普通烟草及其祖先种基因组SSR位点信息分析
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- 英文论文题名：Analysis of SSR Loci in N. tabacum Genome and Its Two Ancestral Species Genome
- 论文作者：童治军 ; 焦芳婵 ; 肖炳光
- 英文论文作者：TONG Zhi-jun ; JIAO Fang-chan ; XIAO Bing-guang ; Key Laboratory of Tobacco Biotechnological Breeding ; National Tobacco Genetic Engineering Research Center ; Yunnan Academy of Tobacco Agricultural Sciences
- 年：2016
- 作者机构：云南省烟草农业科学研究院烟草行业烟草生物技术育种重点实验室国家烟草基因工程研究中心;
- 论文关键词：绒毛状烟草 ; 林烟草 ; 普通烟草 ; 基因组 ; SSR位点
- 英文论文关键词：N.tomentosiformis ; N.sylvestris ; N.tabacum ; genome ; SSR loci
- 会议召开时间：2016-12-01
- 会议录名称：中国烟草学会2016年度优秀论文汇编——烟草农业主题
- 语种：中文
- 分类号：S572
- 学会代码：ZGYG
- 学会名称：中国烟草学会
- 页数：16
- 文件大小：664k
- 原文格式：D
摘要
【目的】通过对普通烟草(Nicotiana tabacum L.;2n=24Ⅱ=48 TTSS)及其两个祖先种-绒毛状烟草(Nicotiana tomentosoformis L.;2n=12Ⅱ=24 TT)和林烟草(Nicotiana sylvestris L.;2n=12Ⅱ=24 SS)基因组SSR位点信息的统计分析,为烟草属植物应用SSR分子标记进行大规模的遗传分析提供基础。【方法】利用公共数据库NCBI(National Center for Biotechnology Information)中普通烟草基因组、绒毛状烟草基因组和林烟草基因组数据,分析其各自SSR位点分布特征,并根据SSR位点的搜索结果,合成了150对引物(每个基因组随机合成了50对),利用分别属于5个不同烟草组的8份烟草材料对所合成的引物进行应用性验证和通用性检测。【结果】在全长分别为1.64Gb、2.05Gb和3.60Gb的绒毛状烟草基因组、林烟草基因组和普通烟草基因组中分别获得了218081、263478和397432个SSR位点,SSR间的平均距离分别为7.52kb、7.78kb和9.06kb。在SSR位点的分布区域上,绝大部分的SSR位点分布在内含子和UTR(尤其是5′-UTR)区域;在SSR基序类型上,主要集中在二、三碱基基序且二者占基因组内SSR位点总数目的 66%以上,其中,二碱基基序类型丰度最高;在SSR基序结构上,基因组中出现频率及数量最高的是含有A(T)n的基序结构;在SSR基序的重复次数上,除单碱基基序类型外,重复次数多在3-10次之间。利用8份烟草材料验证所合成的150对引物,所有合成的引物均能扩增获得目标片段,其中有36对引物存在扩增多态性。【结论】绒毛状烟草、林烟草和普通烟草基因组内SSR分布呈现一定的规律性,初步验证了SSR位点在亲缘关系相对较近的烟草种间具有高度保守性和通用性,基于此三份烟草基因组数据开发SSR引物用于后续的相关遗传研究具有可行性。
【Objective】In order to lay a foundation for the genetic analysis of tobacco species based on the SSR markers, the SSR loci from N. tomentosiformis(2n=12Ⅱ=24; TT), N. sylvestris(2n=12Ⅱ=24; SS) and N. tabacum(2n=24Ⅱ=48; TTSS) genomes were analyzed.【Method】The genomic sequence data of N. tomentosiformis, N. sylvestris and N. tabacum was downloaded from the National Center for Biotechnology Information(NCBI) website and the SSRIT and TRF soft-wares were used to analyze the distribution of genome SSR loci. One hundred and fifty primer pairs, of which 50 primers from each genome according to the searching results of the SSR loci, were randomly synthesized and the 8 tobacco materials which belong to 5 different species of the genus Nicotiana were utilized to examine the application and transferability of designed primers.【Result】A total of 218081, 263478 and 397432 SSR loci were mined out in the 1.64 Gb of N. tomentosiformis genomic sequence, 2.05 Gb of N. sylvestris genomic sequence and 3.60 Gb of N. tabacum genomic sequence, and the average distance between SSR loci was approximately 7.52 kb, 7.78 kb and 9.06 kb, respectively. Almost all the SSR loci were distributed in intron domains and UTR domains(especially the 5'-UTR). Di-nucleotide and tri-nucleotide repeats, totally accounting for more than 66%, were the main motif types and the Di-nucleotide was the most abundance in all SSRs. The highest frequency and the most abundance of SSR motifs configuration were A(T)n. Except the mono-nucleotide motif type, the repeat number of the motif types was between 3 and 10. Three hundred SSR primer pairs synthesized were subjected to PCR using 8 tobacco accessions as materials and the results showed that all the primer pairs could produce clear, stable and target bands, of which 36 SSR primers were polymorphic. 【Conclusion】Some distribution characterization of SSRs in N. tomentosiformis, N. sylvestris and N. tabacum genome were found. The results indicated that SSR loci in the closer genetic relationship among tobacco species were highly conserved and could be used in the following research. It is meaningful to develop SSR primer pairs from the three genomic data for the application of relative genetic analysis.
【Objective】In order to lay a foundation for the genetic analysis of tobacco species based on the SSR markers, the SSR loci from N. tomentosiformis(2n=12Ⅱ=24; TT), N. sylvestris(2n=12Ⅱ=24; SS) and N. tabacum(2n=24Ⅱ=48; TTSS) genomes were analyzed.【Method】The genomic sequence data of N. tomentosiformis, N. sylvestris and N. tabacum was downloaded from the National Center for Biotechnology Information(NCBI) website and the SSRIT and TRF soft-wares were used to analyze the distribution of genome SSR loci. One hundred and fifty primer pairs, of which 50 primers from each genome according to the searching results of the SSR loci, were randomly synthesized and the 8 tobacco materials which belong to 5 different species of the genus Nicotiana were utilized to examine the application and transferability of designed primers.【Result】A total of 218081, 263478 and 397432 SSR loci were mined out in the 1.64 Gb of N. tomentosiformis genomic sequence, 2.05 Gb of N. sylvestris genomic sequence and 3.60 Gb of N. tabacum genomic sequence, and the average distance between SSR loci was approximately 7.52 kb, 7.78 kb and 9.06 kb, respectively. Almost all the SSR loci were distributed in intron domains and UTR domains(especially the 5'-UTR). Di-nucleotide and tri-nucleotide repeats, totally accounting for more than 66%, were the main motif types and the Di-nucleotide was the most abundance in all SSRs. The highest frequency and the most abundance of SSR motifs configuration were A(T)n. Except the mono-nucleotide motif type, the repeat number of the motif types was between 3 and 10. Three hundred SSR primer pairs synthesized were subjected to PCR using 8 tobacco accessions as materials and the results showed that all the primer pairs could produce clear, stable and target bands, of which 36 SSR primers were polymorphic. 【Conclusion】Some distribution characterization of SSRs in N. tomentosiformis, N. sylvestris and N. tabacum genome were found. The results indicated that SSR loci in the closer genetic relationship among tobacco species were highly conserved and could be used in the following research. It is meaningful to develop SSR primer pairs from the three genomic data for the application of relative genetic analysis.
引文
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[28]Moon HS,Nifong JM,Nicholson JS,Heinemann A,Lion K,van der Hoeven R,Hayes AJ,Lewis RS.Microsatellite-based Analysis of Tobacco(Nicotiana tabacum L.)Genetic Resources.Crop Science,2009,49:2149-2159.
[29]SANGUINETTI C J,DIAS N E,SIMPSON A J.Rapid silver staining and recovery of PCR products separated on polyacrylamide gels.Biotechniques,1994,17:915-919.
[30]Lawson M J,Zhang L q.Distinct patterns of SSR distribution in the Arabidopsis thaliana and rice genomes.Genome Biology,2006,7(2):R14.1-R14.11.
[31]Gibum Y,Je M L,Sanghyeob L,et al.Exploitation of pepper EST–SSRs and an SSR-based linkage map.Theoretical and Applied Genetics,2006,114:113-130.
[32]MORGANTE M,OLIVIERI A M.PCR-amplified micro satellites as markers in plant genetics.Plant J,1993,3:175-182.
[33]Tang J F,Samantha J B,Jeanne M J,et al.Large-scale identification of polymorphic micro satellites using an in silico approach.BMC Bioinformatics,2008,9:374-386.
[34]蔡斌,李成慧,姚泉洪,周军,陶建敏,章镇.葡萄全基因组SSR分析和数据库构建.南京农业大学学报,2009,32(4):28-32.Cai B,Li C H,Yao Q H,Zhou J,Tao J M,Zhang Z.Analysis of SSRs in grape genome and development of SSR database.Journal of Nanjing Agricultural University,2009,32(4):28-32.(in Chinese)
[35]陆景标,李杰勤,卢杰,詹秋文.高粱非编码区SSR引物设计以及e-PCR的验证.种子,2010,29(9):1-6,12.Lu J B,Li J Q,Lu J,Zhan Q W.Design of SSR primers and verification of e-PCR in non-coding regions of sorghum genome.Seed,2010,29(9):1-6,12.(in Chinese)
[36]Tóth G,Gáspári Z,Jurka J.Microsatellites in different eukaryotic genomes:survey and analysis.Genome Research,2000,10(7):967-981.
[37]高亚梅,韩毅强,汤辉,孙东梅,王彦杰,王伟东.根瘤菌基因组内简单重复序列的分析.中国农业科学,2008,41(10):2992-2998.Gao Y M,Han Y Q,Tang H,Sun D M,Wang Y J,Wang W D.Analysis of simple sequence repeats in rhizobium genome.Scientia Agricultura Sinica,2008,41(10):2992-2998.(in Chinese)
[38]Sia E A,Kokoska R J,Dominska M,Greenwell P,Petes T D.Microsatellite instability in yeast:dependence on repeat unit size and DNA mismatch repair genes.Molecular and Cellular Biology,1997,17(5):2851-2858.
[39]Harry B,Schl?tterer C.Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times,which cause their genome-wide underrepresentation.Genetics,2000,155:1213-1220.
[40]Gibum Y,Je M L,Sanghyeob L,et al.Exploitation of pepper EST–SSRs and an SSR-based linkage map[J].Theor Appl Genet,2006,114:113-130.
[41]Lee S B,Kaittanis C,Jansen R K,Hosterler J B,Tallon L J,Town C D,Daniell H.The complete chloroplast genome sequence of Gossypium hisutum:organization and phylogenetic relationships to other angiosperms.BMC Genomics,2006,7:61-72.
[42]尚明照,刘芳,华金平,王坤波.陆地棉叶绿体基因组密码子使用偏性的分析.中国农业科学,2011,44(2):245-253.Shang M Z,Liu F,Hua J P,Wang K B.Analysis on codon usage of chloroplast genome of Gossypium hirsutum.Scientia Agricultura Sinica,2011,44(2):245-253.(in Chinese)
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[2]Gerstel DU.Segregation in new allopolyploids of Nicotiana.I.Comparison of 6×(N.tabacum×tomentosiformis)and 6×(N.tabacum×otophora).Genetics,1960,45:1723-1734.
[3]Gerstel DU.Segregation in new allopolyploids of Nicotiana.II.Discordant ratios from individual loci in 6×(N.tabacum×N.sylvestris).Genetics,1963.48:677-689.
[4]Sierro N,Battey J N,Ouadi S,Bakaher N,Bovet L,Willig A,Goepfert S,Peitsch M C,Ivanov N V.The tobacco genome sequence and its comparison with those of tomato and potato.Nature Communications,2014,5:3833,DOI:10.1038/ncomms4833.
[5]Wang Z,Weber J L,Zhong G,Tanksley S D.Survey of plant short tandem DNA repeats.Theoretical and Applied Genetics,1994,88:1-6.
[6]Richards R I,Sutherland G R.Dynamic mutations:a new class of mutation causing human disease.Cell,1992,70(5):709-712.
[7]Levinson G,Gutman G A.Slipped-strand mispairing:a major mechanism for DNA sequence evolution.Molecular Biology and Evolution,1987,4(3):203-221.
[8]Yu J K,Rota M L,Kantety R V,et al.EST derived SSR markers for comparative mapping in wheat and rice.Mol Gen Genomics,2004,271:742-751.
[9]Eujayl I,Sorrells M E,Baum M,et al.Isolation of EST-derived micro satellite markers for genotyping the A and B genomes of wheat.Theor Appl Genet,2002,104:399-407.
[10]Kantety R V,Rota M L,Matthews D E,et al.Data mining for simple sequence repeats in expressed sequence tags from barley,maize,rice,sorghum and wheat.Plant Mol Biol,2002,48:501-510.
[11]Chen H M,Li L Z,Wei X Y,et al.Development,chromosome location and genetic mapping of EST-SSR markers in wheat.Chinese Science Bulletin,2005,50(20):2328-2336.
[12]Li B,Xia Q,Lu C,Zhou Z.Analysis on frequency and density of microsatellites in coding sequences of eudaryotic genomes.Genomics Proteomics,2004,2(1):24-31.
[13]Chen C,Zhou P,Choi Y A,Choi Y A.Mining and characterizing microsatellites from citrus EST.Theoretical and Applied Genetics,2006,112(7):1248-1257.
[14]Bindler G,der Hoeven V R,Gunduz I,Plieske J,Ganal M,Rossi L,Gadani F,and Donini P.A microsatellite marker based linkage map of tobacco.Theoretical and Applied Genetics,2007,114:341-349.
[15]Bindler G,Plieske J,Bakaher N,Gunduz I,Ivanov N,der Hoeven V R,Ganal M,and Donini P.A high density genetic map of tobacco(Nicotiana tabacum L.)obtained from large scale microsatellite marker development.Theoretical and Applied Genetics,2011,123:219-230.
[16]Tong Z J,Yang Z M,Chen X J,Jiao F C,Li X Y,Wu X F,Gao Y L,Xiao B G and Wu W R.Large-scale development of microsatellite markers in Nicotiana tabacum and construction of a genetic map of flue-cured tobacco.Plant Breeding,2012,131(5):674-680.
[17]Tong Z J,Jiao T L,Wang F Q,Li M Y,Len X D,Gao Y L,Li Y P,Xiao B G and Wu W R.Mapping of quantitative trait loci conferring resistance to brown spot in flue-cured tobacco(Nicotiana tabacum L.).Plant Breeding,2012,131(2):335-339.
[18]Vontimitta Vijay and Lewis R S.Mapping of quantitative trait loci affecting resistance to Phytophtora nicotianae in tobacco(Nicotiana tabacum L.)line Beinhart-100.Mol Breeding,2012,29:89-98.
[19]Johnson E S,Wolff M F,Wernsman E A and Rufty R C.Marker-assisted selection for resistance to black shank disease in tobacco.Plant Dis,2002,86:1303-1309.
[20]Moon H S,Nicholson J S and Lewis R S.Use of transferable Nicotiana tabacum L.microsatellite markers for investigating genetic diversity in the genus Nicotiana.Genome,2008,51:547-559
[21]Smith JSC,Simth OS.An evaluation of the utility of SSR loci a molecular marker in maize(Zea mays L.):Comparison with data from RFLPs and pedigree.Theoretical and Applied Genetics,1997,95:163-173.
[22]Rossi L,Bindler G,Pijnenburg H,Isaac P G,Giraud-Henri I,Mahe M,Orvain C,Gadani F.Potential of molecular marker analysis for variety identification in processed tobacco.Plant Varieties Seeds,2001,14:89-101.
[23]Sarala K,Rao RVS.Genetic diversity in Indian FCV and burley tobacco cultivars.Journal of Genetics,2008,87(2):159-163.
[24]Bai D,Reeleder R,Brandle JE.Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco.Theoretical and Applied Genetics,1995,91:1184-1189.
[25]Ren N,Timko MP.AFLP analysis of genetic polymorphism and evolutionary relationships among cultivated and wild Nicotiana species.Genome,2001,44:559-571.
[26]Rossi L,Bindler G,Pijnenburg H,Isaac P G,Giraud-Henri I,Mahe M,Orvain C,Gadani F.Potential of molecular marker analysis for variety identification in processed tobacco.Plant Varieties Seeds,2001,14:89-101.
[27]Moon HS,Nicholson JS,Heineman A,Lion K,van der Hoeven R,Hayes AJ,Lewis RS.Changes in Genetic Diversity of U.S.Flue-Cured Tobacco Germplasm over Seven Decades of Cultivar Development.Crop Science,2009,49:498-508.
[28]Moon HS,Nifong JM,Nicholson JS,Heinemann A,Lion K,van der Hoeven R,Hayes AJ,Lewis RS.Microsatellite-based Analysis of Tobacco(Nicotiana tabacum L.)Genetic Resources.Crop Science,2009,49:2149-2159.
[29]SANGUINETTI C J,DIAS N E,SIMPSON A J.Rapid silver staining and recovery of PCR products separated on polyacrylamide gels.Biotechniques,1994,17:915-919.
[30]Lawson M J,Zhang L q.Distinct patterns of SSR distribution in the Arabidopsis thaliana and rice genomes.Genome Biology,2006,7(2):R14.1-R14.11.
[31]Gibum Y,Je M L,Sanghyeob L,et al.Exploitation of pepper EST–SSRs and an SSR-based linkage map.Theoretical and Applied Genetics,2006,114:113-130.
[32]MORGANTE M,OLIVIERI A M.PCR-amplified micro satellites as markers in plant genetics.Plant J,1993,3:175-182.
[33]Tang J F,Samantha J B,Jeanne M J,et al.Large-scale identification of polymorphic micro satellites using an in silico approach.BMC Bioinformatics,2008,9:374-386.
[34]蔡斌,李成慧,姚泉洪,周军,陶建敏,章镇.葡萄全基因组SSR分析和数据库构建.南京农业大学学报,2009,32(4):28-32.Cai B,Li C H,Yao Q H,Zhou J,Tao J M,Zhang Z.Analysis of SSRs in grape genome and development of SSR database.Journal of Nanjing Agricultural University,2009,32(4):28-32.(in Chinese)
[35]陆景标,李杰勤,卢杰,詹秋文.高粱非编码区SSR引物设计以及e-PCR的验证.种子,2010,29(9):1-6,12.Lu J B,Li J Q,Lu J,Zhan Q W.Design of SSR primers and verification of e-PCR in non-coding regions of sorghum genome.Seed,2010,29(9):1-6,12.(in Chinese)
[36]Tóth G,Gáspári Z,Jurka J.Microsatellites in different eukaryotic genomes:survey and analysis.Genome Research,2000,10(7):967-981.
[37]高亚梅,韩毅强,汤辉,孙东梅,王彦杰,王伟东.根瘤菌基因组内简单重复序列的分析.中国农业科学,2008,41(10):2992-2998.Gao Y M,Han Y Q,Tang H,Sun D M,Wang Y J,Wang W D.Analysis of simple sequence repeats in rhizobium genome.Scientia Agricultura Sinica,2008,41(10):2992-2998.(in Chinese)
[38]Sia E A,Kokoska R J,Dominska M,Greenwell P,Petes T D.Microsatellite instability in yeast:dependence on repeat unit size and DNA mismatch repair genes.Molecular and Cellular Biology,1997,17(5):2851-2858.
[39]Harry B,Schl?tterer C.Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times,which cause their genome-wide underrepresentation.Genetics,2000,155:1213-1220.
[40]Gibum Y,Je M L,Sanghyeob L,et al.Exploitation of pepper EST–SSRs and an SSR-based linkage map[J].Theor Appl Genet,2006,114:113-130.
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