基于名优谷子品种晋谷21全基因组重测序的分子标记开发
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摘要
小米因其营养丰富日益受到重视,而小米的品质是民众选择小米时最为关注的指标。晋谷21米质优异,但由于缺少基因组信息,严重阻碍了其优异米质形成机制的研究。本研究利用高通量测序技术,对晋谷21全基因组进行重测序,获得了14.95 Gb高质量测序数据。进一步将其与豫谷1号参考基因组比较,发掘了169 037个In Del位点和1 167 555个SNP位点,其中长度在13~50 bp之间适于琼脂糖凝胶电泳检测的In Del位点为14 578个。选择其中1个SNP位点和68个In Del位点验证,表明利用二代测序技术开发的In Del和SNP标记真实可靠。基于名优谷子晋谷21重测序数据开发的In Del和SNP分子标记具有通用性,可用于其他谷子、狗尾草和谷莠子等种质资源的相关研究。同时,开发了一个晋谷21特异的In Del标记2G5501976,利用该标记即可快速鉴定待测材料是否为晋谷21及其衍生品种。本研究初步揭示了晋谷21的基因组特征,不仅为深入解析其优异米质形成的分子机制奠定了基础,而且为相关分子标记辅助育种、遗传分析和基因克隆提供了分子标记资源。
Foxtail millet becomes more and more popular for its rich nutrients, and the grain quality is the key concern that consumers would consider when selecting millet brand. Jingu 21 is an elite cultivar with high edible quality. However, the lack of genomic information impedes studies on the molecular mechanisms of millet quality formation. Here, we re-sequenced the whole genome of Jingu 21 using high-throughput sequencing technology, and obtained 14.95 Gb high quality data. By comparing sequence of Jingu 21 with the reference genome of Yugu 1, we identified 169 037 In Dels and 1 167 555 SNPs. Of these In Dels, 14 578 could be detected easily by agarose gel electrophoresis. One SNP and 68 In Del markers were selected to verify the polymorphism between Jingu 21 and Yugu 1, showing that the In Del and SNP markers developed by using next generation sequencing technology were reliable. Although the In Del and SNP markers were generated based on genome re-sequencing data of the elite cultivar Jingu 21, they could also be used for research on other foxtail millet, green foxtail, and giant foxtail. Moreover, a specific In Del marker 2 G5501976 for Jingu 21 was developed, which could be used to identify Jingu 21 and its derivative varieties. Taken all together, the genomic characterization of Jingu 21 not only lays a foundation for elucidating the molecular mechanisms of high quality formation, but also provides a large number of molecular markers for marker-assisted selection of high quality millet, genetic analysis and map-based cloning.
Foxtail millet becomes more and more popular for its rich nutrients, and the grain quality is the key concern that consumers would consider when selecting millet brand. Jingu 21 is an elite cultivar with high edible quality. However, the lack of genomic information impedes studies on the molecular mechanisms of millet quality formation. Here, we re-sequenced the whole genome of Jingu 21 using high-throughput sequencing technology, and obtained 14.95 Gb high quality data. By comparing sequence of Jingu 21 with the reference genome of Yugu 1, we identified 169 037 In Dels and 1 167 555 SNPs. Of these In Dels, 14 578 could be detected easily by agarose gel electrophoresis. One SNP and 68 In Del markers were selected to verify the polymorphism between Jingu 21 and Yugu 1, showing that the In Del and SNP markers developed by using next generation sequencing technology were reliable. Although the In Del and SNP markers were generated based on genome re-sequencing data of the elite cultivar Jingu 21, they could also be used for research on other foxtail millet, green foxtail, and giant foxtail. Moreover, a specific In Del marker 2 G5501976 for Jingu 21 was developed, which could be used to identify Jingu 21 and its derivative varieties. Taken all together, the genomic characterization of Jingu 21 not only lays a foundation for elucidating the molecular mechanisms of high quality formation, but also provides a large number of molecular markers for marker-assisted selection of high quality millet, genetic analysis and map-based cloning.
引文
[1]张超,张晖,李冀新.小米的营养以及应用研究进展.中国粮油学报,2007,22(1):51-55Zhang C,Zhang H,Li J X.Advance of millet research on nutrition and application.J Chin Cereals Oils Assoc,2007,22(1):51-55(in Chinese with English abstract)
[2]刘敏轩,陆平.中国谷子育成品种维生素E含量分布规律及其与主要农艺性状和类胡萝卜素的相关性分析.作物学报,2013,39:398-408Liu M X,Lu P.Distribution of vitamin e content and its correlation with agronomic traits and carotenoids content in foxtail millet varieties in China.Acta Agron Sin,2013,39:398-408(in Chinese with English abstract)
[3]Sharma N,Niranjan K.Foxtail millet:properties,processing,health benefits,and uses.Food Rev Int,2017,34:329-363
[4]杨延兵,管延安,秦岭,石慧,王海莲,张华文.不同地区谷子小米黄色素含量与外观品质研究.中国粮油学报,2012,27(1):14-19Yang Y B,Guan Y A,Qin L,Shi H,Wang H L,Zhang H W.The studies on yellow pigment content and appearance quality of millet from different regions.J Chin Cereals Oils Assoc,2012,27(1):14-19(in Chinese with English abstract)
[5]王润奇,高俊华,关中波,毛丽萍.谷子几种农艺性状基因染色体定位及连锁关系的初步研究.作物学报,2007,33:9-14Wang R Q,Gao J H,Guan Z B,Mao L P.Chromosome location and linkage analysis of a few agronomical important traits in foxtail millet.Acta Agron Sin,2007,33:9-14(in Chinese with English abstract)
[6]张耀元,路阳,张彬,禾璐,刘龙龙,王兴春,李红英,韩渊怀.谷子类胡萝卜素生物合成途径Si LCYB基因与米色形成的关系.分子植物育种,2016,14:1341-1351Zhang Y Y,Lu Y,Zhang B,He L,Liu L L,Wang X C,Li H Y,Han Y H.The relationship between the gene Si LCYB related to carotenoid synthesis and the colored formation of foxtail millet.Mol Plant Breed,2016,14:1341-1351(in Chinese with English Abstract)
[7]刘辉,张敏.不同品种小米的直链淀粉含量与快速黏度分析仪谱特征值关系研究.食品科学,2010,31:31-33Liu H,Zhang M.Relationships between amylose content and gelatinization characteristics of different varieties of millet.Food Sci,2010,31:31-33(in Chinese with English abstract)
[8]张昌泉,赵冬生,李钱峰,顾铭洪,刘巧泉.稻米品质性状基因的克隆与功能研究进展.中国农业科学,2016,49:4267-4283Zhang C Q,Zhao D S,Li Q F,Gu M H,Liu Q Q.Progresses in research on cloning and functional analysis of key genes involving in rice grain quality.Sci Agric Sin.2016,49:4267-4283(in Chinese with English abstract)
[9]He L,Zhang B,Wang X,Li H Y,Han Y H.Foxtail millet:nutritional and eating quality,and prospects for genetic improvement.Front Agric Sci Eng,2015,2:124-133
[10]Bennetzen J L,Schmutz J,Wang H,Percifield R,Hawkins J,Pontaroli A C,Estep M,Feng L,Vaughn J N,Grimwood J,Jenkins J,Barry K,Lindquist E,Hellsten U,Deshpande S,Wang X,Wu X,Mitros T,Triplett J,Yang X,Ye C Y,Mauro-Herrera M,Wang L,Li P,Sharma M,Sharma R,Ronald P C,Panaud O,Kellogg E A,Brutnell T P,Doust A N,Tuskan G A,Rokhsar D,Devos K M.Reference genome sequence of the model plant Setaria.Nat Biotechnol,2012,30:555-561
[11]Quan Z W,Cheng S F,Xu X,Pan S K,Xie M,Zeng P,Yue Z,Wang W L,Tao Y,Bian C L,Han C L,Xia Q J,Peng X H,Cao R,Yang X H,Zhan D L,Hu J C,Zhang Y X,Li H N,Li H,Li N,Wang J Y,Wang C C,Wang R Y,Guo T,Cai Y J,Liu C Z,Xiang H T,Shi Q H,Huang P,Chen Q C,Li Y R,Wang J,Zhao Z H,Wang J.Genome sequence of foxtail millet(Setaria italica)provides insights into grass evolution and biofuel potential.Nat Biotechnol,2012,30:549-554
[12]Zhang S,Tang C,Zhao Q,Li J,Yang L,Qie L,Fan X,Li L,Zhang N,Zhao M,Liu X,Chai Y,Zhang X,Wang H,Li Y,Li W,Zhi H,Jia G,Diao X.Development of highly polymorphic simple sequence repeat markers using genome-wide microsatellite variant analysis in Foxtail millet[Setaria italica(L.)P.Beauv].BMCGenomics,2014,15:78
[13]Fang X,Dong K,Wang X,Liu T,He J,Ren R,Zhang L,Liu R,Liu X,Li M,Huang M,Zhang Z,Yang T.A high density genetic map and QTL for agronomic and yield traits in Foxtail millet[Setaria italica(L.)P.Beauv].BMC Genomics,2016,17:336
[14]Venkata S B,Muthamilarasan M,Misra G,Prasad M.Fm MDb:a versatile database of foxtail millet markers for millets and bioenergy grasses research.PLo S One,2013,8:e71418
[15]Yadav C B,Bonthala V S,Muthamilarasan M,Pandey G,Khan Y,Prasad M.Genome-wide development of transposable elementsbased markers in foxtail millet and construction of an integrated database.DNA Res,2015,22:79-90
[16]Bai H,Cao Y,Quan J,Dong L,Li Z,Zhu Y,Zhu L,Dong Z,Li D.Identifying the genome-wide sequence variations and developing new molecular markers for genetics research by re-sequencing a Landrace cultivar of foxtail millet.PLo S One,2013,8:e73514
[17]Wang J,Wang Z,Du X,Yang H,Han F,Han Y,Yuan F,Zhang L,Peng S,Guo E.A high-density genetic map and QTL analysis of agronomic traits in foxtail millet[Setaria italica(L.)P.Beauv.]using RAD-seq.PLo S One,2017,12:e0179717
[18]Jia G Q,Huang X H,Zhi H,Zhao Y,Zhao Q,Li W J,Chai Y,Yang L F,Liu K Y,Lu H Y,Zhu C R,Lu Y Q,Zhou C C,Fan D,Weng Q J,Guo Y L,Huang T,Zhang L,Lu T T,Feng Q,Hao H F,Liu H K,Lu P,Zhang N,Li Y H,Guo E H,Wang S J,Wang S Y,Liu J R,Zhang W F,Chen G Q,Zhang B J,Li W,Wang Y F,Li HQ,Zhao B H,Li J Y,Diao X M,Han B.A haplotype map of genomic variations and genome-wide association studies of agronomic traits in foxtail millet(Setaria italica).Nat Genet,2013,45:957-961
[19]陈瑛,卫天业,景小兰,张象贤.晋谷21号的选育及推广前景.山西农业科学,1992,(12):7-8Chen Y,Wei T Y,Jing X L,Zhang X X.Breeding and popularization prospect of Jingu 21.J Shanxi Agric Sci,1992,(12):7-8(in Chinese without English abstract)
[20]穆彩琴,张瑞娟,屈聪玲,韩渊怀,王兴春,杨致荣.基于RNA-Seq技术的谷子新基因发掘及基因结构优化.植物生理学报,2016,52:1066-1072Mu C Q,Zhang R J,Qu C L,Han Y H,Wang X C,Yang Z R.Identifi cation of novel genes and optimization of annotated genes in foxtail millet by RNA-Seq technology.Plant Physiol J,2016,52:1066-1072(in Chinese with English abstract)
[21]Stewart C N J,Via L E.A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications.BioTechniques,1993,14:748-750
[22]Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics,2009,25:1754-1760
[23]Mc Kenna A,Hanna M,Banks E,Sivachenko A,Cibulskis K,Kernytsky A,Garimella K,Altshuler D,Gabriel S,Daly M,DePristo M A.The Genome Analysis Toolkit:a Map Reduce framework for analyzing next-generation DNA sequencing data.Genome Res,2010,20:1297-1303
[24]Neff M M,Turk E,Kalishman M.Web-based primer design for single nucleotide polymorphism analysis.Trends Genet,2002,18:613-615
[25]Cingolani P,Platts A,Wang L,Coon M,Nguyen T,Wang L,Land S J,Lu X,Ruden D M.A program for annotating and predicting the effects of single nucleotide polymorphisms,Snp Eff:SNPs in the genome of Drosophila melanogaster strain w1118;iso-2;iso-3.Fly(Austin),2012,6:80-92
[26]Liu B,Wang Y,Zhai W,Deng J,Wang H,Cui Y,Cheng F,Wang X,Wu J.Development of In Del markers for Brassica rapa based on whole-genome re-sequencing.Theor Appl Genet,2013,126:231-239
[27]张体付,戚维聪,顾闽峰,张晓林,李坦,赵涵.藜麦EST-SSR的开发及通用性分析.作物学报,2016,42:492-500Zhang T F,Qi W C,Gu M F,Zhang X L,Li T,Zhao H.Exploration and transferability evaluation of EST-SSRs in quinoa.Acta Agron Sin,2016,42:492-500(in Chinese with English abstract)
[28]Ul Haq S,Kumar P,Singh R K,Verma K S,Bhatt R,Sharma M,Kachhwaha S,Kothari S L.Assessment of functional EST-SSRmarkers(sugarcane)in cross-species transferability,genetic diversity among poaceae plants,and bulk segregation analysis.Genet Res Int,2016,doi:10.1155/2016/7052323:7052323
[2]刘敏轩,陆平.中国谷子育成品种维生素E含量分布规律及其与主要农艺性状和类胡萝卜素的相关性分析.作物学报,2013,39:398-408Liu M X,Lu P.Distribution of vitamin e content and its correlation with agronomic traits and carotenoids content in foxtail millet varieties in China.Acta Agron Sin,2013,39:398-408(in Chinese with English abstract)
[3]Sharma N,Niranjan K.Foxtail millet:properties,processing,health benefits,and uses.Food Rev Int,2017,34:329-363
[4]杨延兵,管延安,秦岭,石慧,王海莲,张华文.不同地区谷子小米黄色素含量与外观品质研究.中国粮油学报,2012,27(1):14-19Yang Y B,Guan Y A,Qin L,Shi H,Wang H L,Zhang H W.The studies on yellow pigment content and appearance quality of millet from different regions.J Chin Cereals Oils Assoc,2012,27(1):14-19(in Chinese with English abstract)
[5]王润奇,高俊华,关中波,毛丽萍.谷子几种农艺性状基因染色体定位及连锁关系的初步研究.作物学报,2007,33:9-14Wang R Q,Gao J H,Guan Z B,Mao L P.Chromosome location and linkage analysis of a few agronomical important traits in foxtail millet.Acta Agron Sin,2007,33:9-14(in Chinese with English abstract)
[6]张耀元,路阳,张彬,禾璐,刘龙龙,王兴春,李红英,韩渊怀.谷子类胡萝卜素生物合成途径Si LCYB基因与米色形成的关系.分子植物育种,2016,14:1341-1351Zhang Y Y,Lu Y,Zhang B,He L,Liu L L,Wang X C,Li H Y,Han Y H.The relationship between the gene Si LCYB related to carotenoid synthesis and the colored formation of foxtail millet.Mol Plant Breed,2016,14:1341-1351(in Chinese with English Abstract)
[7]刘辉,张敏.不同品种小米的直链淀粉含量与快速黏度分析仪谱特征值关系研究.食品科学,2010,31:31-33Liu H,Zhang M.Relationships between amylose content and gelatinization characteristics of different varieties of millet.Food Sci,2010,31:31-33(in Chinese with English abstract)
[8]张昌泉,赵冬生,李钱峰,顾铭洪,刘巧泉.稻米品质性状基因的克隆与功能研究进展.中国农业科学,2016,49:4267-4283Zhang C Q,Zhao D S,Li Q F,Gu M H,Liu Q Q.Progresses in research on cloning and functional analysis of key genes involving in rice grain quality.Sci Agric Sin.2016,49:4267-4283(in Chinese with English abstract)
[9]He L,Zhang B,Wang X,Li H Y,Han Y H.Foxtail millet:nutritional and eating quality,and prospects for genetic improvement.Front Agric Sci Eng,2015,2:124-133
[10]Bennetzen J L,Schmutz J,Wang H,Percifield R,Hawkins J,Pontaroli A C,Estep M,Feng L,Vaughn J N,Grimwood J,Jenkins J,Barry K,Lindquist E,Hellsten U,Deshpande S,Wang X,Wu X,Mitros T,Triplett J,Yang X,Ye C Y,Mauro-Herrera M,Wang L,Li P,Sharma M,Sharma R,Ronald P C,Panaud O,Kellogg E A,Brutnell T P,Doust A N,Tuskan G A,Rokhsar D,Devos K M.Reference genome sequence of the model plant Setaria.Nat Biotechnol,2012,30:555-561
[11]Quan Z W,Cheng S F,Xu X,Pan S K,Xie M,Zeng P,Yue Z,Wang W L,Tao Y,Bian C L,Han C L,Xia Q J,Peng X H,Cao R,Yang X H,Zhan D L,Hu J C,Zhang Y X,Li H N,Li H,Li N,Wang J Y,Wang C C,Wang R Y,Guo T,Cai Y J,Liu C Z,Xiang H T,Shi Q H,Huang P,Chen Q C,Li Y R,Wang J,Zhao Z H,Wang J.Genome sequence of foxtail millet(Setaria italica)provides insights into grass evolution and biofuel potential.Nat Biotechnol,2012,30:549-554
[12]Zhang S,Tang C,Zhao Q,Li J,Yang L,Qie L,Fan X,Li L,Zhang N,Zhao M,Liu X,Chai Y,Zhang X,Wang H,Li Y,Li W,Zhi H,Jia G,Diao X.Development of highly polymorphic simple sequence repeat markers using genome-wide microsatellite variant analysis in Foxtail millet[Setaria italica(L.)P.Beauv].BMCGenomics,2014,15:78
[13]Fang X,Dong K,Wang X,Liu T,He J,Ren R,Zhang L,Liu R,Liu X,Li M,Huang M,Zhang Z,Yang T.A high density genetic map and QTL for agronomic and yield traits in Foxtail millet[Setaria italica(L.)P.Beauv].BMC Genomics,2016,17:336
[14]Venkata S B,Muthamilarasan M,Misra G,Prasad M.Fm MDb:a versatile database of foxtail millet markers for millets and bioenergy grasses research.PLo S One,2013,8:e71418
[15]Yadav C B,Bonthala V S,Muthamilarasan M,Pandey G,Khan Y,Prasad M.Genome-wide development of transposable elementsbased markers in foxtail millet and construction of an integrated database.DNA Res,2015,22:79-90
[16]Bai H,Cao Y,Quan J,Dong L,Li Z,Zhu Y,Zhu L,Dong Z,Li D.Identifying the genome-wide sequence variations and developing new molecular markers for genetics research by re-sequencing a Landrace cultivar of foxtail millet.PLo S One,2013,8:e73514
[17]Wang J,Wang Z,Du X,Yang H,Han F,Han Y,Yuan F,Zhang L,Peng S,Guo E.A high-density genetic map and QTL analysis of agronomic traits in foxtail millet[Setaria italica(L.)P.Beauv.]using RAD-seq.PLo S One,2017,12:e0179717
[18]Jia G Q,Huang X H,Zhi H,Zhao Y,Zhao Q,Li W J,Chai Y,Yang L F,Liu K Y,Lu H Y,Zhu C R,Lu Y Q,Zhou C C,Fan D,Weng Q J,Guo Y L,Huang T,Zhang L,Lu T T,Feng Q,Hao H F,Liu H K,Lu P,Zhang N,Li Y H,Guo E H,Wang S J,Wang S Y,Liu J R,Zhang W F,Chen G Q,Zhang B J,Li W,Wang Y F,Li HQ,Zhao B H,Li J Y,Diao X M,Han B.A haplotype map of genomic variations and genome-wide association studies of agronomic traits in foxtail millet(Setaria italica).Nat Genet,2013,45:957-961
[19]陈瑛,卫天业,景小兰,张象贤.晋谷21号的选育及推广前景.山西农业科学,1992,(12):7-8Chen Y,Wei T Y,Jing X L,Zhang X X.Breeding and popularization prospect of Jingu 21.J Shanxi Agric Sci,1992,(12):7-8(in Chinese without English abstract)
[20]穆彩琴,张瑞娟,屈聪玲,韩渊怀,王兴春,杨致荣.基于RNA-Seq技术的谷子新基因发掘及基因结构优化.植物生理学报,2016,52:1066-1072Mu C Q,Zhang R J,Qu C L,Han Y H,Wang X C,Yang Z R.Identifi cation of novel genes and optimization of annotated genes in foxtail millet by RNA-Seq technology.Plant Physiol J,2016,52:1066-1072(in Chinese with English abstract)
[21]Stewart C N J,Via L E.A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications.BioTechniques,1993,14:748-750
[22]Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics,2009,25:1754-1760
[23]Mc Kenna A,Hanna M,Banks E,Sivachenko A,Cibulskis K,Kernytsky A,Garimella K,Altshuler D,Gabriel S,Daly M,DePristo M A.The Genome Analysis Toolkit:a Map Reduce framework for analyzing next-generation DNA sequencing data.Genome Res,2010,20:1297-1303
[24]Neff M M,Turk E,Kalishman M.Web-based primer design for single nucleotide polymorphism analysis.Trends Genet,2002,18:613-615
[25]Cingolani P,Platts A,Wang L,Coon M,Nguyen T,Wang L,Land S J,Lu X,Ruden D M.A program for annotating and predicting the effects of single nucleotide polymorphisms,Snp Eff:SNPs in the genome of Drosophila melanogaster strain w1118;iso-2;iso-3.Fly(Austin),2012,6:80-92
[26]Liu B,Wang Y,Zhai W,Deng J,Wang H,Cui Y,Cheng F,Wang X,Wu J.Development of In Del markers for Brassica rapa based on whole-genome re-sequencing.Theor Appl Genet,2013,126:231-239
[27]张体付,戚维聪,顾闽峰,张晓林,李坦,赵涵.藜麦EST-SSR的开发及通用性分析.作物学报,2016,42:492-500Zhang T F,Qi W C,Gu M F,Zhang X L,Li T,Zhao H.Exploration and transferability evaluation of EST-SSRs in quinoa.Acta Agron Sin,2016,42:492-500(in Chinese with English abstract)
[28]Ul Haq S,Kumar P,Singh R K,Verma K S,Bhatt R,Sharma M,Kachhwaha S,Kothari S L.Assessment of functional EST-SSRmarkers(sugarcane)in cross-species transferability,genetic diversity among poaceae plants,and bulk segregation analysis.Genet Res Int,2016,doi:10.1155/2016/7052323:7052323