Genetic Dissection of Quantitative Trait in Yeast and Discovery of Single Feature Polymorphisms in Gene Expression Profiling Microarray Data

论文摘要

鉴定控制和影响复杂性状的功能基因是当前遗传学中最具挑战性的领域之一。遗传作图的终极目标是检测和定位产生性状差异的遗传变异,在这个过程中最根本的困难来自于基因定位方法粗糙的解析率。传统的的分析手段通过初始的基因组扫描获得的最小的数量性状位点（QTL）区间都不小于10～30 cM,这样大的一个遗传距离并不适合从分子水平上解析复杂性状的遗传变异。本论文主要包括两个方面的内容,一是数量性状的遗传剖析,另一个是利用基因芯片数据检测遗传多态。第一部分内容以酿酒酵母（Saccharomyces cerevisiae）的乙醇耐受这一复杂性状作为一个遗传模型,试图高精度高解析率地解析复杂性状的多基因遗传结构。我们选择了两个表型差异极其显著的酵母菌株,同时开发了一套在全基因均匀分布的STR/SNP遗传标记。选择的两个酵母菌株杂交后生成传统的F2群体并随后建立了多世代的双向重复选择回交（recurrent selection and backcross,RSB）群体。在F2分离群体用复合区间作图（CIM）方法,定位了5个QTL,它们共同解释了约50%的表型变异;其中第九号染色体上的主效QTL解释了25%的表型变异。RSB作图方法充分利用了一套高密度标记信息,定位到了更多的QTL,QTL区间也进一步缩小,在9号染色体的一个QTL区间已经缩小至仅仅包括少数几个基因,已经能够通过对极少的几个基因进行敲除和替换实验来确认影响乙醇耐性的功能基因。在全基因组范围检测基因编码区的序列多态具有重要的价值,一是有助于了解基因的功能,二是能获得高密度的遗传标记可以用于QTL作图。本论文第二部分的内容提出了一个新的算法,该算法利用基因芯片表达谱数据来检测序列多态。这种在基因芯片上检测的多态已经被命名为单征多态（single feature polymorphism,SFP）。SFP可以用作遗传标记来对杂交群体进行基因分型。与文献报道的4种方法相比较,这种新的SFP算法（1）受基因表达量差异的影响小,（2）RNA表达谱芯片与基因组DNA杂交芯片有近似的检测和基因分型的效力,（3）有较高的交互预测能力,以及（4）较高的假阴性率和较低的假阳性率。我们分别用SFP分型数据和SNP分型数据对一个barley单倍体二倍化（double haploid,DH）群体构建遗传连锁图,发现SFP分型数据构建的连锁图有相当的可靠性。这种SFP检测方法应用于前述的酵母RSB群体,有效地提高了QTL作图的解析率。

论文目录

• Acknowledgements
• LIST OF FIGURES
• LIST OF TABLES
• ABSTRACT
• 摘要
• PART Ⅰ
• CHAPTER 1.INTRODUCTION
• 1.1.Genetic Basis of Phenotype Variation
• 1.2.Identifying Genes Affecting Quantitative Traits
• 1.2.1.Mutagenesis
• 1.2.2.QTL Mapping
• 1.2.2.1.Basis Concepts of QTL mapping
• 1.2.2.2.Molecular Markers
• 1.2.2.3.High Resolution Mapping
• 1.2.2.4.From QTL to Gene
• 1.2.3.Genetical Genomics
• 1.3.Ethanol Tolerance in the Budding Yeast Saccharomyces cerevisiae
• 1.3.1.Yeast as a model organism
• 1.3.2.Ethanol tolerance in yeast
• 1.3.2.1.Ethanol Fermentation with Yeast
• 1.3.2.2.Biochemical/Physiological Determinants of Ethanol Tolerance in Yeast
• 1.3.2.3.Progress in Genetic Dissection of Ethanol Tolerance in Yeast
• 1.4.Objective
• CHAPTER 2.SELECTION OF HIGHLY PHENOTYPICALLY DIVERGENT STRAINS
• 2.1.Reagents and Solutions
• 2.1.1.Reagents
• 2.1.2.Solutions
• 2.2.Materials
• 2.2.1.Strains
• 2.2.2.Plasmids
• 2.3.Methods
• 2.3.1.Phenotype Scoring of Ethanol Tolerance
• 2.3.2.Preparation of Competent Cells
• 2.3.3.Bacteria Transformation
• 2.3.4.Plasmid Extraction
• 2.3.5.Yeast Transformation
• 2.3.6.Yeast DNA Extraction
• 2.3.7.Yeast Tetrad Spores Dissection
• 2.3.8.HO Gene Knockout
• 2.3.9.Diploidization and Mating-type Switch
• 2.3.10.Selection of Parent Strains with Divergent Ethanol Tolerance Phenotype
• 2.3.11.Gene Dosage Assay
• 2.4.Results
• 2.4.1.Phenotype Distribution in 53 Yeast Strains
• 2.4.2.Selection for Parent Strain with Low Ethanol Tolerance
• 2.4.3.Performance of Haploid and Diploid Strains
• 2.5.Discussion
• 2 POPULATION'>CHAPTER 3.GENOMEWIDE SCANNING FOR ET QTL AND CANDIDATE QTL GENES IN F₂POPULATION
• 3.1.Reagents and Materials
• 3.1.1.Reagents
• 3.1.2.Materials
• 3.2.Methods
• 2 Mapping Population'>3.2.1.Establishment of F₂ Mapping Population
• 3.2.2.Screening of Short Tandem Repeat Markers
• 3.2.3.Screening of SNP Markers
• 3.2.4.Genotyping
• 3.2.5.DNA Gel Extraction
• 3.2.6.PCR Purification
• 3.2.7.DNA Sequencing
• 3.2.8.Mapping Ethanol-tolerance QTL
• 3.3.Results
• 2 Mapping Population'>3.3.1.F₂ Mapping Population
• 3.3.2.Molecular Marker
• 3.3.3.Genomewide Scanning for ET QTL and Candidate QTL Genes
• 3.3.4.Multi-locus Association Analysis
• 3.4.Discussion
• CHAPTER 4.FINE MAPPING USING RECURRENT SELECTION AND BACKCROSS BREEDING SCHEME
• 4.1.Background
• 4.2.Theoretical Basis of RSB
• 4.3.Materials
• 4.4.Methods
• 4.4.1.Construction of Parent Strains with Opposite Mating Types
• 4.4.2.RSB Breeding Scheme
• 4.4.3.Genotyping of RSB Individuals
• 4.4.4.Test for Maker-ET Association in RSB Mapping Population
• 4.5.Results
• 4.5.1.Construction of RSB Populations
• 4.5.1.1.Selection of Highly Tolerant Segregants
• 4.5.1.2.Selection of Sensitive Segregants
• 4.5.1.3.Introgression of Donor Genes at Two STR Loci
• 4.5.2.Marker-ET Association
• 4.5.3.Fine Mapping on Chromosome 9 and Functional Evaluation of the Candidate Gene
• 4.6.Discussion
• BIBLIOGRAPHY
• PART Ⅱ
• CHAPTER 5.INTRODUCTION
• 5.1.Backgronud
• 5.2.DNA Microarray
• 5.3.Allelic Variation Scanning Using DNA Microarray
• 5.4.Simultaneous Genotyping and Gene Expression Measurement
• 5.5.Objective
• CHAPTER 6.YEAST AND BARLEY MICROARRAY DATA
• 6.1.Materials and Datasets
• 6.1.1.Yeast Strains and RSB Mapping Population
• 6.1.2.Yeast Genomic DNA Hybridizaiton and RNA Profiling Microarray Data
• 6.1.3.Barley Expression Profiling Microarray Data
• 6.1.4.Sources of Check Data
• 6.2.Experiment Protocols for Yeast Genome 2.0 Arrays
• 6.2.1.Reagents
• 6.2.2.Labeled Genomic DNA Preparation
• 6.2.2.1.Preparation of Yeast Cells for DNA Extraction
• 6.2.2.2.Isolation of DNA from Yeast
• 6.2.2.3.Determination of Yield and Purity of the DNA
• 6.2.2.4.Genomic DNA Fragmentation
• 6.2.2.5.Terminal Labeling and Checking of Labeling Efficiency
• 6.2.2.6.Quantifying Labeled DNA
• 6.2.3.RNA Target Preparation
• 6.2.3.1.Preparation of Yeast Cells for RNA Extraction
• 6.2.3.2.Hot Phenol Extraction of RNA from Yeast
• 6.2.3.3.Purification of Total RNA from Yeast
• 6.2.3.4.Preparation of Poly-A RNA Controls for One-Cycle cDNA Synthesis
• 6.2.3.5.First-Strand cDNA Synthesis
• 6.2.3.6.Second-Strand cDNA Synthesis
• 6.2.3.7.Cleanup of Double-Stranded cDNA
• 6.2.3.8.Synthesis of Biotin-Labeled cRNA
• 6.2.3.9.Cleanup of Biotin-Labeled cRNA
• 6.2.3.10.Quantification of Biotin-Labeled cRNA
• 6.2.3.11.Fragmenting the cRNA for Target Preparation
• 6.2.4.Microarray Hybridization
• 6.2.5.Washing,Staining,and Scanning
• CHAPTER 7.STATISTICAL INFERENCE OF SINGLE FEATURE POLYMORPHISMS
• 7.1 Analytical Model
• 7.2.SFP Genotyping
• 7.3.Calculation of True Discovery Rate and Rate of False Negative
• 7.4.Results
• 7.4.1.Predictability of PM Hybridization Intensity
• 7.4.2.Consistency in SFPs Predicted from Parallel DNA and RNA Datasets
• 7.4.3.Proportion of Differentially Expressed Genes
• 7.4.4.Mutual Predictability among Different Methods
• 7.4.5.Efficiency to Predict Sequence Polymorphisms
• 7.4.6.Genotyping and Genetic Map Construction Using SFP
• CHAPTER 8.DISCUSSION
• BIBLIOGRAPHY
• LIST OF PUBLICATIONS