大豆是世界上重要的油料作物之一,它具有獨特的化學成分、營養(yǎng)價值高、功能健康保健和工業(yè)用途。為了滿足人類對大豆需求的不斷增長,發(fā)掘大豆產量相關基因對于育種和生產均具有十分重要的意義。利用TILLING和EcoTILLING技術挖掘基因變異位點,為連接基因型和表型型提供了直接的功能手段。本研究從1046份大豆品系（自然品種和EMS誘變材料）中對3個產量相關基因進行等位基因發(fā)掘,包括與AtIKU2同源的大豆百粒重相關基因IKU2（Glyma.13g253300）、開花和熟期相關基因E4（GmPhyA2;Glyma.20g22160）及E3（GmPhyA3;Glyma.19g224200）。比較誘發(fā)突變和自然變異的異同點,可以為創(chuàng)造變異和發(fā)掘新的等位基因提供理論依據。主要結果如下:1.明確了3個基因在EMS誘變群體中的變異特點。用TILLING方法檢測476個突變體株系,共發(fā)現16個突變位點,突變頻率為1/418kb。在基因IKU2上檢測到8個變異位點,4個位于基因內含子區(qū),4個外顯子變異位點可以引起1個沉默突變和3個錯義突變;而E4基因中檢測到7個錯義突變和1個沉默突變,但是未檢測到E3基因... 

【文章頁數】：157 頁

【學位級別】：博士

【文章目錄】：
摘要
Abstract
Chapter 1 REVIEW
    1.0 General introduction
    1.1 Soybean origin,classification, domestication and distribution
    1.2 Soybeanplant breeding
        1.2.1 Conventional soybean breeding
        1.2.2 Soybean molecular breeding
    1.3 Advances of soybean plant genetics
    1.4 Novel allele discovery
        1.4.1 TILLING
            1.4.1.1 Employment of TILLING in crops and model plants
            1.4.1.2 Employment of TILLING in soybean
        1.4.2 EcoTILLING
            1.4.2.1 Employment of EcoTILLING in crops and model plants
    1.5 Objectives of the study
Chapter 2 DETECTION OF INDUCED MUTATIONS RELATED TO SOYBEAN SEED SIZE ANDMATURITY BY TILLING
    2.1 Introduction
    2.2 Material and Methods
        2.2.1 Development of mutagenesis population
        2.2.2 TILLING procedure
            2.2.2.1 DNA preparations
            2.2.2.2 Designing PCR primers for target genes for TILLING and EcoTILLING
            2.2.2.3 Preparation of CEL1 for detection heteroduplexes
            2.2.2.4 Fragment separation of digested PCR products
    2.3 Results
        2.3.1 Detection of IKU2 alleles
            2.3.1.1 Identification and description of IKU2 gene of soybean
            2.3.1.2 Optimization of CEL1 digestion condition and estimation of mutation rate
            2.3.1.3 Allele variation of IKU2 gene in the mutagenesis population
            2.3.1.4 The detected haplotypes based nucleotides and amino acids in IKU2 gene
        2.3.2 TILLING of E4 and E3 gene in soybean mutagenesis population
            2.3.2.1 Identification of E3 and E4 gene
            2.3.2.2 CEL1 digestion and fragment separation of E4 and E3 primers
            2.3.2.3 Allele variation of E3 and E4 gene in the mutagenesis population
            2.3.2.4 The detected haplotypes based nucleotides and amino acids in E4 gene
        2.3.3 Summary of detected mutations of IKU2, E3 and E4 in mutagenesis population
    2.4 Discussions
        2.4.1 Development of mutant population
        2.4.2 TILLING of IKU2 gene in mutagenesis soybean
            2.4.2.1 Identification and description of IKU2 gene
            2.4.2.2 Detected mutations in IKU2 in mutagenesis soybean
            2.4.2.3 Identification of induced mutation of IKU2 in mutagenesis soybean
        2.4.3 TILLING of E4 and E3 gene in mutagenesis soybean
            2.4.3.1 CEL1 Digestion of E4 and E3 primers in mutagenesis soybean
    2.5 Conclusion
Chapter 3 ECOTILLING TO DISCOVER NATURAL VARIATIONS OF IKU2,E3 AND E4 CANDIDATE GENES RELATED TO SEED SIZE AND MATURITY IN NATURALSOYBEAN
    3.1 Introduction
    3.2 Material and methods
        3.2.1 Germplasm
        3.2.2 EcoTILLING Procedure
            3.2.2.1 DNA preparations
            3.2.2.2 Designing of primers and PCR amplification
            3.2.2.3 Preparation of CJE for detection heteroduplexes
            3.2.2.4. Fragment separation of digested PCR products
    3.3 Results
        3.3.1 EcoTILLING in IKU2 gene of soybean
            3.3.1.1 CEL1 digestion of IKU2 primers and fragment separation
            3.3.1.2 Identification of DNA nucleotide variations of IKU2 in soybean naturalpopulation
            3.3.1.3 UPGMA Phylogenic tree of 27 soybean accessions
        3.3.2 EcoTILLING in E4 and E3 gene of soybean
            3.3.2.1 CEL1 digestion and fragment separation of E4 and E3 primers utilized innatural population
            3.3.2.2 The detected haplotypes based nucleotides and amino acids in E4 gene
            3.3.2.3 UPGMA Phylogenic tree of 15 soybean accessions
        3.3.3. Summary of the detected mutations of IKU2, E3 and E4 in soybean naturalpopulation through EcoTILLING
    3.4 Discussions
        3.4.1 Germplasm
        3.4.2 EcoTILLING of IKU2 gene in soybean natural population
            3.4.2.1 Identification and description of IKU2 gene
            3.4.2.2 CEL1 Digestion of IKU2 primers in soybean natural population
            3.4.2.3 Identification of nucleotide variations of IKU2 gene in soybean naturalpopulation
            3.4.2.4 UPGMA phylogeny tree of detected variations in soybean naturalpopulation
        3.4.3 EcoTILLING of E4 and E3 gene in soybean natural population
            3.4.3.1 Identification of E4 and E3 gene
    3.5 Conclusion
Chapter 4 COMPARISONS BETWEEN TILLING AND ECOTILLING SCREENS OF IKU2,E3 AND E4 GENES IN MUTAGENESIS AND NATURAL SOYBEANS
    4.1 Introduction
    4.3 Results and discussions
        4.3.1 Detected mutations in mutagenesis and natural populations
        4.3.2 Nucleotide variations in mutagenesis and natural populations
        4.3.3 The 'common' and 'private' nucleotide variations
        4.3.4 The phenotypes of detected mutagenesis and natural soybeans
    4.4 Discussions
        4.4.1 Detected mutations in mutagenesis and natural soybean
        4.4.2 'Common' and ‘private’ nucleotide variations in mutagenesis and untreated soybean
        4.4.3 The phenotypes of detected mutagenesis and natural soybeans
    4.5 Conclusions
Chapter 5 OVERALL CONCLUSION
    5.1 Detection of mutation by Tilling
    5.2 Detection of natural variation by Ecotilling
    5.3 Comparison of TILLING and EcoTILLING
    5.4 Prospect
Reference
ACKNOWLEDGEMENT
RESUME


【參考文獻】：
期刊論文
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