利用EcoTILLING和TILLING技术检测大豆百粒重和熟期的遗传变异
发布时间：2022-12-10 08:28
大豆是世界上重要的油料作物之一,它具有独特的化学成分、营养价值高、功能健康保健和工业用途。为了满足人类对大豆需求的不断增长,发掘大豆产量相关基因对于育种和生产均具有十分重要的意义。利用TILLING和EcoTILLING技术挖掘基因变异位点,为连接基因型和表型型提供了直接的功能手段。本研究从1046份大豆品系（自然品种和EMS诱变材料）中对3个产量相关基因进行等位基因发掘,包括与AtIKU2同源的大豆百粒重相关基因IKU2（Glyma.13g253300）、开花和熟期相关基因E4（GmPhyA2;Glyma.20g22160）及E3（GmPhyA3;Glyma.19g224200）。比较诱发突变和自然变异的异同点,可以为创造变异和发掘新的等位基因提供理论依据。主要结果如下:1.明确了3个基因在EMS诱变群体中的变异特点。用TILLING方法检测476个突变体株系,共发现16个突变位点,突变频率为1/418kb。在基因IKU2上检测到8个变异位点,4个位于基因内含子区,4个外显子变异位点可以引起1个沉默突变和3个错义突变;而E4基因中检测到7个错义突变和1个沉默突变,但是未检测到E3基因...
【文章页数】：157 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]Development and utilization of a new chemically-induced soybean library with a high mutation density[J]. Zhongfeng Li,Lingxue Jiang,Yansong Ma,Zhongyan Wei,Huilong Hong,Zhangxiong Liu,Jinhui Lei,Ying Liu,Rongxia Guan,Yong Guo,Longguo Jin,Lijuan Zhang,Yinghui Li,Yulong Ren,Wei He,Ming Liu,Nang Myint Phyu Sin Htwe,Lin Liu,Bingfu Guo,Jian Song,Bing Tan,Guifeng Liu,Maiquan Li,Xianli Zhang,Bo Liu,Xuehui Shi,Sining Han,Sunan Hua,Fulai Zhou,Lili Yu,Yanfei Li,Shuang Wang,Jun Wang,Ruzhen Chang,Lijuan Qiu. Journal of Integrative Plant Biology. 2017(01)
[2]Progress in TILLING as a tool for functional genomics and improvement of crops[J]. Liang Chen,Liugen Hao,Martin A. J. Parry,Andrew L. Phillips,Yin-Gang Hu. Journal of Integrative Plant Biology. 2014(05)
[3]大豆粒形性状的遗传效应分析[J]. 梁慧珍,李卫东,王辉,方宣钧. 遗传学报. 2005(11)
[4]MIXED LINEAR MODEL APPROACHES FOR ANALYZING GENETIC MODELS OF COMPLEX QUANTITATIVE TRAITS[J]. 朱军. Journal of Zhejiang University Science. 2000(01)
本文编号：3716423
【文章页数】：157 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]Development and utilization of a new chemically-induced soybean library with a high mutation density[J]. Zhongfeng Li,Lingxue Jiang,Yansong Ma,Zhongyan Wei,Huilong Hong,Zhangxiong Liu,Jinhui Lei,Ying Liu,Rongxia Guan,Yong Guo,Longguo Jin,Lijuan Zhang,Yinghui Li,Yulong Ren,Wei He,Ming Liu,Nang Myint Phyu Sin Htwe,Lin Liu,Bingfu Guo,Jian Song,Bing Tan,Guifeng Liu,Maiquan Li,Xianli Zhang,Bo Liu,Xuehui Shi,Sining Han,Sunan Hua,Fulai Zhou,Lili Yu,Yanfei Li,Shuang Wang,Jun Wang,Ruzhen Chang,Lijuan Qiu. Journal of Integrative Plant Biology. 2017(01)
[2]Progress in TILLING as a tool for functional genomics and improvement of crops[J]. Liang Chen,Liugen Hao,Martin A. J. Parry,Andrew L. Phillips,Yin-Gang Hu. Journal of Integrative Plant Biology. 2014(05)
[3]大豆粒形性状的遗传效应分析[J]. 梁慧珍,李卫东,王辉,方宣钧. 遗传学报. 2005(11)
[4]MIXED LINEAR MODEL APPROACHES FOR ANALYZING GENETIC MODELS OF COMPLEX QUANTITATIVE TRAITS[J]. 朱军. Journal of Zhejiang University Science. 2000(01)
本文编号：3716423
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