中国野生大豆6个基因SNP筛选
摘要
大豆是人类主要的经济作物之一,由于种质基础比较狭窄,栽培大豆(Glycine max)遗传多样性程度较低,对于新型病虫害抵抗力弱,极大的影响大豆产量以及世界粮食安全。相对而言野生大豆(Glycine soja)有较高的遗传多样性,是大豆育种上有重要的利用价值的遗传资源,因此研究野生大豆的遗传多样性具有重要的应用价值。单核苷酸多态性(single nucleotide polymorphism, SNP)的筛选及其检测是研究遗传多样性的主要方法。本研究针对六个与几种植物代谢密切相关的基因:铵转运子基因(AMT1),硝酸盐转运子基因(NRT2),天冬氨酸转氨酶基因(AspAT2);抗逆性相关基因:ABA应答转录因子基因(ABP9),甜菜碱醛脱氢酶基因(BADH),钙依赖性蛋白激酶基因(CPK13)对部分中国野生大豆种质(43份)进行单核苷酸多态性(SNP)扫描并分析不同野生大豆之间的差异。通过检测到的位点对不同野生大豆种质之间的遗传多样性进行评估,主要结果如下:
1根据大豆与小麦,高粱,百脉根在进化上的亲缘关系,以这些植物相应基因为查询序列(Query Sequence),在TIGR网站大豆的EST数据库搜索匹配EST拼接完成序列,根据EST拼接成的连接群(Contig)设计引物,从野生大豆的基因组中分离了6个基因的部分序列。
2运用Vector NTI软件将每个基因片段的所有测序结果进行序列比对,发现在AMT1基因中有2个SNP位点,NRT2基因26个SNP位点,AspAT基因3个SNP位点,CPK13基因66个SNP位点,ABP9基因12个SNP位点,BADH基因27个SNP位点,SNP发生的频率远高于在玉米、大豆等植物中报道的SNP发生频率。这些位点中转换与颠换的比例约为2:1,符合以前的研究报道。
3应用DnaSP软件进行数学统计分析,AMT1基因π值为0.00240,θ值为0.00111, Fu and Li's D* test以及Fu and Li's F* test中P>0.10;NRT2基因π值为0.02129,θ值为0.01269,Fu and Li's D* test以及Fu and Li's F* test中P < 0.02;CPK13基因π值为0.02603,θ值为0.02087,Fu and Li's D* test以及Fu and Li's F* test中P > 0.10;BADH基因π值为0.00462,θ值为0.00759,Fu and Li's D* test以及Fu and Li's F* test中P > 0.10;ABP9基因π值为0.00220,θ值为0.00396,Fu and Li's D* test以及Fu and Li's F* test中P> 0.10;AspAT2基因π值为0.00046,θ值为0.00198, Fu and Li's D* test以及Fu and Li's F* test中0.05< P < 0.10。
Soybean (Glycine.max) is one of the main economic crops. Because high-yielding cultivars dominate production but are relatively few in number and are genetically similar, genetic diversity in these crops is presumed to have declined to alarmingly low levels.The reduction of genetic diversity does not bode well for future genetic gains in crop productivity and could result in broad susceptibility to newly emerging diseases or insect pests, thereby threatening long-term food and feed security. Due to the high degree of genetic diversity, Glycine.soja has become a valuable genetic resource for soybean breeding. In this study, three N-metabolism related genes: Ammonium transporter(AMT1), nitrate transporter2(NRT2), aspartate aminotransferase2(AspAT2); three resistence-related genes:, ABRE Binding Protein 9(ABP9), betaine-aldehyde dehydrogenase(BADH), calcium dependent protein kinase 13(CPK13) were isolated and cloned using the approach of molecular biology combined with bioinformatics, the related information of SNPs were obtained and the results were as the following:
1 Using cDNA sequences of some other species as query sequences, the homologous Expressed Sequence Tags(EST) assembled contigs were searched out on TIGR website; the primers were designed according to the contigs sequences, and part of six genes were isolated.
2 All the fragment of the each gene were alingned by using Vector NTI software. 2 SNPs were detected in AMT1, 26 in NRT2, 66 in CPK13, 12 in ABP9, 3 in AspAT, and 27 in BADH. Just the same as reported before, the number of SNPs in non-coding regions is doubled that of SNPs in coding regions.
3 Statistics were generated by using DnaSP software5.0, theπof AMT1, CPK13, BADH , ABP9, AspAT2, and NRT2 are 0.00240, 0.02603, 0.00220, 0.00046, and0.02129. Theθof AMT1, CPK13, BADH , ABP9, AspAT2, and NRT2 are 0.00111, 0.02087, 0.00759, 0.00396, 0.00198, 0.01269.
1根据大豆与小麦,高粱,百脉根在进化上的亲缘关系,以这些植物相应基因为查询序列(Query Sequence),在TIGR网站大豆的EST数据库搜索匹配EST拼接完成序列,根据EST拼接成的连接群(Contig)设计引物,从野生大豆的基因组中分离了6个基因的部分序列。
2运用Vector NTI软件将每个基因片段的所有测序结果进行序列比对,发现在AMT1基因中有2个SNP位点,NRT2基因26个SNP位点,AspAT基因3个SNP位点,CPK13基因66个SNP位点,ABP9基因12个SNP位点,BADH基因27个SNP位点,SNP发生的频率远高于在玉米、大豆等植物中报道的SNP发生频率。这些位点中转换与颠换的比例约为2:1,符合以前的研究报道。
3应用DnaSP软件进行数学统计分析,AMT1基因π值为0.00240,θ值为0.00111, Fu and Li's D* test以及Fu and Li's F* test中P>0.10;NRT2基因π值为0.02129,θ值为0.01269,Fu and Li's D* test以及Fu and Li's F* test中P < 0.02;CPK13基因π值为0.02603,θ值为0.02087,Fu and Li's D* test以及Fu and Li's F* test中P > 0.10;BADH基因π值为0.00462,θ值为0.00759,Fu and Li's D* test以及Fu and Li's F* test中P > 0.10;ABP9基因π值为0.00220,θ值为0.00396,Fu and Li's D* test以及Fu and Li's F* test中P> 0.10;AspAT2基因π值为0.00046,θ值为0.00198, Fu and Li's D* test以及Fu and Li's F* test中0.05< P < 0.10。
Soybean (Glycine.max) is one of the main economic crops. Because high-yielding cultivars dominate production but are relatively few in number and are genetically similar, genetic diversity in these crops is presumed to have declined to alarmingly low levels.The reduction of genetic diversity does not bode well for future genetic gains in crop productivity and could result in broad susceptibility to newly emerging diseases or insect pests, thereby threatening long-term food and feed security. Due to the high degree of genetic diversity, Glycine.soja has become a valuable genetic resource for soybean breeding. In this study, three N-metabolism related genes: Ammonium transporter(AMT1), nitrate transporter2(NRT2), aspartate aminotransferase2(AspAT2); three resistence-related genes:, ABRE Binding Protein 9(ABP9), betaine-aldehyde dehydrogenase(BADH), calcium dependent protein kinase 13(CPK13) were isolated and cloned using the approach of molecular biology combined with bioinformatics, the related information of SNPs were obtained and the results were as the following:
1 Using cDNA sequences of some other species as query sequences, the homologous Expressed Sequence Tags(EST) assembled contigs were searched out on TIGR website; the primers were designed according to the contigs sequences, and part of six genes were isolated.
2 All the fragment of the each gene were alingned by using Vector NTI software. 2 SNPs were detected in AMT1, 26 in NRT2, 66 in CPK13, 12 in ABP9, 3 in AspAT, and 27 in BADH. Just the same as reported before, the number of SNPs in non-coding regions is doubled that of SNPs in coding regions.
3 Statistics were generated by using DnaSP software5.0, theπof AMT1, CPK13, BADH , ABP9, AspAT2, and NRT2 are 0.00240, 0.02603, 0.00220, 0.00046, and0.02129. Theθof AMT1, CPK13, BADH , ABP9, AspAT2, and NRT2 are 0.00111, 0.02087, 0.00759, 0.00396, 0.00198, 0.01269.
引文
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[2]Dong YS, Zhao LM, Liu B, Wang ZW, Jin ZQ, Sun HThe genetic diversity of cultivated soybean grown in China. Theor Appl Genet,2004, 108: 931-936
[3]Guo WT Origins of soybean cultivation in China. Journal of Nanjing Agricultural University (Social Sciences Edition) 2004, 4(1):60-69
[4]李福山,王衍桐.野生大豆分布与环境条件.大豆科学,1989,8(3):245～251.
[5]Zhao TJ, Gai JY The Origin and evolution of cultivated soybean [Glycine max (L) Merr]. Scientia Agricultural Sinica,2004, 37(7): 954-962
[5]董英山.中国野生大豆遗传多样性及核心种质构建.[博士学位论文].哈尔滨:东北师范大学,2000.
[6]李军,庄炳昌.中国野生大豆生物学研究.北京:科学出版社,1999.
[7]Qiu LJ, Cao YS, Chang RZ, Zhou XA, Wang GX, Sun JY, Xie H, Zhang B, Li XH, XuZY, Liu LH Establishment of Chinese soybean ( G max) core collection:Sampling strategy. Scientia Agricultura Sinica ,2003,36:1442-1449
[8]李福山主编.中国野生大豆资源目录.中国农业科学院作物品种资源研究所主编.中国农业出版社,1994.
[9]庄炳昌,徐豹.种子萌发过程中大豆种子蛋白组分变化的研究.作物学报,1988,14(3):232～235.
[10]徐豹,邹淑华,庄炳昌,林忠平,赵玉锦.野生大豆(G..soja.)种子贮藏蛋白组份11S/7S的研究.作物学报,1990,16(3):235～241
[11]王小波,姜治华,林文君,张永晖.四川夏大豆品种资源的蛋白质研究与脂肪含量研究.西南农业学报,1996,9:133～137.
[12]徐豹,徐航,庄炳昌,路琴华,王玉民,李福山.中国野生大豆籽粒性状的遗传多样性及其地理分布.作物学报,1995,21(6):733～739.
[13]邵启全,林章棋,周端敏.中国野生大豆光周期生态类型分析.作物学报,1980,6(1):45～50.
[14]王金陵,孟庆喜,祝其昌.中国南北地区野生大豆光照生态类型分析.遗传学通讯.1973,(3):1～8
[15]徐豹,庄炳昌,路琴华,王玉民,胡传璞,梁岐,郑惠玉,吕景良.中国野生大豆(G..soja)脂肪及脂肪酸组成的研究.吉林农业科学,1993,12(4):269～274.
[16]Cho RJ,Mindrinos M,Riehards DR,et al. Genome-wide mapping with biallelic markers in Arabidopsis thaliana. Nature Geneties,1999,23:203一207.
[17]Bhattraakki D,Dolan M,Hnaafey M,Wineland R,Vaske D,Register J C,Tingey SV,Rafalski A. Insertiondeletion polymorphisms in 3,-Regions of maize genes occur frequentlyand can be used as highly Informative genetic markers. Plant Mol Biol,2002,48:539~547.
[18]Ching A,Caldwell KS, Jung M,MDolna M,Smith O S,Tingey S,Morgant M, Rafalski AJ,2002,SNP frequency,624 21(5).
[19]Goldstein D B.Islnads of linkage disequilibrium. Nat Genet,2001,29:109~111.
[20]Meyers B C,Tingey S V,Morgante M. Abundance,distribution,and Transcriptional activity of repetitive elements in the maize genome.Genome Res,2001,11:1660-1676.
[21]Kruglyak L.The use of a genetic map of biallelic marker in linkage studies. Nature genetics,1997,17:21一24.
[22]Tenaillon M l,Sawkins M C,Long A D,Doebley J F,Gaut BS.Patterns of DNA sequence polymorphism along chromosome 1 of maize(Zea mays ssp.mays L.).Proc Natl Acad Sci USA,2001,98:9161-9166.
[23]The Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering Plant Arbaidopsis thaliana.Nature,2000,408:796~815.
[24]Kruglyak L.Prospects for whole-genome linkage disequilibrium mapping of common disease genes. Nature Genetics,1999,22:139一144.
[25]Taramino G,Tingey S Simple sequence repeats for germplasm analysis and mapping in maize. Genome,1996,39:277一287.
[26]Delanney X,Rodgers D M,Palmer R G. Relative genetic contributions among ancestral lines to North American soybean cultivars. Crop Sci,1983,23:944~949.
[27]LoquéD, von Wirén N. Regulatory levels for the transport of ammonium in plant roots. Journal of Experimental Botany, 2004, 55:1293-1305.
[28]Sohlenkamp C, Shelden M, Howitt S, Udvardi M. Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants. Federation of European Biochemical Societies Letters, 2000, 467: 273-278.
[29]Salvemini F, Marini A M, Riccio A, Patriarca E J, Chiurazzi M. Functional characterization of an ammonium transporter gene from Lotus japonicu. Gene, 2001, 270: 237-243.
[30]Simon-Rosin U, Wood C, Udvardi MK. Molecular and cellular characterization of LjAMT2;1, an ammonium transporter from the model legume Lotus japonicus. Plant Molecular Biology, 2003, 51: 99-108.
[31]Apuzzo E D, Rogato A, Simon-Rosin U, Alaoui H E, Barbulova A, Betti M, Dimou M, Katinakis P, Marquez A, Marini A M, Udvardi M K, and Chiurazzi M. Characterization of three functional high-affinity ammonium transporters in Lotus japonicus with differential transcriptional regulation and spatial expression. Plant Physiology, 2004, 134: 1763-1774.
[32]Pearson J N, Finnemann J and Schjoerring J K. Regulation of the high-affinity ammonium transporter (BnAMT1;2) in the leaves of Brassica napus by nitrogen status. Plant Molecular Biology, 2002, 49: 483-490.
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