culley T M,Sbita S J,Wick A.2007.Population genetic effects of urban habitat fragmentation in the perennial herb Viola pubescens(Violaceae)using ISSR markers.Annals of Botany,100(1):91-100.
Dutta S,Kumawat G,Singh B P,Gupta D K,Singh S,Dogra V,Gaikwad K,Sharma T R,Raje R S,Bandhopadhya T K,Datta S,Singh MN,Bashasab F,Kulwal P,Wanjari K B,Varshney K R,Cook D R,Singh N K.2011.Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea[Cajanus cajan(L.)Millspaugh].BMC Plant Biology,11:17.
Eujayl I,Sorrells M,Banm M,Wolters P,Powell W.2002.Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat.Theor Appl Genet,104(2):399-407.
Guo S,Zheng Y,Joung J G,Liu S Q,Zhang Z H,Crasta O R,Sobral B W,Xu Y,Huang S W,Fei Z G.2010.Transcriptome sequencing and comparative analysis of cucumber flowers with different sex types.BMC Genomics,11(1):384.
Gilles A,Meglecz E,Pech N,Ferreira S,Malausa T,Martin J F.2011.Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing.BMC Genomics,12:245.
Grabherr M G,Haas B J,Yassour M,Levin J Z,Thompson D A,Amit I,Adiconis X,Fan L,Raychowdhury R,Zeng Q D,Chen Z H,Mauceli E,Hacohen N,Gnirke A,Rhind N,Palma F D,Birren B W,Nusbaum C,Lindblad-Toh K,Friedman N,Regev A.2011.Full-length transcriptome assembly from RNA-Seq data without a reference genome.Nature Biotechnology,29:644-652.
Kantety R V,Rota M L,Matthews D E,Sorrells M E.2002.Data mining for simple sequence repeats in expressed sequence tags from barley,maize,rice,sorghum and wheat.Plant Molecular Biology,48:501-510.
Ko M K,Yang J,Jin Y H,Lee C H,Oh B J.1998.Genetic relationships of Viola species evaluated by random amplified polymorphic DNA analysis.Journal of Pomology&Horticultural Science,73(5):601-605.
Kota R,Varshney R K,Thiel T,Dehmer K J,Graner A.2001.Generation and comparison of EST-derived SSRs and SNPs in berley(Hordeum vulgare L.).Hereditas,135(2-3):145-151.
Li Man-tang,Zhang Shi-lin,Deng Peng,Hou Xi-lin,Wang Jian-jun.2015.Analysis on SSR information in transcriptome of onion and the polymorphism.Acta Horticulturae Sinica,42(6):1103-1111.(in Chinese)李满堂,张仕林,邓鹏,侯喜林,王建军.2015.洋葱转录组SSR信息分析及其多态性研究.园艺学报,42(6):1103-1111.
Li X,Gao W,Guo H,Zhang X,Fang D D,Lin Z.2014.Development of EST-based SNP and InDel markers and their utilization in tetraploid cotton genetic mapping.BMC Genomics,15:1046-1047.
Li X,Yu H L,Li Z Y,Liu X P,Fang Z Y,Liu Y M,Yang L M,Zhuang M,Lv H H,Zhang Y Y.2018.Heterotic group classification of 63inbred lines and hybrid purity identification by using SSR markers in winter cabbage(Brassica oleracea L.var.capitata).Horticultural Plant Journal,4(4):158-164.
Li Xiao-mei,Du Xiao-hua,Mu Jin-yan,Liu Hui-chao.2015.Genetic diversity analysis of 41 pansy germplasms based on RSAP markers.Acta Botanica Boreali-Occidentalia Sinica,35(10):1989-1997.(in Chinese)李小梅,杜晓华,穆金燕,刘会超.2015.基于RSAP标记的大花三色堇遗传多样性分析.西北植物学报,35(10):1989-1997.
Li Yan-lin,Yang Xing-xing,Zhang Jia-yin,Huang San-wen,Xiong Xing-yao.2014.Studies on SSR molecular markers based on transcriptome of Taxus chinensis var.mairei.Acta Horticulturae Sinica,41(4):735-745.(in Chinese)李炎林,杨星星,张家银,黄三文,熊兴耀.2014.南方红豆杉转录组SSR挖掘及分子标记的研究.园艺学报,41(4):735-745.
Li Yong-ping,Liu Jian-ting,Chen Min-dong,Zhang Qian-rong,Zhu Hai-sheng,Wen Qing-fang.2018.SSR markers excavation and germplasm analysis using the transcriptome information of Hibiscus esculentus.Acta Horticulturae Sinica,45(3):579-590.(in Chinese)李永平,刘建汀,陈敏氡,张前荣,朱海生,温庆放.2018.利用黄秋葵转录组信息挖掘SSR标记及用于种质分析.园艺学报,45(3):579-590.
Liu Feng,Wang Yun-sheng,Tian Xue-liang,Mao Zhen-chuang,Zhou Xue-xiao,Xie Bing-yan.2012.SSR mining in pepper(Capsicum annuum L.)transcriptome and the polymorphism analysis.Acta Horticulturae Sinica,39(1):168-174.(in Chinese)刘峰,王运生,田雪亮,茆振川,邹学校,谢丙炎.2012.辣椒转录组SSR挖掘及其多态性分析.园艺学报,39(1):168-174.
Long Dang.2014.Large scale development of SSR markers based on transcriptome sequencing of precocious trifoliate orange[M.D.Dissertation].Wuhan:Huazhong Agricultural University.(in Chinese)龙荡.2014.基于转录组測序大规模开发早实枳标记[硕士论文].武汉:华中农业大学.
Lu Min,Yan Xiuqin,Bai Jing,Zhang Huaishan,Wang Daoping,An Huaming.2017.Construction of core collection in wild Rosa roxburghii from Guizhou Province using EST-SSR markers and fruits quality traits.Acta Horticulturae Sinica,44(8):1486-1495.(in Chinese)鲁敏,鄢秀芹,白静,张怀山,王道平,安华明.2017.基于EST-SSR标记与果实品质性状的贵州野生刺梨核心种质构建.园艺学报,44(8):1486-1495.
Nie G,Tang L,Zhang Y,Huang L,Ma X,Cao X,Pan L,Zhang X,Zhang X.2017.Development of SSR markers based on transcriptome sequencing and association analysis with drought tolerance in perennial grass Miscanthus from China.Frontiers in Plant Science,8:801.
Omka A S,Siwińska D,Wolny E B,Kellner K,Kuta E B.2011.Influence of a heavy-metal-polluted environment on Viola tricolor genome size and chromosome number.Acta Biologica Cracoviensia,53(1):7-15.
Shi Xiao-dong,Zhu Xue-hui,Sheng Yu-zhen,Zhuang Guo-qing,Chen Fang.2016.Development of SSR markers based on transcriptome sequence of Phoebe zhennan.Scientia Silvae Sinicae,52(11):71-78.(in Chinese)时小东,朱学慧,盛玉珍,庄国庆,陈放.2016.基于转录组序列的楠木SSR分子标记开发.林业科学,52(11):71-78.
Sun Qing-ming,Ma Wen-chao,Ma Shuai-peng,Zhao Jun-sheng,Bai Li-jun,Chen Jie-zhen,Cai Chang-he,Xiang Xu,Ou Liang-xi.2011.Characteristics of SSRs derived from ESTs and development of EST-SSR markers in litch(Litchi chinensis Sonn).Scientia Agricultura Sinica,44(19):4037-4049.(in Chinese)孙清明,马文朝,马帅鹏,赵俊生,白丽军,陈洁珍,蔡长河,向旭,欧良喜.2011.荔枝EST资源的SSR信息分析及EST-SSR标记开发.中国农业科学,44(19):4037-4049.
Vemmos S N.2015.Characterisation of genetic relationships in pansy(Viola wittrockiana)inbred lines using morphological traits and RAPD markers.Journal of Pomology&Horticultural Science,80(5):529-536.
Wang Guan-lin,Fang Hong-jun.2009.Plant genetic engineering.Beijing:Science Press.(in Chinese)王关林,方宏筠.2009.植物基因工程.北京:科学出版社.
Wang Jian,Bao Man-zhu.2007.Application of RAPD on analysis of genetic relationships between inbred lines and prediction of heterosis in pansy(Viola wittrockiana).Journal of Wuhan Botanical Research,25(1):19-23.(in Chinese)王健,包满珠.2007.RAPD在三色堇(Viola wittrockiana)自交系遗传关系研究及杂种优势预测中的应用.武汉植物学研究,25(1):19-23.
Wang Tao,Xu Jin,Zhang Xi-xi,Zhao Liang-jun.2012.Genetic relationship of 43 cultivars of Viola tricolor and Viola cornuta using SRAP marker.Scientia Agricultura Sinica,45(3):496-502.(in Chinese)王涛,徐进,张西西,赵梁军.2012.43份三色堇、角堇材料亲缘关系的SRAP分析.中国农业科学,45(3):496-502.
Wei Ming-ming.2016.Development and application of SSR markers in eggplant based on transcriptome sequencing[M.D.Dissertation].Beijing:Chinese Academy of Agricultural Sciences.(in Chinese)魏明明.2016.基于转录组测序的茄子SSR标记开发及应用[硕士论文].北京:中国农业科学院.
Xiao Y,Zhou L,Xia W,Mason A S,Yang Y,Ma Z,Peng M.2014.Exploiting transcriptome data for the development and characterization of gene-based SSR markers related to cold tolerance in oil palm(Elaeis guineensis).BMC Plant Biology,14:384.
Yan Xiu-qin,Lu Min,An Hua-ming.2015.Analysis on SSR information in transcriptome and development of molecular markers in Rosa roxburghii.Acta Horticulturae Sinica,42(2):341-349.(in Chinese)鄢秀芹,鲁敏,安华明.2015.刺梨转录组SSR信息分析及其分子标记开发.园艺学报,42(2):341-349.
Yockteng R,Ballard H E J,Mansion G,Dajoz I,Nadot S.2003.Relationships among pansies(Viola,section Melanium)investigated using ITSand ISSR markers.Plant Systematics&Evolution,241(3-4):153-170.
Zhang Huali,Cong Richen,Wang Maoliang,Dong Aixiang,Xin Haibo,Yi Mingfang,Guo Hua.2018.Development of SSR molecular markers based on transcriptome sequencing of Tagetes erecta.Acta Horticulturae Sinica,45(1):159-167.(in Chinese)张华丽,丛日晨,王茂良,董爱香,辛海波,义鸣放,郭华.2018.基于万寿菊转录组测序的SSR标记开发.园艺学报,45(1):159-167.
Zhang J,Liang S,Duan J,Wang J,Chen S,Cheng Z,Zhang Q,Liang X,Li Y.2012.De novo assembly and characterisation of the transcriptome during seed development,and generation of genic-SSR markers in peanut(Arachis hypogaea L.).BMC Genomics,13(1):90.
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Zhang Zhen,Zhang Han-guo,Mo Chi,Zhang Lei.2015.Transcriptome sequencing analysis and development of EST-SSR markers for Pinus koraiensis.Scientia Silvae Sinicae,51(8):114-120.(in Chinese)张振,张含国,莫迟,张磊.2015.红松转录组SSR分析及EST-SSR标记开发.林业科学,51(8):114-120.
Culley T M,Sbita S J,Wick A.2007.Population genetic effects of urban habitat fragmentation in the perennial herb Viola pubescens(Violaceae)using ISSR markers.Annals of Botany,100(1):91-100.
Dutta S,Kumawat G,Singh B P,Gupta D K,Singh S,Dogra V,Gaikwad K,Sharma T R,Raje R S,Bandhopadhya T K,Datta S,Singh MN,Bashasab F,Kulwal P,Wanjari K B,Varshney K R,Cook D R,Singh N K.2011.Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea[Cajanus cajan(L.)Millspaugh].BMC Plant Biology,11:17.
Eujayl I,Sorrells M,Banm M,Wolters P,Powell W.2002.Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat.Theor Appl Genet,104(2):399-407.
Guo S,Zheng Y,Joung J G,Liu S Q,Zhang Z H,Crasta O R,Sobral B W,Xu Y,Huang S W,Fei Z G.2010.Transcriptome sequencing and comparative analysis of cucumber flowers with different sex types.BMC Genomics,11(1):384.
Gilles A,Meglecz E,Pech N,Ferreira S,Malausa T,Martin J F.2011.Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing.BMC Genomics,12:245.
Grabherr M G,Haas B J,Yassour M,Levin J Z,Thompson D A,Amit I,Adiconis X,Fan L,Raychowdhury R,Zeng Q D,Chen Z H,Mauceli E,Hacohen N,Gnirke A,Rhind N,Palma F D,Birren B W,Nusbaum C,Lindblad-Toh K,Friedman N,Regev A.2011.Full-length transcriptome assembly from RNA-Seq data without a reference genome.Nature Biotechnology,29:644-652.
Kantety R V,Rota M L,Matthews D E,Sorrells M E.2002.Data mining for simple sequence repeats in expressed sequence tags from barley,maize,rice,sorghum and wheat.Plant Molecular Biology,48:501-510.
Ko M K,Yang J,Jin Y H,Lee C H,Oh B J.1998.Genetic relationships of Viola species evaluated by random amplified polymorphic DNA analysis.Journal of Pomology&Horticultural Science,73(5):601-605.
Kota R,Varshney R K,Thiel T,Dehmer K J,Graner A.2001.Generation and comparison of EST-derived SSRs and SNPs in berley(Hordeum vulgare L.).Hereditas,135(2-3):145-151.
Li Man-tang,Zhang Shi-lin,Deng Peng,Hou Xi-lin,Wang Jian-jun.2015.Analysis on SSR information in transcriptome of onion and the polymorphism.Acta Horticulturae Sinica,42(6):1103-1111.(in Chinese)李满堂,张仕林,邓鹏,侯喜林,王建军.2015.洋葱转录组SSR信息分析及其多态性研究.园艺学报,42(6):1103-1111.
Li X,Gao W,Guo H,Zhang X,Fang D D,Lin Z.2014.Development of EST-based SNP and InDel markers and their utilization in tetraploid cotton genetic mapping.BMC Genomics,15:1046-1047.
Li X,Yu H L,Li Z Y,Liu X P,Fang Z Y,Liu Y M,Yang L M,Zhuang M,Lv H H,Zhang Y Y.2018.Heterotic group classification of 63inbred lines and hybrid purity identification by using SSR markers in winter cabbage(Brassica oleracea L.var.capitata).Horticultural Plant Journal,4(4):158-164.
Li Xiao-mei,Du Xiao-hua,Mu Jin-yan,Liu Hui-chao.2015.Genetic diversity analysis of 41 pansy germplasms based on RSAP markers.Acta Botanica Boreali-Occidentalia Sinica,35(10):1989-1997.(in Chinese)李小梅,杜晓华,穆金燕,刘会超.2015.基于RSAP标记的大花三色堇遗传多样性分析.西北植物学报,35(10):1989-1997.
Li Yan-lin,Yang Xing-xing,Zhang Jia-yin,Huang San-wen,Xiong Xing-yao.2014.Studies on SSR molecular markers based on transcriptome of Taxus chinensis var.mairei.Acta Horticulturae Sinica,41(4):735-745.(in Chinese)李炎林,杨星星,张家银,黄三文,熊兴耀.2014.南方红豆杉转录组SSR挖掘及分子标记的研究.园艺学报,41(4):735-745.
Li Yong-ping,Liu Jian-ting,Chen Min-dong,Zhang Qian-rong,Zhu Hai-sheng,Wen Qing-fang.2018.SSR markers excavation and germplasm analysis using the transcriptome information of Hibiscus esculentus.Acta Horticulturae Sinica,45(3):579-590.(in Chinese)李永平,刘建汀,陈敏氡,张前荣,朱海生,温庆放.2018.利用黄秋葵转录组信息挖掘SSR标记及用于种质分析.园艺学报,45(3):579-590.
Liu Feng,Wang Yun-sheng,Tian Xue-liang,Mao Zhen-chuang,Zhou Xue-xiao,Xie Bing-yan.2012.SSR mining in pepper(Capsicum annuum L.)transcriptome and the polymorphism analysis.Acta Horticulturae Sinica,39(1):168-174.(in Chinese)刘峰,王运生,田雪亮,茆振川,邹学校,谢丙炎.2012.辣椒转录组SSR挖掘及其多态性分析.园艺学报,39(1):168-174.
Long Dang.2014.Large scale development of SSR markers based on transcriptome sequencing of precocious trifoliate orange[M.D.Dissertation].Wuhan:Huazhong Agricultural University.(in Chinese)龙荡.2014.基于转录组測序大规模开发早实枳标记[硕士论文].武汉:华中农业大学.
Lu Min,Yan Xiuqin,Bai Jing,Zhang Huaishan,Wang Daoping,An Huaming.2017.Construction of core collection in wild Rosa roxburghii from Guizhou Province using EST-SSR markers and fruits quality traits.Acta Horticulturae Sinica,44(8):1486-1495.(in Chinese)鲁敏,鄢秀芹,白静,张怀山,王道平,安华明.2017.基于EST-SSR标记与果实品质性状的贵州野生刺梨核心种质构建.园艺学报,44(8):1486-1495.
Nie G,Tang L,Zhang Y,Huang L,Ma X,Cao X,Pan L,Zhang X,Zhang X.2017.Development of SSR markers based on transcriptome sequencing and association analysis with drought tolerance in perennial grass Miscanthus from China.Frontiers in Plant Science,8:801.
Omka A S,Siwińska D,Wolny E B,Kellner K,Kuta E B.2011.Influence of a heavy-metal-polluted environment on Viola tricolor genome size and chromosome number.Acta Biologica Cracoviensia,53(1):7-15.
Shi Xiao-dong,Zhu Xue-hui,Sheng Yu-zhen,Zhuang Guo-qing,Chen Fang.2016.Development of SSR markers based on transcriptome sequence of Phoebe zhennan.Scientia Silvae Sinicae,52(11):71-78.(in Chinese)时小东,朱学慧,盛玉珍,庄国庆,陈放.2016.基于转录组序列的楠木SSR分子标记开发.林业科学,52(11):71-78.
Sun Qing-ming,Ma Wen-chao,Ma Shuai-peng,Zhao Jun-sheng,Bai Li-jun,Chen Jie-zhen,Cai Chang-he,Xiang Xu,Ou Liang-xi.2011.Characteristics of SSRs derived from ESTs and development of EST-SSR markers in litch(Litchi chinensis Sonn).Scientia Agricultura Sinica,44(19):4037-4049.(in Chinese)孙清明,马文朝,马帅鹏,赵俊生,白丽军,陈洁珍,蔡长河,向旭,欧良喜.2011.荔枝EST资源的SSR信息分析及EST-SSR标记开发.中国农业科学,44(19):4037-4049.
Vemmos S N.2015.Characterisation of genetic relationships in pansy(Viola wittrockiana)inbred lines using morphological traits and RAPD markers.Journal of Pomology&Horticultural Science,80(5):529-536.
Wang Guan-lin,Fang Hong-jun.2009.Plant genetic engineering.Beijing:Science Press.(in Chinese)王关林,方宏筠.2009.植物基因工程.北京:科学出版社.
Wang Jian,Bao Man-zhu.2007.Application of RAPD on analysis of genetic relationships between inbred lines and prediction of heterosis in pansy(Viola wittrockiana).Journal of Wuhan Botanical Research,25(1):19-23.(in Chinese)王健,包满珠.2007.RAPD在三色堇(Viola wittrockiana)自交系遗传关系研究及杂种优势预测中的应用.武汉植物学研究,25(1):19-23.
Wang Tao,Xu Jin,Zhang Xi-xi,Zhao Liang-jun.2012.Genetic relationship of 43 cultivars of Viola tricolor and Viola cornuta using SRAP marker.Scientia Agricultura Sinica,45(3):496-502.(in Chinese)王涛,徐进,张西西,赵梁军.2012.43份三色堇、角堇材料亲缘关系的SRAP分析.中国农业科学,45(3):496-502.
Wei Ming-ming.2016.Development and application of SSR markers in eggplant based on transcriptome sequencing[M.D.Dissertation].Beijing:Chinese Academy of Agricultural Sciences.(in Chinese)魏明明.2016.基于转录组测序的茄子SSR标记开发及应用[硕士论文].北京:中国农业科学院.
Xiao Y,Zhou L,Xia W,Mason A S,Yang Y,Ma Z,Peng M.2014.Exploiting transcriptome data for the development and characterization of gene-based SSR markers related to cold tolerance in oil palm(Elaeis guineensis).BMC Plant Biology,14:384.
Yan Xiu-qin,Lu Min,An Hua-ming.2015.Analysis on SSR information in transcriptome and development of molecular markers in Rosa roxburghii.Acta Horticulturae Sinica,42(2):341-349.(in Chinese)鄢秀芹,鲁敏,安华明.2015.刺梨转录组SSR信息分析及其分子标记开发.园艺学报,42(2):341-349.
Yockteng R,Ballard H E J,Mansion G,Dajoz I,Nadot S.2003.Relationships among pansies(Viola,section Melanium)investigated using ITSand ISSR markers.Plant Systematics&Evolution,241(3-4):153-170.
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