绿豆SSR标记的开发及遗传多样性分析
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摘要
SSR标记以其数量丰富、多态性好、共显性遗传等优点在基础研究和育种工作中发挥了重要作用,但目前绿豆基因组中的SSR标记依然较少。本研究将磁珠富集法和测序技术相结合高通量检测绿豆基因组SSR位点,鉴定出3,275,355个SSR位点,开发了2742个SSR标记。选取其中157个SSR进行PCR验证,发现有90个(57.33%)标记在10份材料中表现出多态性。挑选40个条带清晰、多态性高、染色体上均匀分布的标记对90份绿豆资源进行遗传多样性分析,单个位点检测到的等位变异数为2～8个,平均为3.0个,有效等位基因数为1.31～4.21个,平均为2.16。Nei’s基因多样性指数在0.23～0.76之间,平均为0.51。多态性信息含量为0.22～0.72,平均为0.43。聚类分析将90份材料分为2个类群,包含4个组。第I组主要由北方资源组成,第Ⅱ组种质来源较为分散,第Ⅲ组主要由山东的资源构成,第Ⅳ组包含多数河北的种质资源。本研究开发的多态性SSR标记不仅可以用于绿豆种质资源的遗传多样性分析,也将在高密度遗传图谱构建、基因定位和分子标记辅助育种中发挥重要作用。
SSR markers play an important role in basic research and crop breeding due to their advantages of large number, high polymorphism and co-dominant inheritance. However, there are still few SSR markers available in mung bean. In this study, the magnetic bead enrichment method and sequencing technology were combined to identify the SSR loci of mung bean in high throughput, a total of 3,275,355 SSR loci were found, and 2742 markers were developed. A total of 157 markers were selected for validation by PCR method, 90(57.33%) showed polymorphic among 10 mung bean accessions. Forty SSR markers with clear PCR products, high polymorphism and uniform distribution on chromosomes were selected to evaluate the genetic diversity among 90 mung bean accessions. The number of alleles per marker varied from two to eight, with an average of three. The effective number of alleles ranged from 1.31 to 4.21, with a mean value of 2.16. The Nei's gene diversity was between 0.23 and 0.76,with an average of 0.51. Polymorphism information content was between 0.22 and 0.72, with a mean of 0.43. Cluster analysis distributed 90 materials into two clusters, including four groups. The germplasm of group Ⅱ came from several areas, while those of groups I and Ⅲ were mainly from North China and Shandong province, respectively. Most of the gerplasm from Hebei province were clustered in Group Ⅳ. These polymorphic SSR markers will be valuable for genetic diversity analysis, high-resolution genetic linkage maps construction, gene mapping and marker assisted selection in mung bean breeding.
SSR markers play an important role in basic research and crop breeding due to their advantages of large number, high polymorphism and co-dominant inheritance. However, there are still few SSR markers available in mung bean. In this study, the magnetic bead enrichment method and sequencing technology were combined to identify the SSR loci of mung bean in high throughput, a total of 3,275,355 SSR loci were found, and 2742 markers were developed. A total of 157 markers were selected for validation by PCR method, 90(57.33%) showed polymorphic among 10 mung bean accessions. Forty SSR markers with clear PCR products, high polymorphism and uniform distribution on chromosomes were selected to evaluate the genetic diversity among 90 mung bean accessions. The number of alleles per marker varied from two to eight, with an average of three. The effective number of alleles ranged from 1.31 to 4.21, with a mean value of 2.16. The Nei's gene diversity was between 0.23 and 0.76,with an average of 0.51. Polymorphism information content was between 0.22 and 0.72, with a mean of 0.43. Cluster analysis distributed 90 materials into two clusters, including four groups. The germplasm of group Ⅱ came from several areas, while those of groups I and Ⅲ were mainly from North China and Shandong province, respectively. Most of the gerplasm from Hebei province were clustered in Group Ⅳ. These polymorphic SSR markers will be valuable for genetic diversity analysis, high-resolution genetic linkage maps construction, gene mapping and marker assisted selection in mung bean breeding.
引文
[1]程须珍,王述民.中国食用豆类品种志.北京:中国农业科学技术出版社,2009.pp 19-20.Cheng X Z,Wang S M.Chinese Legumes Variety Pictorial.Beijing:China Agricultural Science and Technology Press,2009.pp 19-20(in Chinese).
[2]Fuller D Q,Harvey E L.The archaeobotany of Indian pulses:identification,processing and evidence for cultivation.Environ Archaeol,2006,11:219-246.
[3]Nair R M,Schafleitner R,Kenyon L,Srinivasan R,Easdown W,Ebert A W,Hanson P.Genetic improvement of mung bean.SABRAO J Breed Genet,2012,44:177-190.
[4]郑卓杰,王述民,宗绪晓.中国食用豆类学.北京:中国农业出版社,1997.pp 3-6.Zheng Z J,Wang S M,Zong X X.Food Legumes in China.Beijing:China Agriculture Press,1997.pp 3-6(in Chinese).
[5]王丽侠,程须珍,王素华.绿豆种质资源、育种及遗传研究进展.中国农业科学,2009,42:1519-1527.Wang L X,Cheng X Z,Wang S H.Advances in research on genetic resources,breeding and genetics of mung bean(Vigna radiata L.).Sci Agric Sin,2009,42:1519-1527(in Chinese with English abstract).
[6]程须珍,王素华.中国绿豆产业发展及科技应用.北京:中国农业科学技术出版社,2002.pp 3-8.Cheng X Z,Wang S H.Indusdustrial Development and Technology Utilization of Mungbean in China.Beijing:China Agricultural Science and Technology Press,2002.pp 3-8(in Chinese).
[7]Kang Y J,Kim S K,Kim M Y,Lestari P,Kim K H,Ha B K,Jun T H,Hwang W J,Lee T,Lee J,Shim S,Yoon M Y,Jang Y E,Han K S,Taeprayoon P,Yoon N,Somta P,Tanya P,Kim K S,Gwag JG,Moon J K,Lee Y H,Park B S,Bombarely A,Doyle J J,Jackson S A,Schafleitner R,Srinives P,Varshney R K,Lee S H.Genome sequence of mung bean and insights into evolution within Vigna species.Nat Commun,2014,5:5443,doi:10.1038/ncomms6443.
[8]刘岩,程须珍,王丽侠,王素华,白鹏,吴传书.基于SSR标记的中国绿豆种质资源遗传多样性研究.中国农业科学,2013,46:4197-4209.Liu Y,Cheng X Z,Wang L X,Wang S H,Bai P,Wu C S.Genetic diversity research of mungbean germplasm resources by SSRmarkers in China.Sci Agric Sin,2013,46:4197-4209(in Chinese with English abstract).
[9]任红晓,程须珍,徐东旭,高运青,尚启兵.应用SSR标记分析中国北方名优绿豆的遗传多样性.植物遗传资源学报,2015,16:395-399.Ren H X,Cheng X Z,Xu D X,Gao Y Q,Shang Q B.Genetic diversity of traditional mungbean varieties in northern China by SSR markers.J Plant Genet Resour,2015,16:395-399(in Chinese with English abstract).
[10]赵丹,程须珍,王丽侠,王素华,马燕玲.绿豆遗传连锁图谱的整合.作物学报,2010,36:932-939.Zhao D,Cheng X Z,Wang L X,Wang S H,Ma Y L.Integration of mungbean(Vigna radiata)genetic linkage map.Acta Agron Sin,2010,36:932-939(in Chinese with English abstract).
[11]Isemura T,Kaga A,Tabata S,Somta P,Srinives P,Shimizu T,Jo U,Vaughan D A,Tomooka N.Construction of a genetic linkage map and genetic analysis of domestication related traits in mungbean(Vigna radiata).PLoS One,2012,7:e41304.
[12]王建花,张耀文,程须珍,王丽侠.绿豆分子遗传图谱构建及若干农艺性状的QTL定位分析.作物学报,2017,43:1096-1102.Wang J H,Zhang Y W,Cheng X Z,Wang L X.Construction of genetic map and identification of QTLs related to agronomic traits in mung bean.Acta Agron Sin,2017,43:1096-1102(in Chinese with English abstract).
[13]王丽侠,程须珍,王素华,刘长友,梁辉.小豆SSR引物在绿豆基因组中的通用性分析.作物学报,2009,35:816-820.Wang L X,Cheng X Z,Wang S H,Liu C Y,Liang H.Transferability of SSR from adzuki bean to mungbean.Acta Agron Sin,2009,35:816-820(in Chinese with English abstract).
[14]钟敏,程须珍,王丽侠,王素华,王小宝.绿豆基因组SSR引物在豇豆属作物中的通用性.作物学报,2012,38:223-230.Zhong M,Cheng X Z,Wang L X,Wang S H,Wang X B.Transferability of mungbean genomic-SSR markers in other vigna species.Acta Agron Sin,2012,38:223-230(in Chinese with English abstract).
[15]Kumar S V,Tan S G,Quah S C,Yusoff K.Isolation and characterization of seven tetranucleotide microsatellite loci in mungbean,Vigna radiata.Mol Ecol Notes,2002,2:293-295.
[16]Miyagi M,Humphry M,Ma Z Y,Lambrides C J,Bateson M,Liu C J.Construction of bacterial artificial chromosome libraries and their application in developing PCR-based markers closely linked to a major locus conditioning bruchid resistance in mung bean(Vigna radiata L.Wilczek).Theor Appl Genet,2004,110:151-156.
[17]Gwag J G,Chung J W,Chung H K,Lee J H,Ma K H.Characterization of new microsatellite markers in mungbean,Vigna radiata(L.).Mol Ecol Notes,2006,6:1132-1134.
[18]Somta P,Musch W,Kongsamai B,Chanprame S,Nakasatien S,Toojinda T,Sorajjapinun W,Seehalak W,Tragoonrung S,Srinives P.New microsatellite markers isolated from mungbean(Vigna radiata(L.)Wilczek).Mol Ecol Resour,2008,8:1155-1157.
[19]Seehalak W,Somta P,Sommanas W,Srinives P.Microsatellite markers for mungbean developed from sequence database.Mol Ecol Resour,2009,9:862-864.
[20]Tangphatsornruang S,Somta P,Uthaipaisanwong P,Chanprasert J,Sangsrakru D,Seehalak W,Sommanas W,Tragoonrung S,Srinives P.Characterization of microsatellites and gene contents from genome shotgun sequences of mungbean(Vigna radiata(L.)Wilczek).BMC Plant Biol,2009,9:137,doi:10.1186/1471-2229-9-137.
[21]Somta P,Seehalak W,Srinives P.Development,characterization and cross-species amplification of mungbean(Vigna radiata)genic microsatellite markers.Conserv Genet,2009,10:1939-1943.
[22]Wang L X,Elbaidouri M,Abernathy B,Chen H L,Wang S H,Lee S H,Jackson S A,Cheng X Z.Distribution and analysis of SSR in mung bean(Vigna radiata L.)genome based on an SSR-enriched library.Mol Breed,2015,35:25,doi:10.1007/s11032-015-0259-8.
[23]Chen H L,Wang L X,Wang S H,Liu C Y,Blair M W,Cheng X Z.Transcriptome sequencing of mung bean(Vigna radiate L.)genes and the identification of EST-SSR markers.PLoS One,2015,10:e0120273.
[24]Liu C Y,Fan B J,Cao Z M,Su Q Z,Wang Y,Zhang Z X,Wu J,Tian J.A deep sequencing analysis of transcriptomes and the development of EST-SSR markers in mungbean(Vigna radiata).JGenet,2016,95:527-535.
[25]孙子奎,陈永灿.一种基于磁珠富集法高通量开发基因组SSR标记的方法.中国专利,2014,ZL201310222359.9.Sun Z K,Chen Y C.A method of developing genome SSRmarkers based on magnetic bead enrichment for NGS,China patent,2014,ZL201310222359.9.
[26]Lindgreen S.AdapterRemoval:easy cleaning of next-generation sequencing reads.BMC Res Notes,2012,5:337,doi:10.1186/1756-0500-5-337.
[27]Mago?T,Salzberg S L.FLASH:fast length adjustment of short reads to improve genome assemblies.Bioinformatics,2011,27:2957-2963.
[28]Untergasser A,Cutcutache I,Koressaar T,Ye J,Faircloth B C,Remm M,Rozen S G.Primer3:new capabilities and interfaces.Nucl Acids Res,2012,40:e115.
[29]Krawczak M,Nikolaus S,von Eberstein H,Croucher P J,El Mokhtari N E,Schreiber S.PopGen:population-based recruitment of patients and controls for the analysis of complex genotype-phenotype relationships.Commun Genet,2006,9:55-61.
[30]Liu K,Muse S V.PowerMarker:An integrated analysis environment for genetic marker analysis.Bioinformatics,2005,21:2128-2129.
[31]吴传书.绿豆SSR标记的开发及高密度分子遗传连锁图谱的构建.甘肃农业大学硕士学位论文,甘肃兰州,2014.Wu C S.Development of SSR Markers and Construction of a Genetic Linkage Map in Mungbean(Vigan radiate L.).MS Thesis of Gansu Agricultural University,Lanzhou,Gansu,China,2014(in Chinese with English abstract).
[32]Gupta S K,Bansal R,Gopalakrishna T.Development and characterization of genic SSR markers for mungbean(Vigna radiata(L.)Wilczek).Euphytica,2014,195:245-258.
[33]Yang T,Bao S Y,Ford R,Jia T J,Guan J P,He Y H,Sun X L,Jiang J Y,Hao J J,Zhang X Y,Zong X X.High-throughput novel microsatellite marker of faba bean via next generation sequencing.BMC Genomics,2012,13:602,doi:10.1186/1471-2164-13-602.
[34]Gao L F,Tang J F,Li H W,Jia J Z.Analysis of microsatellites in major crops assessed by computational and experimental approaches.Mol Breed,2003,12:245-261.
[35]Nicot N,Chiquet V,Gandon B,Amilhat L,Legeai F,Leroy P,Bernard M,Sourdille P.Study of simple sequence repeat(SSR)markers from wheat expressed sequence tags(ESTs).Theor Appl Genet,2004,109:800-805.
[2]Fuller D Q,Harvey E L.The archaeobotany of Indian pulses:identification,processing and evidence for cultivation.Environ Archaeol,2006,11:219-246.
[3]Nair R M,Schafleitner R,Kenyon L,Srinivasan R,Easdown W,Ebert A W,Hanson P.Genetic improvement of mung bean.SABRAO J Breed Genet,2012,44:177-190.
[4]郑卓杰,王述民,宗绪晓.中国食用豆类学.北京:中国农业出版社,1997.pp 3-6.Zheng Z J,Wang S M,Zong X X.Food Legumes in China.Beijing:China Agriculture Press,1997.pp 3-6(in Chinese).
[5]王丽侠,程须珍,王素华.绿豆种质资源、育种及遗传研究进展.中国农业科学,2009,42:1519-1527.Wang L X,Cheng X Z,Wang S H.Advances in research on genetic resources,breeding and genetics of mung bean(Vigna radiata L.).Sci Agric Sin,2009,42:1519-1527(in Chinese with English abstract).
[6]程须珍,王素华.中国绿豆产业发展及科技应用.北京:中国农业科学技术出版社,2002.pp 3-8.Cheng X Z,Wang S H.Indusdustrial Development and Technology Utilization of Mungbean in China.Beijing:China Agricultural Science and Technology Press,2002.pp 3-8(in Chinese).
[7]Kang Y J,Kim S K,Kim M Y,Lestari P,Kim K H,Ha B K,Jun T H,Hwang W J,Lee T,Lee J,Shim S,Yoon M Y,Jang Y E,Han K S,Taeprayoon P,Yoon N,Somta P,Tanya P,Kim K S,Gwag JG,Moon J K,Lee Y H,Park B S,Bombarely A,Doyle J J,Jackson S A,Schafleitner R,Srinives P,Varshney R K,Lee S H.Genome sequence of mung bean and insights into evolution within Vigna species.Nat Commun,2014,5:5443,doi:10.1038/ncomms6443.
[8]刘岩,程须珍,王丽侠,王素华,白鹏,吴传书.基于SSR标记的中国绿豆种质资源遗传多样性研究.中国农业科学,2013,46:4197-4209.Liu Y,Cheng X Z,Wang L X,Wang S H,Bai P,Wu C S.Genetic diversity research of mungbean germplasm resources by SSRmarkers in China.Sci Agric Sin,2013,46:4197-4209(in Chinese with English abstract).
[9]任红晓,程须珍,徐东旭,高运青,尚启兵.应用SSR标记分析中国北方名优绿豆的遗传多样性.植物遗传资源学报,2015,16:395-399.Ren H X,Cheng X Z,Xu D X,Gao Y Q,Shang Q B.Genetic diversity of traditional mungbean varieties in northern China by SSR markers.J Plant Genet Resour,2015,16:395-399(in Chinese with English abstract).
[10]赵丹,程须珍,王丽侠,王素华,马燕玲.绿豆遗传连锁图谱的整合.作物学报,2010,36:932-939.Zhao D,Cheng X Z,Wang L X,Wang S H,Ma Y L.Integration of mungbean(Vigna radiata)genetic linkage map.Acta Agron Sin,2010,36:932-939(in Chinese with English abstract).
[11]Isemura T,Kaga A,Tabata S,Somta P,Srinives P,Shimizu T,Jo U,Vaughan D A,Tomooka N.Construction of a genetic linkage map and genetic analysis of domestication related traits in mungbean(Vigna radiata).PLoS One,2012,7:e41304.
[12]王建花,张耀文,程须珍,王丽侠.绿豆分子遗传图谱构建及若干农艺性状的QTL定位分析.作物学报,2017,43:1096-1102.Wang J H,Zhang Y W,Cheng X Z,Wang L X.Construction of genetic map and identification of QTLs related to agronomic traits in mung bean.Acta Agron Sin,2017,43:1096-1102(in Chinese with English abstract).
[13]王丽侠,程须珍,王素华,刘长友,梁辉.小豆SSR引物在绿豆基因组中的通用性分析.作物学报,2009,35:816-820.Wang L X,Cheng X Z,Wang S H,Liu C Y,Liang H.Transferability of SSR from adzuki bean to mungbean.Acta Agron Sin,2009,35:816-820(in Chinese with English abstract).
[14]钟敏,程须珍,王丽侠,王素华,王小宝.绿豆基因组SSR引物在豇豆属作物中的通用性.作物学报,2012,38:223-230.Zhong M,Cheng X Z,Wang L X,Wang S H,Wang X B.Transferability of mungbean genomic-SSR markers in other vigna species.Acta Agron Sin,2012,38:223-230(in Chinese with English abstract).
[15]Kumar S V,Tan S G,Quah S C,Yusoff K.Isolation and characterization of seven tetranucleotide microsatellite loci in mungbean,Vigna radiata.Mol Ecol Notes,2002,2:293-295.
[16]Miyagi M,Humphry M,Ma Z Y,Lambrides C J,Bateson M,Liu C J.Construction of bacterial artificial chromosome libraries and their application in developing PCR-based markers closely linked to a major locus conditioning bruchid resistance in mung bean(Vigna radiata L.Wilczek).Theor Appl Genet,2004,110:151-156.
[17]Gwag J G,Chung J W,Chung H K,Lee J H,Ma K H.Characterization of new microsatellite markers in mungbean,Vigna radiata(L.).Mol Ecol Notes,2006,6:1132-1134.
[18]Somta P,Musch W,Kongsamai B,Chanprame S,Nakasatien S,Toojinda T,Sorajjapinun W,Seehalak W,Tragoonrung S,Srinives P.New microsatellite markers isolated from mungbean(Vigna radiata(L.)Wilczek).Mol Ecol Resour,2008,8:1155-1157.
[19]Seehalak W,Somta P,Sommanas W,Srinives P.Microsatellite markers for mungbean developed from sequence database.Mol Ecol Resour,2009,9:862-864.
[20]Tangphatsornruang S,Somta P,Uthaipaisanwong P,Chanprasert J,Sangsrakru D,Seehalak W,Sommanas W,Tragoonrung S,Srinives P.Characterization of microsatellites and gene contents from genome shotgun sequences of mungbean(Vigna radiata(L.)Wilczek).BMC Plant Biol,2009,9:137,doi:10.1186/1471-2229-9-137.
[21]Somta P,Seehalak W,Srinives P.Development,characterization and cross-species amplification of mungbean(Vigna radiata)genic microsatellite markers.Conserv Genet,2009,10:1939-1943.
[22]Wang L X,Elbaidouri M,Abernathy B,Chen H L,Wang S H,Lee S H,Jackson S A,Cheng X Z.Distribution and analysis of SSR in mung bean(Vigna radiata L.)genome based on an SSR-enriched library.Mol Breed,2015,35:25,doi:10.1007/s11032-015-0259-8.
[23]Chen H L,Wang L X,Wang S H,Liu C Y,Blair M W,Cheng X Z.Transcriptome sequencing of mung bean(Vigna radiate L.)genes and the identification of EST-SSR markers.PLoS One,2015,10:e0120273.
[24]Liu C Y,Fan B J,Cao Z M,Su Q Z,Wang Y,Zhang Z X,Wu J,Tian J.A deep sequencing analysis of transcriptomes and the development of EST-SSR markers in mungbean(Vigna radiata).JGenet,2016,95:527-535.
[25]孙子奎,陈永灿.一种基于磁珠富集法高通量开发基因组SSR标记的方法.中国专利,2014,ZL201310222359.9.Sun Z K,Chen Y C.A method of developing genome SSRmarkers based on magnetic bead enrichment for NGS,China patent,2014,ZL201310222359.9.
[26]Lindgreen S.AdapterRemoval:easy cleaning of next-generation sequencing reads.BMC Res Notes,2012,5:337,doi:10.1186/1756-0500-5-337.
[27]Mago?T,Salzberg S L.FLASH:fast length adjustment of short reads to improve genome assemblies.Bioinformatics,2011,27:2957-2963.
[28]Untergasser A,Cutcutache I,Koressaar T,Ye J,Faircloth B C,Remm M,Rozen S G.Primer3:new capabilities and interfaces.Nucl Acids Res,2012,40:e115.
[29]Krawczak M,Nikolaus S,von Eberstein H,Croucher P J,El Mokhtari N E,Schreiber S.PopGen:population-based recruitment of patients and controls for the analysis of complex genotype-phenotype relationships.Commun Genet,2006,9:55-61.
[30]Liu K,Muse S V.PowerMarker:An integrated analysis environment for genetic marker analysis.Bioinformatics,2005,21:2128-2129.
[31]吴传书.绿豆SSR标记的开发及高密度分子遗传连锁图谱的构建.甘肃农业大学硕士学位论文,甘肃兰州,2014.Wu C S.Development of SSR Markers and Construction of a Genetic Linkage Map in Mungbean(Vigan radiate L.).MS Thesis of Gansu Agricultural University,Lanzhou,Gansu,China,2014(in Chinese with English abstract).
[32]Gupta S K,Bansal R,Gopalakrishna T.Development and characterization of genic SSR markers for mungbean(Vigna radiata(L.)Wilczek).Euphytica,2014,195:245-258.
[33]Yang T,Bao S Y,Ford R,Jia T J,Guan J P,He Y H,Sun X L,Jiang J Y,Hao J J,Zhang X Y,Zong X X.High-throughput novel microsatellite marker of faba bean via next generation sequencing.BMC Genomics,2012,13:602,doi:10.1186/1471-2164-13-602.
[34]Gao L F,Tang J F,Li H W,Jia J Z.Analysis of microsatellites in major crops assessed by computational and experimental approaches.Mol Breed,2003,12:245-261.
[35]Nicot N,Chiquet V,Gandon B,Amilhat L,Legeai F,Leroy P,Bernard M,Sourdille P.Study of simple sequence repeat(SSR)markers from wheat expressed sequence tags(ESTs).Theor Appl Genet,2004,109:800-805.