Polyploidy: Methods and Protocols

Preface
Acknowledgments
Contents
Contributors
Part I: Comparative Genomics to Study (Paleo-)Polyploidy
Chapter 1: Inference of Ancient Polyploidy from Genomic Data
1 Introduction
2 Whole-Paranome Age Distributions
3 WGD Inference from Empirical Age Distributions
4 Inference of KS-Based Age Distributions Using ``wgd´´
5 Synteny, Collinearity, and Anchor Pairs
6 Other Approaches and Conclusion
References
Chapter 2: Navigating the CoGe Online Software Suite for Polyploidy Research
1 Introduction
2 Materials
2.1 Genome Assemblies
2.2 Online Tools
2.2.1 SynMap
2.2.2 FractBias
2.2.3 GEvo
2.2.4 FeatView and CoGeBlast
2.2.5 SynFind
3 Methods
3.1 SynMap: Basic Default Operation with Ks Calculation
3.2 FractBias: Visualizing Syntenic Depths and Subgenome Biases
3.3 GEvo: Examining Microsynteny Among Genomic Blocks
3.4 SynFind: Discovery of Blocks of Genomes That Are Syntenic to a Window Containing a Target Gene
4 Notes
References
Chapter 3: Inference of Ancient Polyploidy Using Transcriptome Data
1 Introduction
2 Materials
2.1 Plant Genomes and Transcriptomes
3 Methods
3.1 De Novo Assembly of Transcriptomes
3.1.1 Data Preprocessing
3.1.2 Pipeline 1
3.1.3 Pipeline 2
3.1.4 Pipeline 3
3.1.5 BUSCO Evaluation
3.2 Building KS Distributions for the Whole Paranomes
4 Missing Reference Genes and KS Distributions
4.1 Gene Space Reconstructed by Transcriptome Assembly
4.2 Redundant ORFs
5 Gene Family Clustering and KS Distributions
5.1 Presence and Absence of Gene Families
5.2 Size Differences of Gene Families
5.3 Gene Family Sizes and KS Distributions
6 De Novo Assemblies and KS Distributions
7 Discussion
References
Chapter 4: POInT: Modeling Polyploidy in the Era of Ubiquitous Genomics
Abbreviations
1 Polyploidy and the Advent of Genomics
2 Gene Loss, Comparative Genomics, and the Need for Models
3 One Polyploidy or Two?
4 The POInT Computation
4.1 Comments on the POInT Computation
4.2 Example Uses of POInT
5 Future Directions and Concluding Remarks
References
Chapter 5: Applying Machine Learning to Classify the Origins of Gene Duplications
1 Introduction
2 Materials
2.1 Dependencies
2.2 Pipeline Input
3 Methods
3.1 Biologically Informed Approach
3.2 Simulation
3.3 Training
3.4 Testing and Validation
3.5 Empirical Examples
3.6 Summary
References
Part II: Phylogenetics to Study Polyploidy
Chapter 6: Phasing Gene Copies into Polyploid Subgenomes Using a Bayesian Phylogenetic Approach
1 Introduction
1.1 Overview
1.2 homologizer Model and Assumptions
1.3 Practical Considerations
1.4 Getting Started with RevBayes
1.4.1 Installing RevBayes
1.4.2 Running RevBayes
2 Phasing Gene Copies
2.1 Overview
2.1.1 Names in the Sequence Alignment Files
2.1.2 Setting up the Rev File
2.1.3 Running the MCMC
2.1.4 Summarizing the Posterior Distribution
3 Comparing Phasing Models to Distinguish Homeologs from Allelic Variation
3.1 Overview
3.2 Computing Marginal Likelihoods
3.3 Setting up the Alternative homologizer Model
3.4 Comparing the Two homologizer Models
4 Conclusion
References
Chapter 7: Constraining Whole-Genome Duplication Events in Geological Time
1 Introduction
2 Materials
2.1 Required Data
2.2 Annotating Sequences
2.3 Constructing Calibrations
2.4 Examining the Prior
2.5 Running an Analysis
2.6 Interpreting and Visualizing Results
3 Discussion
4 Concluding Remarks
References
Chapter 8: SCORPiOs, a Novel Method to Reconstruct Gene Phylogenies in the Context of a Known WGD Event
1 Introduction
2 Material: Software and Input Data
2.1 SCORPiOs on GitHub and ReadTheDocs
2.2 Structure of the SCORPiOs Package
2.3 A Short Guide to Data Preparation
3 Methods: Improved Gene Trees with SCORPiOs
3.1 Running SCORPiOs in Simple and Iterative Modes
3.2 Visualizing the Corrected Gene Trees
3.3 Summary Statistics After a SCORPiOs Run
3.4 Tracking the Correction History for a Specific Gene Family
4 Conclusion
References
Chapter 9: Inferring Chromosome Number Changes Along a Phylogeny Using chromEvol
1 Introduction
2 Methods
2.1 Input Data
2.2 Model Selection
2.3 Model Adequacy
2.4 Ploidy Inference
2.5 Missing Input
2.5.1 Missing Chromosome Counts
2.5.2 Missing Phylogeny
2.6 Interpreting chromEvol Web Server Results
3 Working Example: Centaurium
4 Notes
References
Chapter 10: PURC Provides Improved Sequence Inference for Polyploid Phylogenetics and Other Manifestations of the Multiple-Cop...
1 Overview
1.1 The Multiple-Copy Problem
1.2 The PURC Approach
1.3 PURC v2.0
2 Materials
2.1 Hardware
2.2 Software
3 Methods
3.1 Installing PURC v2.0
3.2 Preparing Input Files
3.2.1 Barcode File
3.2.2 Reference Sequence File
3.2.3 Map File
3.2.4 Config File
3.3 Running PURC v2.0
3.3.1 Full Run with Demultiplexing and Sequence Inference
3.3.2 Analyses on Previously Demultiplexed Data
4 Conclusions
References
Part III: Analysis of Gene Expression and Regulation in Polyploids
Chapter 11: Analyses of Genome Regulatory Evolution Following Whole-Genome Duplication Using the Phylogenetic EVE Model
1 Introduction
2 Overview of Analytical Pipeline
3 Cross-Species Normalization of Regulatory Phenotypes
4 A Biologist´s Guide to the EVE Model
5 Testing of Evolutionary Hypotheses Using the evemodel
5.1 Testing for WGD-Associated Theta Shift in Regulatory Phenotype Theta
5.2 Beta Shift Following WGD
6 Power Analyses for Shift in Expression Variance or Level
6.1 Shift in Expression Level (Theta)
6.2 Shift in Expression Variance (Beta)
7 Concluding Remarks
References
Chapter 12: Beyond Transcript Concentrations: Quantifying Polyploid Expression Responses per Biomass, per Genome, and per Cell...
1 Introduction
2 Overview of Procedures and Potential Branch Points
3 Detailed Procedures and Recommendations
3.1 Tissue Collection
3.2 Estimating Number of Cells per Sample
3.3 Applying Exogenous Spike-Ins
3.4 Library Construction and Sequencing
3.5 Sequence Data Analysis
4 Summary
References
Chapter 13: A Robust Methodology for Assessing Homoeolog-Specific Expression
1 Introduction
2 Materials
3 Methods
3.1 Ortholog/Homoeolog Identification
3.2 Reference Sequence Biases
3.3 Bayesian Inference of Homoeolog-Specific Expression
References
Part IV: Population Genomics Approaches to Study Polyploidy
Chapter 14: Analyzing Autopolyploid Genetic Data Using GenoDive
1 Introduction
2 Getting the Data in
3 Genetic Diversity and Hardy-Weinberg Equilibrium
4 Quantifying Population Structure
5 Detecting Population Structure
6 Distances
7 Conclusions
References
Chapter 15: Inference of Polyploid Origin and Inheritance Mode from Population Genomic Data
1 Introduction
1.1 Polyploid ``Types´´ and Inference of Inheritance Patterns
1.1.1 Cytogenetic Inferences
1.1.2 Phylogenetic Inferences
1.1.3 Segregation Patterns in Offspring and Gametes
1.1.4 Conflict Between Inferences
1.2 Sources of Mixed Inheritance Patterns
1.2.1 Homeologous Exchanges
1.2.2 Rediploidization
1.2.3 Interspecific Introgression
2 Materials
3 Methods
4 Outlook
5 Notes
References
Chapter 16: Population Genomic Analysis of Diploid-Autopolyploid Species
1 Introduction
2 Variant (SNP) Calling and Filtering
3 Allele Frequency Estimation and Inference of Genetic Diversity
4 Analysis of Population Genetic Structure
4.1 Population Differentiation
4.2 Principal Component Analysis
4.3 Clustering Approaches
4.4 Tree and Network Reconstruction Algorithms
4.5 Interpretation and Example Application in A. arenosa
4.6 Best Practices
5 Inference of Population Demographic History
5.1 Best Practices
6 Population Genomic Inference of Selection
6.1 Assumptions and Limitations
6.2 Best Practices
References
Chapter 17: Inferring the Demographic History and Inheritance Mode of Tetraploid Species Using ABC
1 Introduction
2 Methods
2.1 Demography Inferred from Population Genomic Data
2.2 From Tetraploid Sequences to First Inferences
2.2.1 Defining the Models to be Explored
2.2.2 Processing of Observed and Simulated Sequences
2.2.3 Comparison Between Observation and Simulations
3 Conclusion
References
Part V: Experimental Approaches to Study Polyploidy
Chapter 18: Studying Whole-Genome Duplication Using Experimental Evolution of Chlamydomonas
1 Introduction
2 Selecting the Experimental Strains
3 Determining Ploidy Level
4 Creating Allopolyploid Strains
5 Creating Autopolyploid Strains
6 Designing an Evolution Experiment with Chlamydomonas
7 Quantifying Evolution
7.1 Phenotype
7.2 Genotype
8 Concluding Statement
References
Chapter 19: Studying Whole-Genome Duplication Using Experimental Evolution of Spirodela polyrhiza
1 Introduction
2 Strain Selection
3 Determining Ploidy
4 Making Polyploids
5 Designing Spirodela Evolution Experiments
5.1 General Designs
5.2 Replication
5.3 Growth Conditions
5.4 Preservation
6 Quantifying Phenotypic Change
6.1 Fitness
6.1.1 Growth Rate
6.1.2 Competition Assays
6.1.3 Stress Resistance
6.2 General Morphology
6.3 Pigments and Photosynthetic Parameters
7 Quantifying Transcriptomic Change
8 Quantifying Genetic Change
9 Concluding Statement
References
Chapter 20: Experimental Approaches to Generate and Isolate Human Tetraploid Cells
1 Introduction
2 Material
2.1 Induction of Cytokinesis Failure by DCD Followed by Clone Isolation
2.2 Instruments
2.3 Induction of Mitotic Slippage by Monastrol Followed by FACS Sorting
2.4 Instruments
3 Methods
3.1 Generation of Tetraploid Cells by Cytokinesis Failure
3.2 Growth of Post-Tetraploid Cells and the Analysis of DNA Content
3.3 Generation of Tetraploid Cells by Inducing Mitotic Slippage and Synchronization in G1
3.4 FACS Sorting to Isolate Diploid and Polyploid Populations
4 Notes
References
Chapter 21: Measuring Cellular Ploidy In Situ by Light Microscopy
1 Introduction
2 Materials
2.1 Siliconized Coverslips
2.2 Solutions
2.3 Squashing Supplies
2.4 Staining Supplies
3 Methods
3.1 Tissue Squash Protocol for Measuring Nuclear Ploidy from Decondensed Interphase Nuclei
3.1.1 Tissue Preparation
3.1.2 Imaging and Ploidy Measurement
3.2 Tissue Squash Protocol for Measuring Nuclear Ploidy from Condensed Chromosomes
3.2.1 Tissue Preparation
3.2.2 Imaging and Chromosome Visualization
4 Notes
References
Chapter 22: Using Mosaic Cell Labeling to Visualize Polyploid Cells in the Drosophila Brain
1 Introduction
2 Materials
3 Methods
3.1 Fly Husbandry and Cell Labeling In Vivo
3.1.1 To Label Cells During Development ``Early-FLP´´: This Will Allow Detection of Fusion Events in the Adult Brain
3.1.2 To Label Cells in the Adult ``Late-FLP´´: This Will Allow Detection of Cell Cycle Reentry Events in the Adult Brain
3.2 Dissection, Fixation, Immunostaining (for Nuclear Lamina or Cell Type-Specific Markers), and Imaging
3.3 Preparing Brain Tissues for Flow Cytometry Analysis
4 Notes
References
Part VI: Sequencing, Assembling, Editing and Engineering Polyploid Genomes
Chapter 23: Sequencing and Assembly of Polyploid Genomes
1 Introduction
2 Overview of Polyploid Genomes
2.1 Early History of Polyploid Genome Assembly
2.2 Rapid Development of Polyploid Genome Assembly in Recent Years
2.3 Lack of High-Quality Reference Genomes of Polyploid Organisms
3 Sequencing of Polyploid Genomes
3.1 Overview of the Sequencing Technologies
3.2 Long-Read Sequencing
3.3 Linked-Read Sequencing
3.4 Long-Range Technologies for Genome Scaffolding
4 Algorithmic Challenges During a Polyploid Genome Assembly
5 Computational Approaches of Assembly of Polyploid Genomes
5.1 Resolving Haplotype Assembly by Reference-Based Phasing
5.2 De Novo Haplotype Assembly by Local Phasing
5.3 Chromosome-Scale Haplotype-Resolved Assembly
6 Role of High-Quality Polyploid Genome Assembly and Prospects
References
Chapter 24: Genome Editing by CRISPR/Cas9 in Polyploids
1 Introduction
2 Materials
2.1 Sequenced Genome Databases
2.2 Tools for sgRNA Selection
2.3 CRISPR/Cas9 Vector Requirements
2.4 Tools and Approaches for CRISPR/Cas9-Mediated Genome Editing Screening
3 Methods
3.1 Target Gene Sequences Retrieving
3.2 sgRNA Prediction
3.2.1 CRISPOR
3.2.2 CRISPR MultiTargeter
3.3 sgRNA Selection
3.4 Electrophoresis-Based Detection of Mutations
3.4.1 Large InDels Detection by PCR
3.4.2 Detection of Genome Editing by Restriction Analysis of the PCR Product
3.5 Homoeolog-Specific Primer Design
3.6 Analysis of Genome Editing by Sequencing
3.7 Sequences Alignment by MEGA
3.8 CRISPR/Cas9 Edited Line Selection
4 Notes
References
Chapter 25: Developing a CRISPR System in Nongenetic Model Polyploids
Abbreviations
1 Introduction
2 Materials
2.1 Molecular Cloning Reagents
2.2 Protoplast Transient Assay
2.3 Agrobacterium-Mediated Transformation
3 Methods
3.1 sgRNA Design and CRISPR Plasmid Construction
3.2 Protoplast Isolation and Transformation
3.2.1 Prepare Plant Material
3.2.2 Protoplast Isolation
3.2.3 Protoplast Transformation
3.3 Tragopogon Transformation
3.3.1 Agrobacterium Culture
3.3.2 Transformation
4 Notes
References
Chapter 26: Efficiently Editing Multiple Duplicated Homeologs and Alleles for Recurrent Polyploids
1 Introduction
2 Materials
2.1 RNA Extraction
2.2 PCR Cloning
2.3 FISH
2.4 Gene Editing with CRISPR/Cas9
2.5 Histologic Section
2.6 qPCR
2.7 Western Blot
2.8 Immunofluorescence Staining
3 Methods
3.1 RNA Extraction
3.2 cDNA Synthesis
3.3 PCR Cloning and Sequence Analysis
3.4 FISH
3.4.1 Preparation of Chromosome Metaphases
3.4.2 Probe Labeling
3.4.3 Hybridization
3.4.4 Post-Hybridization Washing and Signal Detection
3.5 Gene Editing with CRISPR/Cas9
3.5.1 Target sgRNA Sequence Selection and Design
3.5.2 Synthesis of Target sgRNA Primers
3.5.3 Preparation of sgRNA
3.5.4 Preparation of Cas9 mRNA
3.5.5 Microinjection
3.5.6 Generation and Identification of Mutant Lines
3.6 Histological Comparison Among WT and Mutant Lines
3.6.1 Paraffin Section Making
3.6.2 Hematoxylin and Eosin (H & E) Staining
3.7 Expression Changes Among WT and Mutant Lines by qPCR
3.8 Expression Changes Among WT and Mutant Lines by Western Blot
3.8.1 Extraction of Total Protein from Tissue
3.8.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3.8.3 Transferring the Protein Gel to a PVDF Membrane
3.8.4 Immunodetection of Western Blot
3.9 Expression Changes Between WT and Mutant Lines by Immunofluorescence Staining of Paraffin Sections
4 Notes
References
Index
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