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作物学报 ›› 2012, Vol. 38 ›› Issue (01): 43-49.doi: 10.3724/SP.J.1006.2012.00043

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用SSCP技术分析甘蓝型油菜10个功能基因序列差异

李媛媛1,2，陈庆芳2，傅廷栋2，马朝芝2,*   

  1. 1 潍坊学院生物工程学院, 山东潍坊 261061；2 华中农业大学/作物遗传改良国家重点实验室, 湖北武汉 430070
  • 收稿日期:2011-05-27 修回日期:2011-09-13 出版日期:2012-01-12 网络出版日期:2011-11-07
  • 通讯作者: 马朝芝, E-mail: yuanbeauty@hzau.edu.cn
  • 基金资助:

    本研究由山东省优秀中青年科学家科研奖励基金项目(BS2009NY017)和国家重点基础研究计划(973计划)项目(2007CB1090)资助。

Polymorphism Analysis of Ten Functional Genes in Brassica napus Using SSCP Method

LI Yuan-Yuan1,2, CHEN Qing-Fang2, FU Ting-Dong2,MA Chao-Zhi2,*   

  1. 1 Department of Bioengineering, Weifang University, Weifang 261061, China; 2 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
  • Received:2011-05-27 Revised:2011-09-13 Published:2012-01-12 Published online:2011-11-07
  • Contact: 马朝芝, E-mail: yuanbeauty@hzau.edu.cn

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1377

被引次数

12

摘要： 以甘蓝型油菜SI-1300和Eagle为材料，利用DNA单链构象多态性(single-strand conformation polymorphism, SSCP)技术，对10对功能基因特异性引物进行多态性分析，每对引物均检测到1个多态性位点。随后随机挑选10个多态性片段进行测序，并利用bl2seq软件比较测序序列与基因原始序列。结果显示测序序列与所对应的基因原始序列之间相似程度平均高达98%，差异碱基数平均仅为2.3个。进一步选取5对引物比较分析两个材料间的差异扩增片段序列，发现差异扩增片段在2个材料中高度保守，平均相似度达97%；在所测序的5对引物扩增序列中，共存在39个单核苷酸多态性(single-nucleotide polymorphisms, SNPs)和5个插入/缺失突变(insertion-deletions, INDELs)，SNP和INDEL的发生频率分别为1 SNP/30 bp和1 INDEL /233 bp。结果表明，SSCP标记能够真实代表原始功能基因，甘蓝型油菜功能基因序列在不同材料间高度保守，其遗传变异类型主要来源于SNP。

关键词: 甘蓝型油菜, 功能基因, SSCP, 序列分析

Abstract: A sensitive technology is very necessary to detect the polymorphisms of functional genes in different cultivars, for the coding sequences of functional genes tend to be conserved even between species. Single-strand conformational polymorphism (SSCP) is a desirable method for DNA polymorphism analysis because of its high sensitivity and cost effectiveness. In previous publications, we developed 177 functional markers corresponding to 111 differentially expressed genes between the parents of a Brassica napus hybrid. And, 45 functional markers involved in 39 genes or expressed sequence tags (ESTs) were linked to the QTLs of 12 yield-related traits in the F2 population from SI-1300×Eagle using SSCP analysis. In the present research, we sequenced some polymorphic bands detected by SSCP analysis to confirm the high sensitivity of SSCP analysis. Firstly, a total of ten primer pairs, which were designed according to ten B. napus functional genes or ESTs, were used to survey polymorphisms between SI-1300 and Eagle. All primers showed polymorphisms, resulting ten polymorphic loci. Subsequently, ten polymorphic bands were randomly selected, sequenced and aligned with the gene sequences for primers designed using the bl2seq software. The results indicated that the average identity was 98%, and the average number of different bases was only 2.3 between the sequenced fragments and their functional genes. Furthermore, the sequence comparison of polymorphic fragments amplified by five primer pairs was performed between SI-1300 and Eagle. The polymorphic fragments are highly conserved between SI-1300 and Eagle, and there were 39 single-nucleotide polymorphisms (SNPs) and five insertion-deletions (INDELs) in the DNA fragments amplified by the five primer pairs. The average frequency of sequence polymorphism was estimated to be one SNP every 30 bp and one INDEL every 233 bp. In conclusion, the sequences of functional genes, which could be really amplified by specific primers, are highly conversed among different cultivars in B. napus, and SNP is the most basic genetic variation for functional genes. This study will provide a foundation for investigating the molecular basis of important traits in rapeseed using comparative genomics.

Key words: Brassica napus, Functional genes, SSCP, Sequencing

[1]Dumolin L S, Bodénès C, Petit R J. Detection of rare polymorphism in mitochondrial DNA of oaks with PCR-RFLP combined to SSCP analysis. For Genet, 1996, 3: 227–230
[2]Sato Y, Nishio T. Mutation detection in rice waxy mutants by PCR-RF-SSCP. Theor Appl Genet, 2003, 107: 560–567
[3]Shirasawa K, Maeda H, Monna L, Kishitani S, Nishio T. The number of genes having different alleles between rice cultivars estimated by SNP analysis. Theor Appl Genet, 2007, 115: 1067–1074
[4]Li Y, Ma C, Fu T, Yang G, Tu J, Chen Q, Wang T, Zhang X, Li C. Construction of a molecular functional map of rapeseed (Brassica napus L.) using differentially expressed genes between hybrid and its parents. Euphytica, 2006, 152: 25–39
[5]Li Y, Shen J, Wang T, Chen Q, Zhang X, Fu T, Meng J, Tu J, Ma C. QTL analysis of yield-related traits and their association with functional markers in Brassica napus L. Aust J Agric Res, 2007, 58: 759–766
[6]Li J(李佳), Shen B-Z(沈斌章), Han J-X(韩继祥), Gan L(甘莉). An effective procedure for extracting total DNA in rape, J Huazhong Agric Univ (华中农业大学学报), 1994, 13: 521–523 (in Chinese with English abstract)
[7]Slabaugh M B, Huestis G M, Leonard J, Holloway J L, Rosato C, Hongtrakul V, Martini N, Toepfer R, Voetz M, Schell J, Knapp S J. Sequence-based genetic markers for genes and gene families: single-strand conformational polymorphisms for the fatty acid synthesis genes of Cuphea. Theor Appl Genet, 1997, 94: 400–408
[8]Lu G-Y(陆光远), Yang G-S(杨光圣), Fu T-D(傅廷栋). An effective SSR detection system in rapeseed. Chin J Oil Crop Sci (中国油料作物学报), 2003, 25: 79–81 (in Chinese with English abstract)
[9]Cavell A C, Lydiate D J, Parkin I A P, Dean C, Trick M. Collinearity between a 30-centimorgan segment of Arabidopsis thaliana chromosome 4 and duplicated regions within the Brassica napus genome. Genome, 1998, 41: 62–69
[10]Westermeier P, Wenzel G, Mohler V. Development and evaluation of single-nucleotide polymorphism markers in allotetraploid rapeseed (Brassica napus L.). Theor Appl Genet, 2009, 119: 1301–1311
[11]Tenaillon M I, Sawkins M C, Long A D, Gaut R L, Doebly J F, Gaut B S. Patterns of DNA sequence polymorphism along chromosome 1 of maize (Zea mays ssp. mays L.). Proc Natl Acad Sci USA, 2001, 98: 9161–9166
[12]Monna L, Ohta R, Masuda H, Koike A, Minobe Y. Genome-wide searching of single-nucleotide polymorphisms among eight distantly and closely related rice cultivars (Oryza sativa L.) and a wild accession (Oryza rufipogon Griff.). DNA Res, 2006, 13: 43–51
[13]Fusari C M, Lia V V, Hopp H E, Heinz R A, Paniego N B. Identification of single nucleotide polymorphisms and analysis of linkage disequilibrium in sunflower elite inbred lines using the candidate gene approach. BMC Plant Biol, 2008, 8: 7
[14]Buckler E S, Thornsberry J M. Plant molecular diversity and applications to genomics. Curr Opin Plant Biol, 2002, 5: 107–111
[15]Mackay T F C. Quantitative trait loci in drosophila. Nat Rev Genet, 2001, 2: 11–20
[16]Inoue H, Nishio T. Efficiency of PCR-RF-SSCP marker production in Brassica oleracea using Brassica EST sequences. Euphytica, 2004, 137: 233–242
[17]Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J. Positional cloning of the wheat vernalization gene VRN1. Proc Natl Acad Sci USA, 2003, 100: 6263–6268
[18]Brown G G, Formanová N, Jin H, Wargachuk R, Dendy C, Patil P, Laforest M, Zhang J, Cheung W Y, Landry B S. The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats. Plant J, 2003, 35: 262–272
[19]Mayerhofer R, Wilde K, Mayerhofer M, Lydiate D, Bansal V K, Good A G, Parkin I A. Complexities of chromosome landing in a highly duplicated genome: toward map-based cloning of a gene controlling blackleg resistance in Brassica napus. Genetics, 2005, 171: 1977–1988
[20]Qin B, Cao A, Wang H, Chen T, You F M, Liu Y, Ji J, Liu D, Chen P, Wang X E. Collinearity-based marker mining for the fine mapping of Pm6, a powdery mildew resistance gene in wheat. Theor Appl Genet, 2011, 123: 207–218
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