Plant Epigenetics and Epigenomics: Methods and Protocols (Methods in Molecular Biology, 2093)

Preface
Contents
Contributors
Part I: Detection and Analysis of Epigenetic Marks in Plant Genomes
	Chapter 1: An Overview of Current Research in Plant Epigenetic and Epigenomic Phenomena
		1 Introduction
		2 The Evolving Definition of Epigenetics: What Exactly Do We Mean?
		3 Epigenetic Phenomena in the Context of Plant Science
		4 Techniques for Addressing Challenges in Plant Epigenetic and Epigenomic Research
		References
	Chapter 2: Approaches to Whole-Genome Methylome Analysis in Plants
		1 Introduction
		2 Limitations of Current Analysis Methods for Methylome Studies
		3 Gene Body Methylation
		4 Newly Emerging Methylation Analysis Programs
		5 Strategies for Methylome Dataset Validation
		6 Methylation vs. Gene Expression Data
		7 Concluding Comments
		References
	Chapter 3: Understanding DNA Methylation Patterns in Wheat
		1 Introduction
			1.1 The Use of Epigenetics in Plant Breeding
			1.2 Challenges of Epigenomic Analysis in Wheat
		2 Methods
			2.1 Sequence Capture for Epityping and Genotyping
			2.2 Bioinformatic Analysis for Non-bisulfite-Treated Sequence Data
			2.3 Bioinformatic Analysis after Bisulfite Sequencing
			2.4 Bioinformatic Analyses Specific to an Allopolyploid
		3 Implications
		4 Notes
		References
	Chapter 4: MCSeEd (Methylation Context Sensitive Enzyme ddRAD): A New Method to Analyze DNA Methylation
		1 Introduction
		2 Materials
			2.1 DNA Extraction
			2.2 Primer and Adapter Preparation
			2.3 Restriction/Ligation reaction
			2.4 Purification with PEG8000
			2.5 Purification with AMpure Beads
			2.6 Size Selection and Gel Extraction
			2.7 PCR Enrichment
		3 Methods
			3.1 DNA Extraction
			3.2 Adapter Preparation
			3.3 Double Digestion and Adapter Ligation
			3.4 Purification with PEG8000
			3.5 Purification with AMpure Beads (1.1x) to Remove Fragments Shorter Than 250 bp
			3.6 Size Selection and Gel Purification
			3.7 Purification with 0.8x AMpure Beads
			3.8 Qubit Quantification
			3.9 Enrichment PCR
			3.10 Purification with AMpure Beads (1x)
			3.11 Sequencing
			3.12 Bioinformatic Analysis
		4 Notes
		References
	Chapter 5: Plant-RRBS: DNA Methylome Profiling Adjusted to Plant Genomes, Utilizing Efficient Endonuclease Combinations, for M...
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Enzymes
			2.3 Solutions
			2.4 Buffers
			2.5 Kits
			2.6 Products
			2.7 Equipment
			2.8 Data Sets
			2.9 Software
		3 Methods
			3.1 Genomic DNA Isolation and Digestion
				3.1.1 DNA Isolation, Quality, and Quantity Evaluation
				3.1.2 In Silico Digestion
				3.1.3 Digestion with Restriction Enzymes
			3.2 Library Preparation for Illumina Sequencing and Quality Control
			3.3 Bioinformatics Pipeline
				3.3.1 Reads Analysis
				3.3.2 Methylation Detection
		4 Notes
		References
	Chapter 6: Rice Histone Propionylation and Generation of Chemically Derivatized Synthetic H3 and H4 Peptides for Identificatio...
		1 Introduction
		2 Materials
			2.1 Rice Histone Extraction
			2.2 Propionylation of Synthetic and Biological Peptides
			2.3 Fmoc-Based Solid-Phase Synthesis of Peptides
			2.4 Workup of Synthesized Peptides
			2.5 LC-MS/MS
		3 Methods
			3.1 Extraction of Rice Histones
			3.2 Double Propionylation of Biological Peptides
			3.3 Synthesis of Rice Histone H3 and H4 Synthetic Peptides
			3.4 Double Propionylation of Recovered Synthetic Peptides
			3.5 Characterization of Doubly Propionylated Peptides Using LC-MS/MS
			3.6 Identification and Quantification of Extracted Rice Histone Peptides Using Synthetic Peptide Information
		4 Notes
		References
Part II: Epigenetics and Plant Chromatin Structure
	Chapter 7: Preparing Chromatin and RNA from Rare Cell Types with Fluorescence-Activated Nuclear Sorting (FANS)
		1 Introduction
		2 Materials
			2.1 Nuclei Isolation
			2.2 Fluorescence-Activated Nuclei Sorting (FANS)
			2.3 RNA Extraction and Quality Control
			2.4 DNA Extraction and Quantification
		3 Methods
			3.1 Nuclei Isolation
			3.2 Fluorescence-Activated Nuclei Sorting (FANS)
			3.3 RNA Extraction and Quantification
			3.4 DNA Extraction and Quantification
		4 Notes
		References
	Chapter 8: Measurement of Arabidopsis thaliana Nuclear Size and Shape
		1 Introduction
		2 Materials
			2.1 Isolation of Nuclei (Adapted from)
			2.2 Image Acquisition and Analysis
		3 Methods
			3.1 Isolation of Nuclei
			3.2 Spreading and Staining with DAPI
			3.3 Image Acquisition
			3.4 Semiautomated Image Analysis
		4 Notes
		References
	Chapter 9: Study of Cell-Type-Specific Chromatin Organization: In Situ Hi-C Library Preparation for Low-Input Plant Materials
		1 Introduction
		2 Materials
			2.1 Tissue Fixation
			2.2 Nuclei Isolation and Flow Cytometry
			2.3 Chromatin Digestion, Ligation, and DNA Purification
			2.4 DNA Manipulation and Library Amplification
		3 Methods
			3.1 Tissue Fixation
			3.2 Nuclei Isolation and Flow Cytometry
			3.3 Chromatin Digestion, Ligation, and DNA Purification
			3.4 DNA Manipulation and Library Amplification
		4 Notes
		References
	Chapter 10: Chromatin Analysis of Metabolic Gene Clusters in Plants
		1 Introduction
		2 Materials
			2.1 Growth Media
			2.2 Measurement of mRNA Levels
				2.2.1 RNA Isolation
				2.2.2 Removal of Genomic DNA
				2.2.3 cDNA Preparation
				2.2.4 qPCR
			2.3 Chromatin Immunoprecipitation
				2.3.1 Stock Solutions and Reagents
				2.3.2 Buffers
			2.4 Metabolite Extraction and Analysis
				2.4.1 Instruments and Equipment
				2.4.2 Solvents and Chemicals
		3 Methods
			3.1 Measurement of mRNA Levels
				3.1.1 RNA Isolation
				3.1.2 Removal of Genomic DNA
				3.1.3 cDNA Preparation
				3.1.4 Quantitative PCR
			3.2 Chromatin Immunoprecipitation
				3.2.1 Chromatin Extraction
				3.2.2 Immunoprecipitation and DNA Recovery
				3.2.3 Quantification
			3.3 Metabolite Analysis
				3.3.1 Sample Preparation for GC-MS Analysis
				3.3.2 GC-MS Analysis Method (See Note 9)
				3.3.3 Sample Preparation for LC-MS Analysis
				3.3.4 Method for LC-MS Analysis (See Note 11)
				3.3.5 Metabolomics Analysis
				3.3.6 Quantitative Analysis (See Note 12)
		4 Notes
		References
	Chapter 11: Characterization of Plant 3D Chromatin Architecture, In Situ Hi-C Library Preparation, and Data Analysis
		1 Introduction
		2 Materials
			2.1 Formaldehyde Fixation
			2.2 Nuclei Isolation
			2.3 In Situ Digestion, Biotin End-Repair, and Ligation
			2.4 On-Bead Illumina TruSeq Library Preparation
			2.5 Hi-C Data Analysis
		3 Methods
			3.1 Formaldehyde Fixation
			3.2 Nuclei Preparation
			3.3 In Situ Restriction Enzyme Digestion
			3.4 On-Bead Illumina TruSeq Library Preparation
			3.5 Data Analysis
				3.5.1 Map Hi-C Data to a Reference Genome
				3.5.2 Chromosome-Wide A/B Compartment Calling
				3.5.3 Local A/B Compartment Calling
				3.5.4 Domain and Loop Calling
		References
Part III: Applications and Novel Insights into Epigenetics and Epigenomics in Plants
	Chapter 12: The Gene Balance Hypothesis: Epigenetics and Dosage Effects in Plants
		1 Gene Dosage Effects
			1.1 Introduction
			1.2 Dosage Effects of Gene Expression
			1.3 Dosage Involvement in Quantitative Traits
			1.4 Evolutionary Genomics
			1.5 Global Dosage Effects in Plants
		2 Generating Ratio Distributions and Scatter Plots to Analyze Dosage Effects
			2.1 Ratio Distribution Plots
			2.2 Scatter Plots
		3 Conclusions
		Software Implementation
		References
	Chapter 13: Identification and Comparison of Imprinted Genes Across Plant Species
		1 Introduction
			1.1 Assessing and Comparing Imprinted Expression Across Species: A Brief Overview
			1.2 Overview of the Imprinting Analysis Pipeline
			1.3 Obtaining Data
			1.4 Imprinting Criteria
			1.5 Comparing Imprinting Between Species
		2 Challenges and Considerations
			2.1 Identifying SNPs
			2.2 Distinguishing Imprinting and Strain Bias
			2.3 Minimizing Mapping Bias
			2.4 Contamination from Other Tissues
			2.5 Leveraging Data from Replicates
		3 Methods
			3.1 Installation Instructions
			3.2 Required Input Files
			3.3 Genome Preparation
			3.4 Initial Read Quality Filtering and Alignment
			3.5 Identifying Imprinted Genes from Alignment Data
			3.6 Comparing Imprinting Between Species
		4 Illustrative Example Using Real Data
			4.1 Identifying Imprinted Genes in an Arabidopsis thaliana Dataset Starting from Raw Sequencing Reads
			4.2 Identifying Imprinted Genes in a Zea mays Dataset Starting from Count Data
			4.3 Comparing Imprinting Between A. thaliana and Z. mays
		5 Conclusion
		References
	Chapter 14: Epigenetic Approaches in Non-Model Plants
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents
		3 Methods
			3.1 Genomic DNA Isolation, Quality Check, and Quantification
			3.2 Digestion of Genomic DNA
			3.3 Ligation of Barcoded Adapters
			3.4 Per Species Samples Pooling, Pool Clean-Up, and Concentration
			3.5 Fragment Size Selection of Digested-Ligated DNA
			3.6 Nick Translation
				3.6.1 Optional Test GBS PCR
			3.7 Bisulfite Conversion
			3.8 Library Amplification (Final epiGBS PCR)
			3.9 Quantification and Assessment of the Quality of the epiGBS Library
		4 Notes
		References
	Chapter 15: Techniques for Small Non-Coding RNA Analysis in Seeds of Forest Tree Species
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 RNA Extraction
			2.3 Quantitative Real-Time RT-PCR
		3 Methods
			3.1 RNA Extraction
			3.2 RNA Concentration Measurement and Quality Check
				3.2.1 RNA Quantity Measurements
				3.2.2 Checking RNA Quality by Electrophoresis
			3.3 sRNA Library Construction and Sequencing
			3.4 Small RNA Data Analysis
			3.5 Validating sRNA-mRNA Pairs via Quantitative Real-Time RT-PCR
		4 Notes
		References
	Chapter 16: Epigenetic Barcodes for Detection of Adulterated Plants and Plant-Derived Products
		1 Introduction
		2 Materials
			2.1 DNA Extraction
			2.2 Agarose Gel Electrophoresis
			2.3 MS-AFLP Analysis of the Saffron Flower Parts
			2.4 Fluorescent Analysis of MS-AFLP Fragments
		3 Methods
			3.1 Sample Set
			3.2 DNA Extraction and Agarose Gel Electrophoresis
			3.3 DNA Amplificability Via PCR Analysis
			3.4 MS-AFLP Analysis of the Saffron Flower Parts
		4 Notes
		References
	Chapter 17: Plant Epigenetic Stress Memory Induced by Drought: A Physiological and Molecular Perspective
		1 Introduction
		2 Mechanisms of Drought Stress Memory
			2.1 Plant Physiological Perspective
			2.2 Cross-Tolerance Between Different Stresses
			2.3 Reprogramming of Transcriptional Memory Induced by Drought Stress
			2.4 Histone Modifications Associated with Drought Stress Memory Genes
		3 Possible Interactions Between Drought Stress Signals and Chromatin-Mediated Stress Memory
		4 Conclusions and Future Perspectives
		References
	Chapter 18: A Critical Guide for Studies on Epigenetic Inheritance in Plants
		1 Introduction
		2 Materials
			2.1 Biological Material
			2.2 Computational Tools
		3 Methods
			3.1 Study Design
				3.1.1 Stress Treatment
				3.1.2 Control Treatments
				3.1.3 Replication and Sampling
				3.1.4 Treatments with Chemical Compounds Acting on Epigenetic Mechanisms
				3.1.5 Resolution of Epigenetic Changes
			3.2 Data Analysis
				3.2.1 Differential DNA Methylation
				3.2.2 Differential Analysis of Histone Modifications
			3.3 Data Interpretation
		References
Index