Plant Cytogenetics and Cytogenomics: Methods and Protocols

Description

This book provides an up-to-date account of the most widespread methods used by specialists in the field of plant cytogenetics and the emerging field of cytogenomics that will likely soon be adapted by more labs. From the classical basic karyological approaches to the most recent genomics-informed and computational methods, the volume explores genome size and ploidy level estimation, chromosome fixation, preparation, and manipulation, banding and staining techniques, in situ hybridization, as well as numerous methods that integrate cytogenetics with bioinformatics and computational genomics. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Plant Cytogenetics and Cytogenomics: Methods and Protocols serves as an ideal resource for plant scientists interested in molecular and evolutionary biology, breeding, systematics, and plant -omics in general. Preface Contents Contributors Part I: Introduction Chapter 1: Plant Cytogenetics: From Chromosomes to Cytogenomics 1 What Cytogenetics Can Answer 2 Studying Chromosomes: From Simple Staining to Multicolor Fluorescence Cytogenetics 2.1 Chromosome Banding 2.2 In Situ Hybridization 3 Preparing Chromosomes: Metaphase, Interphase, Meiosis, and Fibers 4 Dyes, Clones, and Synthetic Probes 4.1 Fluorescent Dyes 4.2 Cloned or PCR-Amplified FISH Probes 4.3 Genome and Chromosome Painting 5 Chromosome Biology and Genomics 6 Conclusion References Part II: Sizing the Nucleus: Methods for Genome Size and Ploidy Level Estimation Chapter 2: The Use of Flow Cytometry for Estimating Genome Sizes and DNA Ploidy Levels in Plants 1 Introduction 1.1 Terminology of Genome Size and Ploidy Level 1.2 The Importance of Ploidy Level and Genome Size 2 Materials 2.1 Plant Material 2.1.1 Plant Material Suitable for DNA Content Estimation 2.1.2 Seeds 2.1.3 Plant Material Preservation: Desiccated Tissues 2.1.4 Plant Material Preservation: Other Approaches 2.2 Reference Standards 2.2.1 Standards for Ploidy Analysis 2.2.2 Standards for the Estimation of Genome Size 2.3 Equipment 2.4 Reagents 2.4.1 Nuclei Isolation Buffers 2.4.2 Fluorochromes 2.4.3 Other Reagents 2.5 Equipment Setup 3 Methods 3.1 Field Sampling, Transport, and Storage of Plant Material 3.2 Isolation and Staining of Plant Nuclei 3.2.1 Isolation and Staining of Plant Nuclei from Fresh Plant Material Using the One-Step Protocol 3.2.2 Isolation and Staining of Plant Nuclei from Fresh Plant Material Using the Two-Steps Protocol 3.2.3 Isolation of Plant Nuclei from Desiccated Material 3.2.4 Isolation of Plant Nuclei from Seeds 3.3 Flow Cytometric Analysis of Isolated Nuclei 3.3.1 Estimating Nuclear DNA Content in Absolute Units 3.3.2 Estimating the DNA-Ploidy Level Estimating DNA-Ploidy Level Using Internal Standardization Estimating DNA-Ploidy Level Using External Standardization 4 Notes References Chapter 3: Nuclear DNA Content Estimation of Seaweed by Fluorimetry Analysis 1 Introduction 2 Materials 2.1 Laboratory Expendables 2.2 Solutions and Reagents 2.3 Chicken Erythrocytes (RBC) 2.4 Devices 2.5 Software 3 Methods 3.1 Sample Collection and Fixation 3.2 Sample Preparation for Fluorimetry 3.3 RBC Preparation for Fluorimetry 3.4 Sample and RBC Staining with DAPI 3.5 Fluorimetry Observations and Sample Measurements 3.6 Image Analysis 3.7 Estimation of Nuclear DNA Content (in Picograms and Mbp) 3.8 Ploidy Peak Identification and Interpretation 4 Notes References Chapter 4: K-Mer-Based Genome Size Estimation in Theory and Practice 1 Introduction 2 The Theory of K-Mer-Based Genome Size Analysis 2.1 Genome Size Terminology 2.2 K-mer Analysis: Terminology and Theory 2.2.1 Average Genome Coverage 2.2.2 K-mers and K-mer Histograms 2.3 K-mer Analysis of Real-Life Sequencing Data 2.3.1 Parameters That Affect the K-mer Distribution of Real-Life Sequencing Data 2.3.2 The Many Other Peaks in K-mer Histograms 3 Methods 3.1 Acquisition and Preprocessing of Short-Read Data 3.2 Generating K-mer Frequency Histograms 3.3 Genome Size Prediction Using K-mer Frequency Histograms 4 Notes References Chapter 5: A Bioinformatic Pipeline to Estimate Ploidy Level from Target Capture Sequence Data Obtained from Herbarium Specime... 1 Introduction 2 Data and Software Installation 2.1 Data 2.2 Software 3 Bioinformatic Pipeline 3.1 Map to Reference 3.2 BAM Transformation 3.3 nQuire Analysis 3.4 Calculating the Allelic Ratios and Estimating the Level of Ploidy 4 Notes References Part III: Getting to the Chromosomes: Methods for Chromosome Fixation, Preparation, and Manipulation Chapter 6: Nitrous Oxide-Induced Metaphase Arrest: A Technique for Somatic Chromosome Analysis 1 Introduction 2 Materials 2.1 Preparation 2.2 Nitrous Oxide Treatment 2.3 Root Fixation and Storage 3 Methods 3.1 Root Preparation 3.2 Nitrous Oxide Treatment 3.3 Root Fixation and Storage 4 Notes References Chapter 7: Preparation of Mitotic Chromosomes with the Squash Technique 1 Introduction 2 Materials 2.1 Solutions 2.2 Laboratory Devices and Other Equipment 3 Methods 3.1 General Procedure 3.2 Reusing the Preparations for Further Assays (Fluorochrome Banding, FISH) 4 Notes References Chapter 8: Preparation of Mitotic Chromosomes with the Dropping Technique 1 Introduction 2 Materials 2.1 Plant Material 2.2 Solutions 2.3 Equipment 3 Methods 3.1 Fixation of Meristematic Tissue 3.2 Enzymatic Digestion 3.3 Purification of Nuclei 3.4 Dropping the Nuclei onto the Slides 3.5 Optimize the Procedure 4 Notes References Chapter 9: Laser Capture Microdissection: From Genomes to Chromosomes, from Complex Tissue to Single-Cell Analysis 1 Introduction 2 Materials 2.1 Root Synchronization 2.2 Chromosome Dropping 2.3 Tissue Preparation 2.4 Laser Capture Microdissection 3 Methods 3.1 Root Synchronization 3.2 Chromosome Dropping 3.3 Tissue Preparation 3.4 Laser Capture Microdissection 4 Notes References Chapter 10: Flow Cytometric Analysis and Sorting of Plant Chromosomes 1 Introduction 2 Materials 2.1 Plant Material 2.2 Reagents and Solutions 2.2.1 Reagents and Solutions for Cell Cycle Synchronization and Accumulation of Cells at Mitotic Metaphase 2.2.2 Reagents and Solutions for the Preparation of Chromosome Suspensions and Chromosome Sorting 2.2.3 Reagents and Solutions for Fluorescence In Situ Hybridization in Solution (FISHIS) 2.2.4 Reagents and Solutions for Fluorescence In Situ Hybridization (FISH) 2.3 Equipment 2.4 Consumables and Disposables 3 Methods 3.1 Seed Germination and Root Growth 3.2 Induction of Cell Cycle Synchrony in Root Tip Meristems 3.3 Metaphase Accumulation (See Note 4) 3.4 Preparation of Chromosome Suspensions 3.5 Microsatellite Labeling Using FISH in Suspension (FISHIS) 3.6 Chromosome Analysis and Sorting Using Flow Cytometry 3.7 Estimation of Purity in Sorted Fractions Using FISH (See Note 13) 4 Notes References Part IV: Putting on Color: Banding and Staining Techniques Chapter 11: C-Banding of Plant Chromosomes 1 Introduction 2 Materials 2.1 Solutions 2.2 Equipment 3 Methods 3.1 Seed Germination 3.2 Pretreatment and Fixation 3.3 N.L. Darvey C-Banding Protocol for Root Tips in Cereals 3.4 ``Spanish´´ Procedure for Meiotic and Mitotic Chromosomes in Cereals 3.5 T.R. Endo Protocol for Wheat Root Tips 4 Notes References Chapter 12: CMA/DAPI Banding of Plant Chromosomes 1 Introduction 2 Materials 2.1 Equipment and Supplies 2.2 Chemicals 2.3 Solutions 3 Methods 3.1 Collecting Material 3.2 Slide Preparation 3.3 CMA/DAPI Staining 4 Notes References Chapter 13: Silver Nitrate Staining of Nucleolar Organizer Regions (Ag-NORs) in Plant Chromosomes 1 Introduction 2 Materials 2.1 Mitotic Metaphases 2.2 Solutions for NOR Techniques 2.3 Interphase Nuclei for Nucleolar Technique 2.4 Solutions for Nucleolar Technique 3 Methods 3.1 Techniques for Ag-NOR Based on Goodpasture and Bloom (1975) 3.2 Techniques for Ag-NOR Based on Hizume et al. (1980) and Rufas et al. (1982) 3.3 Techniques for Ag-NOR Based on Rufas et al. (1982) 3.4 Techniques for Ag-NOR Based on Howell and Black (1980) and Mehra et al. (1985) 3.5 Technique for Nucleolus Staining Based on Fernndez-Gómez (1969) 4 Notes References Chapter 14: Chromatin Immunostaining of Plant Nuclei 1 Introduction 2 Materials 2.1 Plant Materials 2.2 Abiotic Treatment Solutions 2.3 Immunostaining Reagents 2.4 Equipment 2.5 Imaging Equipment 3 Methods 3.1 Treatment of Abiotic Stress to Rice Samples 3.2 3D-Chromatin Immunostaining of Whole Root Tissue 3.3 2D-Chromatin Immunostaining for Single Nucleus 3.4 Imaging of Microscope 3.5 Quantitative Image Analysis 4 Notes References Part V: Labeling DNA: In Situ Hybridization and Other Methods Using Fluorescent Labels Chapter 15: Critical Steps in DAPI and FISH Imaging of Chromosome Spread Preparations 1 Introduction 2 Materials 3 Methods 3.1 DAPI Image Capture and Imaging 4 Notes References Chapter 16: Formamide-Free Genomic In Situ Hybridization (ff-GISH) 1 Introduction 2 Materials 2.1 Citrate Buffer pH 4.8 2.2 Enzyme Cocktail 2.3 20 x SSC pH 7.0-7.2 2.4 2 x SSC pH 7.0-7.2 2.5 DNase-Free RNaseA (See Note 2) 2.6 Pepsin 2.7 Hybridization Mix 2.8 Blocking Solution 2.9 Detection Solution 2.10 Equipment 3 Methods 3.1 Chromosome Preparation 3.2 Extraction and Labeling of Parental Genomic DNAs 3.3 Formamide-Free Genomic In Situ Hybridization (ff-GISH) (see Note 4) 4 Notes References Chapter 17: The Use of Ribosomal DNA for Comparative Cytogenetics 1 Introduction 1.1 Cloned rDNA Probes 1.2 Oligonucleotide Probes 2 Materials 2.1 Amplifying and Cloning the rDNA Sequences from Genomic DNA 2.1.1 PCR Amplification of the rDNA Sequences from the Genomic DNA 2.1.2 Gel Electrophoresis and Purification of the DNA Fragments 2.1.3 Ligation and Transformation 2.2 Labeling the Cloned Probes 2.3 Fluorescence In Situ Hybridization 3 Methods 3.1 Amplifying and Cloning the rDNA Sequences from the Genomic DNA 3.1.1 PCR Amplification of the rDNA Sequences from the Genomic DNA 3.1.2 Gel Electrophoresis and Purification of the DNA Fragments 3.1.3 Ligation and Transformation 3.2 Probe Labeling 3.2.1 Nick-Translation Labeling 3.2.2 Ethanol Precipitation of the DNA Probes 3.2.3 PCR Labeling 3.3 Fluorescence In Situ Hybridization 3.3.1 FISH with 35S rDNA Sequences After Silver Staining 3.3.2 FISH with the 35S and 5S rDNA Sequences After CMA3/DAPI Staining 3.3.3 Reprobing of FISH Preparations 3.4 The Microscopic Image of rDNA Loci 3.5 The Comparative Analyses of rDNA Loci 4 Notes References Chapter 18: Identification of the Sequence and the Length of Telomere DNA 1 Introduction 1.1 Telomere Motif Identification 1.2 Telomere Length Measurement 2 Materials 2.1 High Molecular Weight DNA (HmwDNA) Preparation in Low-Melting Temperature Agarose Plugs 2.2 BAL31 Treatment 2.3 Extraction of DNA from Agarose Plugs Using β-Agarase I 2.4 Data Processing with Tandem Repeat Finder (TRFi) 2.5 Digestion of DNA by Restriction Enzyme(s) 2.5.1 Low Molecular Weight DNA 2.5.2 High Molecular Weight DNA 2.6 Separation of Digested DNA by Agarose Gel Electrophoresis 2.6.1 Conventional Horizontal Agarose Gel Electrophoresis 2.6.2 Pulse-Field Agarose Gel Electrophoresis 2.7 Southern Blot 2.8 Radioactive Labeling of Telomeric Probe 2.8.1 End Labeling of Telomeric Oligonucleotide 2.8.2 Random Labeling of Telomeric Probe 2.9 Hybridization of DNA Immobilized on Nylon Membrane 2.10 Radioactive Signal Detection 2.11 Image Analysis 3 Methods 3.1 High Molecular Weight DNA (HmwDNA) Preparation in Low-Melting Temperature Agarose Plugs 3.2 BAL31 Treatment 3.3 Extraction of DNA from Agarose Plugs Using β-Agarase I 3.4 Next-Generation Sequencing of BAL31 Treated and Untreated HmwDNA 3.5 Tandem Repeats Finder (TRFi) and Custom-Made Scripts Collectively Called Tandem Repeat Merger 3.6 Digestion of DNA by Restriction Enzyme(s) 3.6.1 Digestion of Low Molecular Weight DNA 3.6.2 Digestion of High Molecular Weight DNA 3.7 Separation of Digested DNA by Agarose Gel Electrophoresis 3.7.1 Conventional Horizontal Agarose Gel Electrophoresis 3.7.2 Pulse-Field Agarose Gel Electrophoresis (PFGE) 3.8 Southern Blot 3.9 Radioactive Labeling of Telomere Probes 3.9.1 End Labeling of the Telomere Oligonucleotide 3.9.2 Random Labeling of the Telomere Probe 3.10 Hybridization of the DNA Immobilized on Nylon Membrane 3.11 Radioactive Signal Detection 3.12 Image Analysis-Web-Based Analyzer of the Length of Telomeres (WALTER) 4 Notes References Chapter 19: Chromosome Painting Using Chromosome-Specific BAC Clones 1 Introduction 2 Materials 2.1 Chromosome Preparation Pretreatment 2.2 Probe Labeling 2.3 Probe Preparation and In Situ Hybridization 2.4 Fluorescence Detection of Hybridized Probes 3 Methods 3.1 Chromosome Preparation Pretreatment 3.2 Probe Labeling by Nick Translation 3.3 Probe Preparation and In Situ Hybridization 3.4 Post-hybridization Washing and Fluorescence Detection of Hybridized Probes 4 Notes References Chapter 20: CRISPR-FISH: A CRISPR/Cas9-Based In Situ Labeling Method 1 Introduction 2 Materials 2.1 For Chromosome Preparation and CRISPR-FISH 2.1.1 Plant Material 2.1.2 Buffers, Chemicals, Reagents, and Enzymes 2.1.3 CRISPR Components 2.1.4 Equipment 2.1.5 Imaging Equipment 2.2 CRISPR-FISH onto Formaldehyde-Fixed Chromosomes 2.3 CRISPR-FISH onto Plant Tissue Sections 3 Methods 3.1 Mitotic Chromosomes-A Steam Dropping Chromosome Preparation Method 3.2 Meiotic Chromosomes-Spreading Method for Chromosomes Fixed in Ethanol/Acetic Acid (3:1) 3.3 Preparation of the Ribonucleoprotein (RNP: Cas9+sgRNA) 3.3.1 sgRNA Formation 3.3.2 RNP Complex Assembly 3.4 CRISPR-FISH onto Ethanol: Acetic Acid (3:1) Fixed Chromosomes and Nuclei 3.5 CRISPR-FISH onto Formaldehyde-Fixed Chromosomes 3.5.1 Slide Preparation with Formaldehyde-Fixed Cells Using a Cytospin 3.5.2 Slide Preparation with Formaldehyde-Fixed Cells Without Using a Cytospin 3.6 CRISPR-FISH onto Formaldehyde-Fixed Chromosomes 3.7 Combination of CRISPR-FISH and Immunostaining 3.8 CRISPR-FISH onto Plant Tissue Sections 3.8.1 Fixation of Plant Tissues 3.8.2 Clearing of Plant Tissues by ePro-ClearSee Method 3.8.3 Sectioning of Tissue 3.8.4 Decrosslinking of Tissue 3.8.5 CRISPR-FISH 3.8.6 Immunohistochemistry After CRISPR-FISH of Tissue Sections 4 Notes References Chapter 21: Preparation of Male Meiotic Chromosomes for Fluorescence In Situ Hybridization and Immunodetection with Major Focu... 1 Introduction 2 Materials 2.1 Plant Material 2.2 Materials and Reagents for Fluorescence In Situ Hybridization (FISH) 2.2.1 Chromosome Preparation by Spreading 2.2.2 Prehybridization Step 2.2.3 Probe Labeling Using Nick Translation Mix 2.2.4 Post-hybridization Step 2.3 Immunolabeling 2.3.1 Chromosome Preparation by Spreading 2.3.2 Antibodies Dilution 3 Methods 3.1 Fluorescence In Situ Hybridization (FISH) 3.1.1 Chromosome Preparation by Spreading 3.1.2 Labeling the Probes 3.1.3 Prehybridization Step 3.1.4 Fluorescence In Situ Hybridization 3.1.5 Washing the Probes 3.2 Immunolabeling 3.2.1 Chromosome Preparation by Spreading 3.2.2 Processing the Slides and Applying Antibodies 4 Notes References Chapter 22: Extended DNA Fibers for High-Resolution Mapping 1 Introduction 2 Materials 2.1 Isolation of Nuclei 2.2 DNA Fiber Preparation 2.3 Probe Labeling 2.4 In Situ Hybridization 3 Methods 3.1 Isolation of Nuclei 3.2 Preparation of DNA Fibers 3.3 FISH to DNA Fibers 3.3.1 FISH to DNA Fibers (Standard) 3.3.2 FISH to DNA Fibers Using Separate Denaturation of Probe and Target DNA 3.4 Post-hybridization Wash 4 Notes References Chapter 23: Visualizing Chromosome Territories and Nuclear Architecture of Large Plant Genomes Using Alien Introgressions 1 Introduction 2 Materials 2.1 Probe Preparation 2.2 Blocking DNA Preparation 2.3 Fixation of Plant Material 2.4 Sample Preparation, Flow Sorting 2.5 Embedding Nuclei in Acrylamide Gel 2.6 3D-Fish/Gish 2.7 Imaging, Data Analysis in Imaris 3 Methods 3.1 Probe Preparation 3.2 Blocking DNA Preparation 3.3 Fixation of Plant Material 3.4 Sample Preparation, Flow Sorting 3.5 Embedding Nuclei in Acrylamide Gel 3.6 3D-Fish/Gish 3.7 Imaging, Data Analysis in Imaris 4 Notes References Chapter 24: Visualization of the Nucleolus Using 5′ Ethynyl Uridine 1 Introduction 2 Materials 2.1 Plant Growth 2.2 Labeling and Fixation 2.3 Nuclei Isolation 2.4 EU Detection 3 Methods 3.1 Labeling and Fixation 3.2 Nuclei Isolation 3.3 EU Detection in Roots (See Note 3 and Fig. 3) 3.4 EU Detection in Nuclei (See Fig. 4) 4 Notes References Part VI: Leveraging Data onto Nuclei: Genomics-Informed Methods Chapter 25: Bioinformatic Prediction of Bulked Oligonucleotide Probes for FISH Using Chorus2 1 Introduction 2 Materials 2.1 Dependencies and Software Installation 2.2 Preparation of Target Genome Sequences 2.3 Preparation of Whole-Genome Sequencing (WGS) Data 3 Methods 3.1 Design of Genome-Scaled Oligo Probes for Rice 3.2 Find Conserved Oligos Between Genetically Related Species 3.3 Design Probes for Closely Related Species without a Reference Genome 3.4 Probes Selection for Target Regions 4 Notes References Chapter 26: Visualization of Oligonucleotide-Based Probes Along Pseudochromosomes Using RIdeogram, KaryoploteR, and Circlize (... 1 Introduction 2 Materials 2.1 Input Data 2.1.1 Design of Individual Oligonucleotide-Based Probes 2.1.2 Designed Probes Available 2.1.3 Input Data Used in This Chapter for Exemplary Representation 2.2 Chorus2 2.3 Blast 2.4 RIdeogram 2.5 KaryoploteR 2.6 Circlize (Circos Package for R) 3 Methods 3.1 General Usage of the Provided Code 3.2 Designing and Selection of Oligonucleotide-Based Probes 3.2.1 Downloading the Reference Genome Files 3.2.2 Downloading Short Read Data Files 3.2.3 Probe Design, Filtering, and Selection Using Chorus2 3.3 Determining Positions of Oligonucleotide-Based Probes 3.3.1 Download the Reference of the Second Species 3.3.2 Convert BED to FASTA (bed2fasta.py) 3.3.3 Similarity Search with BLAST 3.4 Data Preparation 3.4.1 Create Reference-Related Files (ref_related.py) 3.4.2 Convert BED to GFF (bed2gff.py) 3.4.3 Convert M6 to GFF (m62gff.py) 3.4.4 Define Oligonucleotide-Base Probe Regions (spots) 3.5 Visualization of Oligonucleotide-Based Probes 3.5.1 Linear Plots Using RIdeogram 3.5.2 Linear Plots Using KaryoploteR 3.5.3 Circular Plots Using Circlize 4 Notes References Chapter 27: Bulked Oligo-FISH for Chromosome Painting and Chromosome Barcoding 1 Introduction 2 Materials 2.1 Material Provided by Daicel Arbor Biosciences Company (Formerly MYcroarray) 2.2 Emulsion PCR 2.3 Debubbling PCR 2.4 In Vitro Transcription 2.5 Reverse Transcription 2.6 Enzymatic RNA Removal 2.7 Chromosome Spread Preparation 2.8 Fluorescence In Situ Hybridization (FISH) 2.9 Equipment 3 Methods 3.1 Preparing Single-Stranded Labeled Probes from MYtags Immortal Libraries 3.1.1 Emulsion PCR-Part 1 (Option 1) 3.1.2 Debubbling PCR-Part 1 (Option 2) 3.1.3 In Vitro Transcription-Part 2 3.1.4 Reverse Transcription-Part 3 3.1.5 Enzymatic RNA Removal-Part 4 3.2 Mitotic Metaphase Chromosome Spread Preparation (Option 1) 3.3 Mitotic Metaphase Chromosome Spread Preparation (Option 2-Dropping Technique) 3.4 Meiotic Pachytene Chromosome Spread Preparation 3.5 Fluorescence In Situ Hybridization (FISH) 4 Notes References Chapter 28: Flow Sorting-Assisted Optical Mapping 1 Introduction 2 Materials 2.1 Plant Material 2.2 Reagents and Solutions 2.2.1 Reagents and Solutions for Preparation of Nuclei Suspensions and Nuclei Sorting 2.2.2 Reagents and Solutions for Preparation of HMW DNA 2.2.3 Reagents and Solutions for HMW DNA Quantification and Quality Control 2.3 Equipment 2.4 Consumables and Disposables 3 Methods 3.1 Preparation of the Material for Flow Sorting 3.1.1 Preparation of Nuclei Suspensions from Root Tips 3.1.2 Preparation of Nuclei Suspensions from Leaves 3.2 Nuclei Purification Using Flow Cytometry 3.3 Preparation of Agarose Miniplugs 3.4 DNA Quality Control by Pulsed-Field Gel Electrophoresis (Optional) 3.4.1 Control of Intact DNA 3.4.2 DNA Accessibility Test 3.5 Plug Washes and RNase Treatment 3.6 DNA Release and Drop Dialysis 3.7 DNA Quantification by Qubit BR Assay Kit 3.8 DLS Labeling and Data Collection 4 Notes References Chapter 29: Chromosome Conformation Capture of Mitotic Chromosomes 1 Introduction 2 Materials 2.1 Chromosome Sample Preparation 2.2 Flow Sorting 2.3 Hi-C Library Preparation 2.4 Sequencing Library Preparation 2.5 Equipment 3 Methods 3.1 Chromosome Sample Preparation 3.2 Chromosome Flow Sorting 3.3 Hi-C Library Construction 3.4 Sequencing Library Preparation 3.5 Data Analysis 4 Notes References Chapter 30: Analysis of 5S rDNA Genomic Organization Through the RepeatExplorer2 Pipeline: A Simplified Protocol 1 Introduction 2 Materials 2.1 De Novo Sequencing 2.2 Sequencing Data Extracted from Public Databases 3 Methods 3.1 Read Preprocessing for the RepeatExplorer2 Pipeline 3.2 RepeatExplorer2 Cluster Graphs Reveal Organization of 5S rDNA in Plant Genomes 3.3 Wet-Lab Verification of 5S rDNA Organization by Southern Blot Hybridization 3.4 Quantification of Homoeologous 5S rRNA Gene Families 3.5 Identification of 5S rRNA Gene Families in Hybrids Using Clustering Analysis 3.6 Other Elements Occasionally Found in the 5S rDNA Clusters 4 Caveats and Final Remarks References Part VII: Software and Online Plant Cytogenetics & Genomics Resources Chapter 31: Tools for Drawing Informative Idiograms 1 Introduction 1.1 KaryoMeasure: Overall Software Design and Architecture 2 Materials 3 Methods 3.1 Karyotype Analysis 3.2 Idiogram Generation 4 Notes References Chapter 32: Using ChromEvol to Determine the Mode of Chromosomal Evolution 1 Introduction 1.1 Background: The Importance of Chromosomal Evolution 1.2 Before ChromEvol Modeling: The Limitations of Parsimony 1.3 A New Probabilistic Model to Infer Chromosome Number Evolution: ChromEvol v.1.0 1.4 New Parameters to Model Polyploid Series from a Base Number: ChromEvol v.2.0 1.5 Complex Models: Multiple Chromosome Number Evolution Models in the Phylogeny 1.6 Complex Models: Chromosome State Speciation and Extinction Model (ChromoSSE) 2 Experimental Examples 2.1 Using ChromEvol v.1.0 and ChromEvol v.2.0 on Genus Centaurium (Gentianaceae) 2.2 Experimental Example on Genus Centaurium (Gentianaceae) Using Bichrom Model 2.3 Experimental Example on the Genus Centaurium (Gentianaceae) Using ChromoSSE Model 3 Future Perspectives References Chapter 33: Online Resources Useful for Plant Cytogenetics and Cytogenomics Research 1 Introduction 2 Databases 2.1 Chromosome Numbers and Special Chromosomes 2.1.1 Taxa-Specific Databases 2.2 Genome Size and Flow Cytometry 2.3 Cytogenetics 2.4 Miscellanea 3 Analysis Tools 3.1 Online Applications and Tools 3.2 Repositories References Index Index