Accelerated Breeding of Cereal Crops

Preface
Contents
Contributors
Chapter 1: Recent Advances in Sequencing of Cereal Genomes
	1 Introduction
	2 Genome Sequencing in Cereal Grasses: Methods and Challenges
		2.1 Sequencing
			2.1.1 Short-Read Sequencing
			2.1.2 Special Applications of Short-Read Sequencing
			2.1.3 Long-Read Sequencing and Optical Mapping
		2.2 Analysis of Genome Sequencing  Data
			2.2.1 Read Processing
			2.2.2 Genome Assembly and Comparison
			2.2.3 Genome Annotation
			2.2.4 Genome Databasing
	3 Status Update on Genome Sequencing and Assembly for Cereals
		3.1 Rice
		3.2 Maize
		3.3 Wheat
		3.4 Rye, Barley, Oat, Sorghum, Millet, Triticale, and Other Poaceae
			3.4.1 Barley
			3.4.2 Rye and Triticale
			3.4.3 Oat
			3.4.4 Sorghum, Millet, and Other Members of the Poaceae
	4 Application of Reference Genomes
		4.1 Transitioning Historic Genomics Research to Available Genome Assemblies
		4.2 Facilitating Breeding through Marker-Assisted Selection, Genome-Wide Association Studies, and Genomic-Assisted Selection
		4.3 Gene Cloning and Mutagenesis
		4.4 Moving Past the Reference Genome and Towards the Future: Pan-Genomes of Cereal Crops
	5 Conclusions and Future Directions
	References
Chapter 2: Mapping Quantitative Trait Loci in Wheat: Historic Perspective, Tools, and Methods for Analysis
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Kits and Reagents
		2.3 Computational Tools and Scripts
	3 Methods
		3.1 Selection of Parents and Generation of Homozygous Mapping Population
		3.2 Phenotyping of Parents and Population
		3.3 Genotyping and Linkage Mapping
			3.3.1 Genotyping
			3.3.2 Linkage Mapping
		3.4 QTL Mapping
	4 Notes
	References
Chapter 3: Introduction to Marker-Assisted Selection in Wheat Breeding
	1 Introduction
		1.1 Breeding for Field-Ready Cultivars and its Challenges
		1.2 MAS as an Assistive Technology
		1.3 Cost-Effective Implementation of MAS
		1.4 Trait Consideration and Marker Implementation
		1.5 Specialized Knowledge and Instrumentation
		1.6 Efficiencies of MAS over Traditional Breeding
	2 Marker Technologies
		2.1 A Brief History of Breeding Markers
		2.2 Molecular Markers
			2.2.1 DArT
			2.2.2 Scar
			2.2.3 ESTs
			2.2.4 SSRs
			2.2.5 SNPs
			2.2.6 Significance of KASP Assay and Primer Design
				Copy Number Variation (CNV)
		2.3 Targeted Versus Genome-Wide Genotyping
			2.3.1 GBS
	3 Marker Development Pipeline
		3.1 Identify Genomic Loci from Biparental and GWAS
		3.2 Marker Validation on Breeding Populations
	4 Implementation of MAS on Breeding Populations and Trait Considerations in each Population
		4.1 Parent Panel
		4.2 Early Generation
		4.3 Advanced Generation
		4.4 DH Population
	5 Genetic, Genomic, and Agronomic Considerations
		5.1 Agronomic Traits
		5.2 Disease
		5.3 Abiotic Stresses
		5.4 Quality Traits
	6 Conclusions
	References
Chapter 4: Genome-Wide Association Studies (GWAS) in Cereals
	1 Introduction
	2 Methodology
		2.1 Plant Material and Phenotyping
		2.2 High-Throughput Genotyping
		2.3 Estimation of Population Structure
		2.4 Linkage Disequilibrium
		2.5 Genome-Wide Association Analysis
	3 Notes
	References
Chapter 5: CropGBM: An Ultra-Efficient Machine Learning Toolbox for Genomic Selection-Assisted Breeding in Crops
	1 Introduction
	2 Example Dataset
		2.1 Hardware Configuration of the Server
		2.2 Software Environment and Preinstalled Libraries
	3 Analytical Procedure of Running CropGBM
		3.1 Preprocessing of Genotype and Phenotype  Data
		3.2 Genotype Data Preprocessing
			3.2.1 Filter and View Overall  Data
			3.2.2 Extract and Remove Specific ID of Samples and SNP  Data
		3.3 Conversion of Character-Based Genotypes to Digits
		3.4 Running the CropGBM with Command-Line Parameters as a Pipeline
	4 Phenotype Data Preprocessing
		4.1 Extract and Visualize Phenotype  Data
		4.2 Extract and Visualize the Phenotype Data According to Sample  ID
		4.3 Normalization of Phenotype Data with Z-Score
		4.4 Conversion of Phenotype Data
	5 Population Structure Analysis
		5.1 PCA and K-Means Clustering Analysis and Visualization of Population Structure
		5.2 t-SNE and OPTICS Clustering Analysis and Visualization
	6 Constructing Genomic Selection GS Model with Training Samples
		6.1 Cross-Validation Analysis
		6.2 Training Model
	7 Feature Selection Functionality
		7.1 Prediction of the Phenotypes of Testing Samples
	8 Concluding Remarks
	References
Chapter 6: Doubled Haploid Production through Microspore Culture
	1 Introduction
	2 Androgenesis Induction through IMC
		2.1 Handling of Spikes for IMC
		2.2 Isolation of Microspores
		2.3 Media Composition and Tissue Culture
	3 Epigenetic Factors to Consider for Androgenesis
	4 Cell Death Considerations
	5 Problem with Albino Plant Regeneration
	6 Summary
	References
Chapter 7: Chemical and Physical Mutagenesis Approaches for Identification of Herbicide and Drought Tolerance Traits in Wheat
	1 Introduction
	2 Materials
		2.1 EMS Mutagenesis
		2.2 Gamma Irradiation
		2.3 Screening and propagation of Mutants
	3 Methods
		3.1 Mutagenesis
			3.1.1 EMS Mutagenesis
			3.1.2 Gamma Irradiation
		3.2 Screening and Propagation of Mutants
			3.2.1 Screening Procedure
			3.2.2 Back Crosses to Reduce Background Mutations
	4 Identification and Characterization of Mutations
	5 Notes
	References
Chapter 8: Using TILLING Lines for Functional Genomics in Wheat
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Equipment
	3 Methods
		3.1 Chemical Mutagenesis and Development of M1 and M2 Generations
		3.2 DNA Preparation and Pooling of Individuals
		3.3 Detection of Mutations in a Targeted Sequence
			3.3.1 Design of Genome-Specific PCR Primers
			3.3.2 PCR Amplification of Target Regions and Heteroduplex Formation
			3.3.3 Heteroduplex Digestion
			3.3.4 Detection of Digestion Products by Agarose Gel
		3.4 Identification of Mutation in Mixed Pool by High-Resolution Melting (HRM) System
		3.5 Analysis of the Mutant Phenotype
			3.5.1 Predicting Effects of Mutation on Gene Function
			3.5.2 Mutation Phenotype Observation
	4 Functional Genomics and Crop Improvement
	5 Prospect of TILLING
	6 Notes
	References
Chapter 9: A Transposon-Based Activation Tag System for Functional Genomics in Cereals: Detection of Mping-Based Activation Ta...
	1 Introduction
		1.1 Insertional Mutagenesis
		1.2 Transposon Tagging in Monocots
			1.2.1 Ac/Ds
			1.2.2 Tos17
			1.2.3 mPing/Ping/Pong
		1.3 Methods for Detecting Transposition
			1.3.1 Reporter Genes
			1.3.2 PCR Analysis
			1.3.3 Sequencing
	2 Materials
		2.1 DNA Extraction
		2.2 Polymerase Chain Reaction
	3 Methods
		3.1 DNA Purification
		3.2 PCR Analysis
	4 Notes
	References
Chapter 10: A Modular Cloning Toolkit for Genome Editing in Cereals
	1 Introduction
	2 Materials
		2.1 Cut-Ligations
		2.2 E. coli Transformation
		2.3 Plasmid Extraction and Verification
	3 Methods
		3.1 General Golden Gate Cut-Ligation Reaction Protocol
		3.2 Assembly of CRISPR/Cas Constructs with sgRNAs/crRNAs Expressed under Individual Pol III Promoters
			3.2.1 Subcloning of the Guide Sequence into the Guide RNA Backbone Acceptor Vector
			3.2.2 Generation of a Transcription Unit for Guide RNA Expression
			3.2.3 Generation of a Nuclease Transcription Unit (Optional)
			3.2.4 Generation of a Multi-Expression Unit L2 Construct
		3.3 CRISPR/Cas Construct Assembly for Multiple Targets Using the Polycistronic tRNA-sgRNA System
			3.3.1 Subcloning of CRISPR/Cas Guide Sequences into Acceptor Vectors
			3.3.2 Generation of a Polycistronic tRNA-sgRNA Transcription  Unit
			3.3.3 Generation of a Multi-Expression Unit L2 Construct
	4 Notes
	References
Chapter 11: Genome Editing and Identification of Targeted Heritable Mutations in Wheat
	1 Introduction
	2 Materials
	3 Methods
		3.1 Overall Construct Design
		3.2 Wheat Target Gene Sequence Verification
		3.3 Guide Design and Selection
		3.4 Construct Design and Construction
		3.5 Wheat Transformation
		3.6 Verification of Transformed T0 Plants
		3.7 Identification of Edits in Transformed Plants
		3.8 Plant Growth and Induction of Additional Edits in Transformed Lines
		3.9 Generation of T-DNA-Free ``Clean´´ Homozygous Lines
	4 Notes
	References
Chapter 12: A Genotyping Protocol to Identify CRISPR/Cas9-Edited Events in Hexaploid Wheat
	1 Introduction
	2 Materials
	3 Methods
		3.1 Growing Wheat Seedlings
		3.2 DNA Extraction
		3.3 Primer Design
		3.4 First Round of  PCR
		3.5 Second Round of PCR
		3.6 Sample Preparation for Capillary Electrophoresis
		3.7 Finishing  up
	4 Notes
	References
Chapter 13: qPCR Methods for the Quantification of Transgene Insert Copy Number and Zygosity Using the Comparative Ct Method i...
	1 Introduction
	2 Materials
		2.1 Miscellaneous Supplies and Equipment
		2.2 Urea-Based gDNA Extraction Buffer (Derived and Modified from) for High-Molecular-Weight gDNA Extraction
			2.2.1 Stock Solutions
			2.2.2 Working Solutions
		2.3 Rapid gDNA Extraction for  qPCR
		2.4 Serial Dilution of gDNA for qPCR Efficiency
		2.5 Determination of Transgene Copy Number
	3 Methods
		3.1 Determination of Transgene Copy Number and Zygosity
			3.1.1 Urea-Based High-Molecular-Weight gDNA Extraction (Modified from for Southern Blotting and qPCR
			3.1.2 Tissue Collection and Processing (see Note 1)
			3.1.3 DNA Extraction
		3.2 Rapid gDNA Extraction Using the DNeasy Plant Mini  Kit
		3.3 Serial Dilution of Transgenic gDNA for qPCR Efficiency Determination
			3.3.1 Preparing the Dilution Series
			3.3.2 Carrying out the Standard Curve Experiment
		3.4 Determination of Transgene Copy Number in Unknown Samples (T0 Transgenics)
		3.5 Determination of Zygosity in T1 Segregants
	4 Notes
	References
Chapter 14: A Road Map for Undertaking Quantitative Proteomics in Plants: New Opportunities for Cereal Crops
	1 Introduction
	2 Protein Extraction and Digestion
	3 Quantification Technologies and Approaches
		3.1 Label-Free Quantitation Approaches
		3.2 Label-Based Quantitation Approaches
	4 Increasing Proteome Resolution and Protein Post-Translational Modifications
		4.1 Increasing Depth of Analysis through Offline Peptide Fraction
		4.2 PTM Enrichment Technologies and Approaches
	5 Approaches to Protein Quantification
		5.1 MaxQuant
		5.2 Progenesis QI
		5.3 Thermo Proteome Discoverer
	6 Emerging Quantitative Proteomic Technologies
		6.1 Data-Independent Acquisition (DIA)
		6.2 Direct Data-Independent Acquisition (directDIA)
	7 Application of Quantitative Proteomics to Cereal Systems
		7.1 Proteomics in Wheat
		7.2 Proteomics in Barley
	8 Considerations for Cereal Agriculture into the Future
	References
Chapter 15: DNA Affinity Purification Sequencing (DAP-Seq) for Mapping Genome-Wide Transcription Factor Binding Sites in Plants
	1 Introduction
	2 Materials
		2.1 DNA Library Preparation
		2.2 Vector Construction and Protein Expression
		2.3 Affinity Purification
		2.4 PCR Enrichment
		2.5 DNA Quantification and Size Selection
	3 Methods
		3.1 DNA Library Construction
		3.2 In Vitro Protein Expression and Purification
		3.3 DNA Affinity Purification
		3.4 DNA Elution, PCR Enrichment, and Pooling
		3.5 Auxiliary Protocol for ampDAP-Seq
		3.6 DNA Sequencing and Data Analysis
	4 Notes
	References
Chapter 16: Basics of Sensor-Based Phenotyping in Wheat
	1 Introduction
	2 High-Throughput Phenotyping Platforms
	3 Traits of Interest in Wheat Phenotyping
		3.1 Biochemical Contents
			3.1.1 Chlorophyll Content
			3.1.2 Nutrient Status
		3.2 Plant Water Relations
			3.2.1 Leaf Water Content and Potential
			3.2.2 Stomatal Conductance
			3.2.3 Canopy Temperature
		3.3 Biomass and Morphological Traits
			3.3.1 In Situ Biomass Production
			3.3.2 Leaf Area and Senescence
			3.3.3 Crop Development and Morphology
		3.4 Yield and Grain Quality Traits
			3.4.1 Grain Quality
		3.5 Root Phenotyping
			3.5.1 Root Architecture
	4 Conclusions
	References
Index