大豆EST-SNP的挖掘鉴定及其CAPS标记的开发

doi:10.3724/SP.J.1006.2010.00574

摘要/Abstract

摘要：

采用生物信息学方法将大豆EST序列联配到大豆基因组序列上，挖掘到大豆EST-SNP位点537个。对其靶向基因进行功能注释分析，发现它们主要参与亚细胞定位、蛋白质结合与催化以及代谢等与大豆重要农艺性状形成相关的生物过程。同时开发了简便易行的SNP检测方法，利用EMBOSS软件筛选导致酶切位点改变的EST-SNP，分别以大豆绥农14、合丰25、Acher、Evans、Peking、PI209332、固新野生大豆、科丰1号、南农1138-2的DNA及其混合的DNA为模板，设计引物进行PCR扩增，发现44个PCR产物中有36个测序峰图在预期的EST-SNP位点表现出多态性。酶切分析发现26个PCR产物具有酶切多态性，可以作为CAPS标记。结果表明该EST-SNP挖掘体系及其CAPS标记转化系统具有高效率、低成本等优点，有利于促进大豆的遗传育种研究。

关键词: 大豆, 表达序列标签, 基因组序列, 单核苷酸多态性, 酶切扩增多态性序列

Abstract:

SNPs widely distribute throughout genomes from non-coding regions to coding regions, constituting the most abundant molecular markers used in animal and plant genetic breeding. With the rapidly growing genome sequencing projects, a large amount of genomic and EST sequences has become available to the public. Many SNPs are identified by comparing genome sequences or ESTs obtained from genetically diverse lines or individuals in plants. However the SNP assay always relies on expensive equipments or reagent, which has limited the application of SNPs in genetics and breeding especially in plants. The CAPS marker, also known as PCR-RFLP marker, is the technique combining PCR and restriction enzymes digestion to detect the restriction fragment length polymorphisms. With the development of high-throughput sequencing technology, more and more SNPs are identified, among them many mutations have altered the restriction enzymes recognition sites. This provides an opportunity for high-through development of CAPS markers. With soybean genome sequences becoming available, high-throughput SNP marker development will significantly improve genetic mapping, map based cloning in soybean. To discover new SNPs in soybean, we aligned the ESTs with whole genome sequences in different soybean varieties and identified 537 EST-SNPs. The function of genes targeted by these EST-SNPs was analysed, the results showed that these genes participated in subcellular localization, protein binding or catalyzing, metabolic process and cell rescue, defense and disease resistance, etc. Most of these functions are involving in various physiological and biochemical processes influencing important agronomic traits. To develop easy assay method for these EST-SNPs, we identified the EST-SNPs which alter the restrict enzyme recognition sites by software EMBOSS, and 48 pair primers were designed to detect these EST-SNPs. forty-four pair primers amplified single bands (400–800 bp) from genomic DNA of Suinong 14 widely planted in the Northeast China. To verify the SNP polymorphisms, we used these primer pairs for PCR amplification from genomic DNA of Suinong 14, Hefeng 25, Acher, Evans, Peking, PI209332, Guxin wild soybean, Kefeng 1, Nannong 1138-2 and pool DNA of the nine soybean varieties. The PCR amplicons were sequenced, the traces of the 36 discordant ones were detected as candidate SNPs, which were then validated by re-sequencing the individuals. SNPs were identified using restriction enzymess of 26 pair primers with unequivocal restriction pattern were identified as CAPS markers. The SNPs discovery and CAPS markers conversion system developed in this study is fast, low cost and effecient, and holds great promise for molecular assisted breeding of soybean. , and the product

Key words: Soybean, EST, Genome sequences, SNP, CAPS

束永俊，李勇，吴娜拉胡，柏锡，才华，纪巍，朱延明. 大豆EST-SNP的挖掘鉴定及其CAPS标记的开发[J]. 作物学报, 2010, 36(4): 574-579.

SHU Yong-Jun,LI Yong,WU Na-La-Hu,BAI Xi,CAI Hua,JI Wei,ZHU Yan-Ming. Mining and Identification of SNP from EST Sequences and Convertion of CAPS Markers in Soybean[J]. Acta Agron Sin, 2010, 36(4): 574-579.

参考文献

[1] Ganal M W, Altmann T, Roder M S. SNP identification in crop plants. Curr Opin Plant Biol, 2009, 12: 211–217

[2] Wiltshire T, Pletcher M T, Batalov S, Barnes S W, Tarantino L M, Cooke M P, Wu H, Smylie K, Santrosyan A, Copeland N G, Jenkins N A, Kalush F, Mural R J, Glynne R J, Kay S A, Adams M D, Fletcher C F. Genome-wide single-nucleotide polymorphism analysis defines haplotype patterns in mouse. Proc Natl Acad Sci USA, 2003, 100: 3380–3385

[3] Feltus F A, Wan J, Schulze S R, Estill J C, Jiang N, Paterson A H. An SNP resource for rice genetics and breeding based on subspecies indica and japonica genome alignments. Genome Res, 2004, 14: 1812–1819

[4] Jander G, Norris S R, Rounsley S D, Bush D F, Levin I M, Last R L. Arabidopsis map-based cloning in the post-genome era. Plant Physiol, 2002, 129: 440–450

[5] Picoult-Newberg L, Ideker T E, Pohl M G, Taylor S L, Donaldson M A, Nickerson D A, Boyce-Jacino M. Mining SNPs from EST databases. Genome Res, 1999, 9: 167–174

[6] Snelling W M, Casas E, Stone R T, Keele J W, Harhay G P, Bennett G L, Smith T P. Linkage mapping bovine EST-based SNP. BMC Genomics, 2005, 6: 74

[7] Batley J, Barker G, O’Sullivan H, Edwards K J, Edwards D. Mining for single nucleotide polymorphisms and insertions/deletions in maize expressed sequence tag data. Plant Physiol, 2003, 132: 84–91

[8] Somers D J, Kirkpatrick R, Moniwa M, Walsh A. Mining single-nucleotide polymorphisms from hexaploid wheat ESTs. Genome, 2003, 46: 431–437

[9] Mao X-G(毛新国), Tang J-F(汤继凤), Zhou R-H(周荣华), Jing R-L(景蕊莲), Jia J-Z(贾继增). Wheat cSNP mining based on full-length cDNA sequences. Acta Agron Sin(作物学报), 2006, 32(12): 1836–1840 (in Chinese with English abstract)

[10] Kota R, Rudd S, Facius A, Kolesov G, Thiel T, Zhang H, Stein N, Mayer K, Graner A. Snipping polymorphisms from large EST collections in barley (Hordeum vulgare L.). Mol Genet Genomics, 2003, 270: 24–33

[11] Kota R, Varshney R, Prasad M, Zhang H, Stein N, Graner A. EST-derived single nucleotide polymorphism markers for assembling genetic and physical maps of the barley genome. FunctIntegr Genomics, 2008, 8: 223–233

[12] Komori T, Nitta N. Utilization of the CAPS/dCAPS method to convert rice SNPs into PCR-based markers. Breed Sci, 2005, 55: 93–98

[13] Cregan P B, Jarvik T, Bush A L, Shoemaker R C, Lark K G, Kahler A L, Kaya N, VanToai T T, Lohnes D G, Chung J, Specht J E. An integrated genetic linkage map of the soybean genome. Crop Sci, 1999, 39: 1464–1490

[14] Song Q J, Marek L F, Shoemaker R C, Lark K G, Concibido V C, Delannay X, Specht J E, Cregan P B. A new integrated genetic linkage map of the soybean. Theor Appl Genet, 2004, 109: 122–128

[15] Tian A G, Wang J, Cui P, Han Y J, Xu H, Cong L J, Huang X G, Wang X L, Jiao Y Z, Wang B J, Wang Y J, Zhang J S, Chen S Y. Characterization of soybean genomic features by analysis of its expressed sequence tags. Theor Appl Genet, 2004, 108: 903–913

[16] Yang Z-Y(杨振宇), Ma X-P(马晓萍), Yamanaka N(山中直树). Polymorphism of phosphoenolpyruvate carboxylase gene in soybean cultivars from northeastern China and Japan. Acta Agron Sin(作物学报), 2005, 31(9): 1233–1235 (in Chinese with English abstract)

[17] Rice P, Longden I, Bleasby A. EMBOSS: The european molecular biology open software suite. Trends Genet, 2000, 16: 276–277

[18] Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol, 2000, 132: 365–386

[19] Garg K, Green P, Nickerson D A. Identification of candidate coding region single nucleotide polymorphisms in 165 human genes using assembled expressed sequence tags. Genome Res, 1999, 9: 1087–1092

[20] Guryev V, Berezikov E, Malik R, Plasterk R H, Cuppen E. Single nucleotide polymorphisms associated with rat expressed sequences. Genome Res, 2004, 14: 1438–1443

[21] Kerstens H H, Kollers S, Kommadath A, Del Rosario M, Dibbits B, Kinders S M, Crooijmans R P, Groenen M A. Mining for single nucleotide polymorphisms in pig genome sequence data. BMC Genomics, 2009, 10: 4

[22] Torjek O, Berger D, Meyer R C, Mussig C, Schmid K J, Rosleff Sorensen T, Weisshaar B, Mitchell-Olds T, Altmann T. Establishment of a high-efficiency SNP-based framework marker set for Arabidopsis. Plant J, 2003, 36: 122–140

[23] Yamamoto N, Tsugane T, Watanabe M, Yano K, Maeda F, Kuwata C, Torki M, Ban Y, Nishimura S, Shibata D. Expressed sequence tags from the laboratory-grown miniature tomato (Lycopersicon esculentum) cultivar Micro-Tom and mining for single nucleotide polymorphisms and insertions/deletions in tomato cultivars. Gene, 2005, 356: 127–134

[24] Dantec L L, Chagné D, Pot D, Cantin O, Garnier-Géré P, Bedon F, Frigerio J M, Chaumeil P, Léger P, Garcia V, Laigret F, de Daruvar A, Plomion C. Automated SNP detection in expressed sequence tags: Statistical considerations and application to maritime pine sequences. Plant Mol Biol, 2004, 54: 461–470

[25] Cordeiro G M, Eliott F, McIntyre C L, Casu R E, Henry R J. Characterisation of single nucleotide polymorphisms in sugarcane ESTs. Theor Appl Genet, 2006, 113: 331–343

[26] Choi I Y, Hyten D L, Matukumalli L K, Song Q, Chaky J M, Quigley C V, Chase K, Lark K G, Reiter R S, Yoon M S. A soybean transcript map: Gene distribution, haplotype and single-nucleotide polymorphism analysis. Genetics, 2007, 176: 685–696

[27] Zhu Y L, Song Q J, Hyten D L, Van Tassell C P, Matukumalli L K, Grimm D R, Hyatt S M, Fickus E W, Young N D, Cregan P B. Single-nucleotide polymorphisms in soybean. Genetics, 2003, 163: 1123–1134

相关文章 15

[1]	陈玲玲, 李战, 刘亭萱, 谷勇哲, 宋健, 王俊, 邱丽娟. 基于783份大豆种质资源的叶柄夹角全基因组关联分析[J]. 作物学报, 2022, 48(6): 1333-1345.
[2]	杨欢, 周颖, 陈平, 杜青, 郑本川, 蒲甜, 温晶, 杨文钰, 雍太文. 玉米-豆科作物带状间套作对养分吸收利用及产量优势的影响[J]. 作物学报, 2022, 48(6): 1476-1487.
[3]	王炫栋, 杨孙玉悦, 高润杰, 余俊杰, 郑丹沛, 倪峰, 蒋冬花. 拮抗大豆斑疹病菌放线菌菌株的筛选和促生作用及防效研究[J]. 作物学报, 2022, 48(6): 1546-1557.
[4]	于春淼, 张勇, 王好让, 杨兴勇, 董全中, 薛红, 张明明, 李微微, 王磊, 胡凯凤, 谷勇哲, 邱丽娟. 栽培大豆×半野生大豆高密度遗传图谱构建及株高QTL定位[J]. 作物学报, 2022, 48(5): 1091-1102.
[5]	李阿立, 冯雅楠, 李萍, 张东升, 宗毓铮, 林文, 郝兴宇. 大豆叶片响应CO₂浓度升高、干旱及其交互作用的转录组分析[J]. 作物学报, 2022, 48(5): 1103-1118.
[6]	彭西红, 陈平, 杜青, 杨雪丽, 任俊波, 郑本川, 罗凯, 谢琛, 雷鹿, 雍太文, 杨文钰. 减量施氮对带状套作大豆土壤通气环境及结瘤固氮的影响[J]. 作物学报, 2022, 48(5): 1199-1209.
[7]	王好让, 张勇, 于春淼, 董全中, 李微微, 胡凯凤, 张明明, 薛红, 杨梦平, 宋继玲, 王磊, 杨兴勇, 邱丽娟. 大豆突变体ygl2黄绿叶基因的精细定位[J]. 作物学报, 2022, 48(4): 791-800.
[8]	李瑞东, 尹阳阳, 宋雯雯, 武婷婷, 孙石, 韩天富, 徐彩龙, 吴存祥, 胡水秀. 增密对不同分枝类型大豆品种同化物积累和产量的影响[J]. 作物学报, 2022, 48(4): 942-951.
[9]	杜浩, 程玉汉, 李泰, 侯智红, 黎永力, 南海洋, 董利东, 刘宝辉, 程群. 利用Ln位点进行分子设计提高大豆单荚粒数[J]. 作物学报, 2022, 48(3): 565-571.
[10]	周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析[J]. 作物学报, 2022, 48(3): 590-596.
[11]	王娟, 张彦威, 焦铸锦, 刘盼盼, 常玮. 利用PyBSASeq算法挖掘大豆百粒重相关位点与候选基因[J]. 作物学报, 2022, 48(3): 635-643.
[12]	董衍坤, 黄定全, 高震, 陈栩. 大豆PIN-Like (PILS)基因家族的鉴定、表达分析及在根瘤共生固氮过程中的功能[J]. 作物学报, 2022, 48(2): 353-366.
[13]	张国伟, 李凯, 李思嘉, 王晓婧, 杨长琴, 刘瑞显. 减库对大豆叶片碳代谢的影响[J]. 作物学报, 2022, 48(2): 529-537.
[14]	禹桃兵, 石琪晗, 年海, 连腾祥. 涝害对不同大豆品种根际微生物群落结构特征的影响[J]. 作物学报, 2021, 47(9): 1690-1702.
[15]	宋丽君, 聂晓玉, 何磊磊, 蒯婕, 杨华, 郭安国, 黄俊生, 傅廷栋, 汪波, 周广生. 饲用大豆品种耐荫性鉴定指标筛选及综合评价[J]. 作物学报, 2021, 47(9): 1741-1752.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed