Preface
Contents
Contributors
Part I: Omics Approaches to Study Male and Female Germlines in Plants
Chapter 1: Measurement of Flower Metabolite Concentrations Using Gas Chromatography-Mass Spectrometry and High-Performance Liq...
1 Introduction
2 Materials
2.1 Plant Material, Harvest, Sampling
2.2 Metabolite Extraction
2.3 Derivatization
2.4 GC-TOF-MS Analysis of Primary Metabolites
2.5 LC-MS Analysis of Secondary Metabolites
3 Methods
3.1 Sample Preparation
3.2 Sample Extraction
3.3 Sample Derivatization and Data Acquisition by GC-MS
3.4 Sample Preparation and Data Acquisition by LC-MS
3.5 Data Visualization
4 Notes
References
Chapter 2: Proteomic-, Phosphoproteomic-, and Acetylomic-Based Mass Spectrometry to Identify Tissue-Specific Protein Complexes...
1 Introduction
2 Materials
2.1 Meiotic Stage Anther Collection
2.2 Total Protein Extraction
2.3 Trypsin Digestion
2.4 Phosphopeptide Enrichment by TiO2 Microcolumn
2.5 Affinity Enrichment of Lysine-Acetylated Peptides
2.6 Desalting and Nano UHPLC-MS/MS Analysis
2.7 Database Search and Data Analysis
3 Methods
3.1 Meiotic Stage Anther Collection
3.2 Total Protein Extraction
3.3 Trypsin Digestion
3.4 Phosphopeptide Enrichment by TiO2 Microcolumn
3.5 Affinity Enrichment of Lysine-Acetylated Peptides
3.6 Desalting
3.7 Nano UHPLC-MS/MS Analysis
3.8 Database Search and Data Analysis
3.9 Bioinformatic Analysis
4 Notes
References
Chapter 3: Identifying small RNAs and Analyzing Their Association with Gene Expression Using Isolated Arabidopsis Male Meiocyt...
1 Introduction
2 Materials
2.1 Arabidopsis Meiocyte Isolation
2.2 Total RNA Extraction from Arabidopsis Meiocytes
2.3 Bioinformatics Analysis of small-RNA Seq and RNA-Seq Data
3 Methods
3.1 Arabidopsis Meiocyte Isolation
3.2 Total RNA Extraction from Arabidopsis Meiocytes
3.3 Bioinformatics Analysis of small-RNA Seq and RNA-Seq Data
3.3.1 Quality Check of the Raw Data
3.3.2 Adapter Trimming and Low-Quality Read Removal
3.3.3 Filtering Out Annotated microRNAs
3.3.4 Generating sRNA Clusters
3.3.5 sRNA Cluster Genomic Feature Distribution
3.3.6 Mapping Cleaned RNA-Seq Data to the Arabidopsis Genome Using STAR
3.3.7 Transcript Assembly and Gene Abundance Calling by Stringtie
3.3.8 Calling Differential Expressed Gene by DESeq2
3.3.9 Analyzing sRNA Distribution Around Expressed Genes
3.3.10 Analyzing Gene Expression Value of sRNA Associated Genes
3.3.11 GO analysis of sRNA Associated DEGs
3.3.12 Snapshot of sRNA and sRNA-wAssociated DEGs
4 Notes
References
Chapter 4: CTAB DNA Extraction and Genotyping-by-Sequencing to Map Meiotic Crossovers in Plants
1 Introduction
2 Materials
2.1 Plant Growth, Tissue Collection, and DNA Extraction
2.2 DNA Library Preparation
3 Methods
3.1 Tissue Collection and DNA Extraction
3.2 DNA Library Preparation
3.2.1 DNA Fragmentation
3.2.2 End Repair
3.2.3 A-Tailing
3.2.4 Adaptor Ligation
3.2.5 Pooling Samples
3.2.6 Size Selection to Enrich for DNA Fragments Between 300 bp and 600 bp
3.2.7 PCR Amplification and DNA Purification
3.3 DNA Concentration and Quality Control of Library Preparation
4 Notes
References
Chapter 5: Interrogating Global Chromatin Interaction Network by High-Throughput Chromosome Conformation Capture (Hi-C) in Pla...
1 Introduction
2 Materials
2.1 Cross-Linking
2.2 Nuclei Extraction
2.3 Restriction Digestion
2.4 Biotin Marking and Ligation
2.5 Cross-Link Reversal and DNA Purification
2.6 DNA Shearing and Size Selection
2.7 Streptavidin Pull-Down
2.8 End Repair
2.9 3โฒ A-Tailing
2.10 Adaptor Ligation
2.11 PCR Amplification
3 Methods
3.1 Cross-Linking (See Note 1)
3.2 Nuclei Extraction
3.3 Digestion
3.4 Biotin Marking and Ligation
3.5 Cross-Linking Reversal and DNA Purification
3.6 DNA Shearing and Size Selection
3.7 Streptavidin Pull-Down
3.8 End Repair
3.9 Deoxynucleotide A-Tailing
3.10 Adaptor Ligation
3.11 PCR Amplification and Sequencing
4 Notes
References
Part II: Microscopy Approaches to Study Gametogenesis in Plants
Chapter 6: Cytogenetic Techniques for Analyzing Meiosis in Hexaploid Bread Wheat
1 Introduction
2 Materials
2.1 Excision of Anthers and Preparation of Slides (General)
2.2 Preparation of Slides: Basic Cytological Technique
2.3 Preparation of Slides: Immunocytological Technique
2.4 Fluorescence In Situ Hybridization (FISH) Analysis
2.5 DNA Probes for FISH
2.6 BrdU Meiotic Time Course
3 Methods
3.1 Excision of Anthers for Slide Preparation
3.2 Preparation of Slides: Basic Cytological Technique
3.3 Preparation of Slides: Immunocytological Technique
3.4 Fluorescence In Situ Hybridization Analysis
3.4.1 Preparation of Slides
3.4.2 Fluorescence In Situ Hybridization
3.5 BrdU Meiotic Time Course
4 Notes
References
Chapter 7: Super-resolution Chromatin Visualization Using a Combined Method of Fluorescence In Situ Hybridization and Structur...
1 Introduction
2 Materials
2.1 Plant Growth and Tissue Collection
2.2 Chromosome Spread
2.3 Fluorescence In Situ Hybridisation
2.4 Structured Illumination Microscopy
3 Methods
3.1 Fixation of Tissue and Chromosome Spreading
3.2 Fluorescence In Situ Hybridization
3.3 Image Capture Using Structured Illumination Microscopy
4 Notes
References
Chapter 8: Application of Chemical Inhibitors in Live Cell Imaging of Plant Meiosis Using Light Sheet Fluorescence Microscopy
1 Introduction
2 Materials
2.1 Seeds and Soil
2.2 List of Solutions
2.3 Sample Preparation
2.4 Sample Mounting
2.5 Microscope
2.6 Software
3 Methods
3.1 Seed Sterilization
3.2 Soil Preparation
3.3 Plant Preparation
3.4 Sample Preparation
3.5 Sample Mounting
3.6 Imaging
3.7 Application of an Inhibitor
3.7.1 Inhibitor Application from Bottom (Buffer Exchange Method)
3.7.2 Inhibitor Application from Top (Buffer Addition Method)
3.8 Image Processing
4 Notes
References
Chapter 9: Bimolecular Fluorescence Complementation to Test for Protein-Protein Interactions and to Uncover Regulatory Mechani...
1 Introduction
2 Materials
2.1 Generation of Entry and Expression Clones
2.1.1 Entry Clone Generation
2.1.2 Expression Clone Generation
2.1.3 E. coli Transformation and Plasmid DNA Isolation
2.2 Transformation of Agrobacteria and Agroinfiltration of N. benthamiana Plants
2.2.1 Transformation of Agrobacteria
2.2.2 Agroinfiltration of N. benthamiana Plants
2.3 Fluorescence Microscopy Analysis
3 Method
3.1 Generation of Entry and Expression Clones
3.1.1 Entry Clone Generation
3.1.2 Expression Clone Generation
3.1.3 E. coli Transformation and Plasmid DNA Isolation
3.2 Transformation of Agrobacteria and Agroinfiltration of N. benthamiana Plants
3.2.1 Transformation (Electroporation) of A. tumefaciens
3.2.2 Agroinfiltration of N. benthamiana Plants
3.3 Fluorescence Microscopy Analysis
4 Notes
References
Chapter 10: Quantifying Meiotic Crossover Recombination in Arabidopsis Lines Expressing Fluorescent Reporters in Seeds Using S...
1 Introduction
2 Materials
3 Methods
3.1 Image Acquisition
3.2 CellProfiler: SeedScoring Pipeline
4 Notes
References
Part III: Plant Manipulation and Genome Editing
Chapter 11: Generation of Mutants by Combined Treatment of Physical and Chemical Mutagens in Rice
1 Introduction
2 Materials
3 Methods
3.1 Physical Mutagenesis Treatment (Gamma Rays)
3.2 Chemical Mutagenesis Treatment (EMS)
3.2.1 Presoaking
3.2.2 EMS Incubation
3.2.3 Post Treatment
3.3 Planting and Analysis of Mutant Plants
4 Notes
References
Chapter 12: Generation of Mutant Plants by Gamma Ray Exposure and Development of Low-Cost TILLING Population in Chickpea (Cice...
1 Introduction
2 Materials
2.1 Mutation Induction
2.2 Phenotypic Evaluation
2.3 Low-Cost TILLING
2.4 Genomic DNA Quantification and Normalization
2.5 Primer Testing and Enzymatic Digestion
3 Methods
3.1 Determination of LD50 and RD50 (See Note 5)
3.2 Raising M1 and M2 Generations
3.2.1 Raising M1 Generation
3.2.2 Raising M2 Generation
3.3 Screening of M2 Generation
3.3.1 Efficiency Assessment
3.3.2 Agronomic Assessment
3.4 Genomic Low-Cost DNA Extraction
3.5 Genomic DNA Quantification
3.5.1 Agarose Gel
3.5.2 Nanodrop Spectrophotometer
3.5.3 Concentration of Genomic DNA
3.6 Database Search for Candidate Genes
3.7 Primer Testing
3.8 Enzymatic Digestion (Enzymatic Mismatch Cleavage)
3.9 Sequence Validation of Low-Cost TILLING Results
4 Notes
References
Chapter 13: Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis...
1 Introduction
2 Materials
2.1 Plant Growth, Transformation, and Selection
2.2 Bacteria Strains, Media and Bacteria Transformation
2.3 Molecular Cloning, PCR, and Plant Genotyping
2.4 Specific Lab Equipment
3 Methods
3.1 Growing Arabidopsis Plants for Agrobacterium-Mediated Transformation
3.2 Designing a Pair of Single-Guide RNAs
3.3 Generation of sgRNA Expression Cassettes
3.4 Generation of CRISPR/Cas9 Binary Vector Construct
3.4.1 Preparation of pFGC-I2Cas9 Vector for sgRNA Cassettes Cloning
3.4.2 Preparation of sgRNA Expression Cassettes
3.4.3 Cloning sgRNA Cassettes into the Binary Vector
3.5 Transformation of CRISPR/Cas9 Construct to Agrobacterium tumefaciens
3.6 Agrobacterium-Mediated Transformation of Arabidopsis thaliana via Floral Dip
3.7 Selection of T1 Transformants Using Epifluorescent Stereomicroscope
3.8 Selection of T2 Lines Carrying Homozygous Mutations
4 Notes
References
Chapter 14: Generation of Deletion Lines in Allohexaploid Bread Wheat
1 Introduction
2 Materials
2.1 Plant Material
2.2 PCR
2.3 Droplet Digital PCR
3 Methods
3.1 Production of Deletion Lines Via the 2C Gametocidal System
3.2 Selection of Plants Carrying Deletion on the Chromosome of Interest
3.2.1 Screening Using PCR and Agarose Gel Electrophoresis
3.2.2 Screening Using PCR with M13 Fluorescent Markers and Capillary Electrophoresis
3.3 Characterization of Deletion Sizes
3.3.1 Characterization Using Molecular Markers
3.3.2 Characterization Using High Throughput SNP Genotyping Array
3.4 Selection of Deletion Lines with the Chromosome of Interest in Disomic Constitution
4 Notes
References
Chapter 15: Post-transcriptional Gene Silencing Using Virus-Induced Gene Silencing to Study Plant Gametogenesis in Tomato
1 Introduction
2 Materials
2.1 RNA Isolation and cDNA Synthesis
2.2 Construct Cloning
2.3 Agrobacterium Preparation
2.4 Plant Preparation
2.5 RNA Analyses for PCR/qPCR
3 Methods
3.1 Preparation of Recombinant Construct
3.1.1 Preparation of tomato RNA
3.1.2 cDNA Synthesis
3.1.3 Amplification of Target Sequence of Selected Gene
3.1.4 Clone Target Sequence into pTRV2
3.2 Agrobacterium Transformation, Culturing, and Induction
3.3 Tomato Infiltration
3.4 RNA Analysis to Confirm Viral Vector Expression and Target Gene Downregulation
3.4.1 Semiquantitative RT-PCR
3.4.2 Real Time Quantitative PCR
4 Notes
References
Chapter 16: Protocols for In Vivo Doubled Haploid (DH) Technology in Maize Breeding: From Haploid Inducer Development to Haplo...
1 Introduction
2 Materials
2.1 Haploid Inducer Development and Improvement
2.1.1 For Gel-Based Marker Analysis
2.1.2 For High Resolution Melting (HRM) Analysis (Optional)
2.2 Haploid Induction
2.3 Haploid Selection
2.3.1 Haploid Selection Using Seed Color Marker
2.3.2 Haploid Selection Using Root Color Marker
2.3.3 Haploid Selection Using QSorter
2.4 Artificial Genome Doubling of Haploids Using Colchicine
2.5 Identifying Genotypes with SHGD Ability
3 Methods
3.1 Haploid Inducer Development
3.2 Haploid Induction
3.3 Haploid Selection
3.3.1 Haploid Selection Using R1-nj and C1 Seed Color Marker
3.3.2 Haploid Selection Using Purple Plant 1 (Pl1) Root Color Marker
3.3.3 Haploid Selection Based on Embryo Oil Content Using QSorter
Select Seed Materials and Measure Oil Content
NIR Spectrum and RGB Image Capturing Using QSorter
Select Training Data and Train Classification Model in MATLAB to Identify Correct Seed Orientation
Annotate Training Data and Train Segmentation Model in MATLAB to Identify Embryo
Determination of Seed Orientation and Relative Embryo Size on the Rest of the Data (~92-95%)
Preprocessing NIR Data and Develop Binary Classification Models to Predict Oil Content
Model Deployment on QSorter
3.4 Artificial Genome Doubling of Haploids Using Colchicine
3.5 Identifying Genotypes with SHGD Ability
4 Notes
References
Chapter 17: Estimation of Heterografting Associated DNA Methylation Changes in Tree Crops by MSAP Analysis
1 Introduction
2 Materials
2.1 Development of Genetically Uniform Seedlings Through Induction of Zygotic Polyembryony
2.2 Grafting
2.3 MSAP Analysis for the Detection of DNA Methylation Polymorphisms in Heterografted Plants
2.4 Identification and Annotation of the Differentially Methylated Genomic Region
3 Methods
3.1 Development of Genetically Uniform Seedlings Through Induction of Zygotic Polyembryony
3.2 Grafting
3.2.1 Establishment of Seedling Nursery
3.2.2 Establishment of BudWood Nursery
3.2.3 Grafting Procedure (Green Budding)
3.3 MSAP Analysis for the Detection of DNA Methylation Polymorphisms in Heterografted Plants
3.4 Identification and Annotation of the Differentially Methylated Genomic Region
4 Notes
References
Chapter 18: In Vitro Fertilization System Using Wheat Gametes by Electric Fusion
1 Introduction
2 Materials
2.1 Isolation and Transfer of Gametes
2.2 Fusion of Gametes
2.3 Culture of Zygotes into Compact Embryonic Calli and Regenerated Plantlets
3 Methods
3.1 Isolation of Wheat Gametes
3.2 Fusion of Gametes
3.3 Culture of Zygotes into Embryo-Like Structure and Plantlets
4 Notes
References
Part IV: Bioinformatics and Data Analysis
Chapter 19: High-Throughput Fluorescent Pollen Tetrad Analysis Using DeepTetrad
1 Introduction
2 Materials
2.1 Pollen Tetrad Imaging
2.2 Install DeepTetrad
3 Methods
3.1 FTL Plant Growth
3.2 Phenotyping for Measurable FTLs
3.3 Pollen Tetrad Imaging
3.4 Running DeepTetrad and Data Analysis
4 Notes
References
Chapter 20: Barley (Hordeum Vulgare) Anther and Meiocyte RNA Sequencing: Mapping Sequencing Reads and Downstream Data Analyses
1 Introduction
2 Materials
2.1 Materials, Consumables, and Chemicals
2.2 Equipment
2.3 Datasets
2.4 Software and Tools
2.4.1 RNA-Seq Data Processing
2.4.2 Differential Gene Expression and Alternative Splicing Analysis
2.4.3 Further Downstream Analysis and Visualization
3 Methods
3.1 Anther and Meiocyte Collection
3.2 RNA Extraction, Quality Control and RNA-Seq Libraries
3.3 Bioinformatics Analysis
3.3.1 Mapping Sequencing Reads
3.3.2 Downstream Analysis
3.4 Further Downstream Analysis and Visualization
3.4.1 GO Enrichment
3.4.2 Visualizing Relationships Between Contrast Groups
3.4.3 Visualization of Differential Alternatively Spliced Transcripts Using Heatmaps or Line Graphs
4 Notes
References
Chapter 21: Profiling m6A RNA Modifications in Low Amounts of Plant Cells Using Maize Meiocytes
1 Introduction
2 Materials
2.1 Plant Material and Equipment
2.2 Buffers, Solutions and Chemicals
3 Methods
3.1 Sample Preparation: Isolating Maize Meiocytes
3.2 RNA Extraction, RNA Quality Control, and Quantification
3.3 m6A Capture, Library Preparation and Sequencing
3.4 Bioinformatic Analyses of m6A-Seq Data
3.4.1 Initial Quality Control
3.4.2 Mapping Reads to the Reference Genome, Peak Calling, and Visualization
3.4.3 Functional Analysis of m6A Peaks
4 Notes
References
Chapter 22: Assembling Plant Genomes with Long-Read Sequencing
1 Introduction
2 Materials
3 Methods
3.1 Pipeline Overview
3.2 Data Collection
3.3 QC and Filtering
3.4 Assembly with Flye
3.5 Polishing with Medaka
3.6 Evaluating Assembly Quality
4 Notes
References
Chapter 23: Mapping-by-Sequencing of Point and Insertional Mutations with Easymap
Abbreviations
1 Introduction
2 Materials
2.1 Plants
2.2 Extraction of Genomic DNA
2.3 Hardware and Software
2.4 Input Files
3 Methods
3.1 Experimental Design
3.1.1 Linkage Analysis Mapping of Point Mutations
3.1.2 Tagged Sequence Mapping of Large Insertions
3.2 Plant Material Collection, DNA Extraction, and Sample Preparation
3.3 Massive Sequencing
3.4 Read Quality Assessment and Preprocessing
3.5 Mapping Analysis Using Easymap
3.5.1 Analysis Using Easymap Through the Web Interface
3.5.2 Analysis Using Easymap Through the Command Line Interface
3.6 Interpretation of the Results
3.6.1 Linkage-Analysis Mapping Report
3.6.2 Tagged-Sequence Mapping Report
3.7 Troubleshooting
4 Notes
References
Chapter 24: Characterization of the Different Levels of Variation in 45S rRNA Genes
1 Introduction
2 Materials
2.1 Software Requirements
2.1.1 Create a Virtual Environment and Install Required Software with Conda
2.2 Data Requirements
2.2.1 Download TAIR10 Reference Genome and 45S rRNA Reference Gene
2.2.2 Download Example Dataset
3 Methods
3.1 Estimation of rDNA Copy Number and Detection of rDNA Variants
3.1.1 Processing Reference Genomes
3.1.2 Processing Short-Reads
3.1.3 Alignment of Short-Reads to the 45S rDNA Reference Gene, and Generation of Files Containing Statistics Necessary for Var...
3.1.4 Alignment of Short-Reads to the TAIR10 Reference Genome
3.1.5 Estimation of rDNA Copy Number
3.1.6 Detection of rDNA Variants
3.1.7 Plot the Frequencies of Alternative Alleles Along the 45S rRNA Gene for Each Sample
3.1.8 Plot the Proportion of Individuals in a Population with an Alternative Allele Frequency Above a Certain Threshold
3.2 Assignment of rDNA Variants to Specific rDNA Clusters
4 Notes
References
Index