Plant Cytogenetics: Methods and Protocols (Methods in Molecular Biology, 1429)

✍ Scribed by Shahryar F. Kianian (editor), Penny M. Avoles Kianian (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 203
• Category: Library

✦ Synopsis

This volume covers a range of methods used in plant cytogenetics, beginning with basic analysis of chromosomes and visualizing gene locations, to manipulating and dissecting chromosomes, and then focusing on less understood features of chromosomes such as recombination initiation sites and epigenomic marks. The methods described in Plant Cytogenetics: Methods and Protocols build on each other and provide, those new to the field, with a comprehensive platform to support their research endeavours, while also introducing advanced techniques to experienced researchers. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting edge and thorough, Plant Cytogenetics: Methods and Protocols, is a valuable resource for anyone who is interested in the diverse and wonderfully complex field of cytogenetics.

✦ Table of Contents

Foreword: The Modern Cytogenetics Tool Box—A Picture Is Still Worth a Thousand Words
Preface
Contents
Contributors
Chapter 1: C-Banding of Plant Chromosomes
1 Introduction
2 Materials
2.1 Solutions
2.2 Materials
3 Methods
3.1 Root-Tip Metaphase Chromosome Pretreatment
3.2 Chromosome Preparations
3.3 C-Banding Protocol
4 Notes
References
Chapter 2: Chromosome Painting by GISH and Multicolor FISH
1 Introduction
2 Materials
2.1 Equipment and Supplies
2.2 Chemicals
2.3 Solutions
3 Methods
3.1 GISH Procedure
3.1.1 Isolation of Genomic DNA for Probe and Blocking
3.1.2 Probe Labeling
3.1.3 Blocking DNA Preparation
3.1.4 Chromosome Preparation
3.1.5 Slide Pretreatment
3.1.6 Hybridization
3.1.7 Post-­hybridization Wash and Signal Detection
3.2 Multicolor FISH Procedure
3.2.1 Isolation of Genomic DNA for Blocking and the DNA for Probe
3.2.2 Probe Labeling
3.2.3 Blocking DNA Preparation
3.2.4 Slide Preparation
3.2.5 Slide Pretreatment
3.2.6 Hybridization
3.2.7 Post-­hybridization Slide Wash and Signal Detection
4 Notes
References
Chapter 3: Fluorescent In Situ Hybridization on Extended Chromatin Fibers for High-Resolution Analysis of Plant Chromosomes
1 Introduction
2 Materials
2.1 Supplies and Equipment
3 Methods
3.1 Isolation of Plant Nuclei
3.2 Preparation of Extended DNA Fibers
3.3 Labeling of DNA Probes for FISH
3.3.1 Labeling of DNA Probes for FISH by PCR
3.3.2 Labeling of DNA Probes for FISH by Nick Translation
3.3.3 Assessment of the Labeling Quality
3.4 Fluorescent In Situ Hybridization on DNA Fibers
3.4.1 Hybridization of the Probe
3.4.2 Post-hybridization Washing
3.4.3 Antibody Reaction
3.5 UV Microscopy
4 Notes
References
Chapter 4: Tyramide Signal Amplification: Fluorescence In Situ Hybridization for Identifying Homoeologous Chromosomes
1 Introduction
2 Materials
2.1 Probe Generation
2.1.1 Probe DNA Isolation, Electrophoresis, and Gel Extraction
2.1.2 Probe Labeling
2.1.3 Solutions for DNA Concentration, Washing, and Preservation
2.2 Generation of Chromosome Preparations for FISH Using TSA
2.2.1 Metaphase Spreads
2.2.2 TSA-FISH
3 Methods
3.1 Probe Generation
3.1.1 PCR Primer Design
3.1.2 Genomic DNA Extraction
3.1.3 PCR Amplification
3.1.4 Agarose Electrophoresis
3.1.5 DNA Extraction from the Agarose Gel
3.1.6 Nick Translation Probe Labeling
3.2 Preparation of FISH Chromosome Spreads
3.2.1 Collection of Root Tips
3.2.2 Chromosome Preparation
3.3 TSA-FISH
3.3.1 Pretreatment of Chromosome Preparations
3.3.2 Hybridization Mixture
3.3.3 Denaturation and Hybridization
3.3.4 Washing and Detection of the Amplified Hybridization Signal
4 Notes
References
Chapter 5: Localization of Low-Copy DNA Sequences on Mitotic Chromosomes by FISH
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 Metaphases
2.2.2 Reagents and Solutions for Root Fixation and Slide Preparation
2.2.3 Reagents and Solutions for Preparation of FISH Probes
2.2.4 Reagents and Solution for Post-fixation and FISH
2.3 Laboratory Devices and Other Equipment
3 Methods
3.1 Seed Germination, Cell Cycle Synchronization, and Metaphase Accumulation (See Note 1)
3.2 Squash Preparation
3.3 Probe Preparation
3.4 Slide Post-
3.5 Fluorescence In Situ Hybridization
3.6 Microscopy (See Note 21)
4 Notes
References
Chapter 6: Immunolabeling and In Situ Labeling of Isolated Plant Interphase Nuclei
1 Introduction
2 Materials
2.1 Plant Material Preparation
2.2 Cytofunnel Preparation
2.3 Immuno
2.4 In Situ Hybridization
3 Methods
3.1 Preparation of Plant Material
3.2 Preparation of Fixative
3.3 Assembly of Cytofunnel Unit
3.4 Immuno
3.5 In Situ Hybridization Procedure
3.6 Combined Immunofluorescence and In Situ Hybridization Procedure
4 Notes
References
Chapter 7: Manipulation of Homologous and Homoeologous Chromosome Recombination in Wheat
1 General Comments
2 Changing Crossover Density in Designated Homologous Segments
3 Recombination of Homoeologues
4 The Two-Step Approach to Engineering Alien Chromosome Segments into Wheat
References
Chapter 8: Dissecting Plant Chromosomes by the Use of Ionizing Radiation
1 Introduction
2 Materials
3 Methods
3.1 Seed Preparation
3.2 Seed Tempering
3.2.1 Tempering Process
3.3 Sample Irradiation
3.4 Optimal Irradiation Dosage
3.5 Seed Survival Applied to Population Development
4 Notes
References
Chapter 9: Optical Nano-mapping and Analysis of Plant Genomes
1 Introduction
2 Materials
2.1 Consumables and Equipment
2.2 Buffers
2.3 Safety
3 Methods
3.1 Nuclei Isolation and Embedding
3.1.1 Homogenize Leaf Tissue
3.1.2 Pellet and Rinse Nuclei
3.1.3 Embed Nuclei into Agarose Plugs
3.1.4 Proteinase K Treatment
3.1.5 RNaseA Treatment
3.2 DNA Extraction and Cleanup
3.2.1 Melt Agarose Plug(s)
3.2.2 Clean Up DNA via Drop Dialysis
3.2.3 Quantify DNA
3.3 Nick, Label, Repair, and Stain (NLRS) Reactions (See Note 13)
3.3.1 Nick DNA
3.3.2 Label DNA
3.3.3 Repair DNA
3.3.4 Stain DNA
3.3.5 Quantify NLRS DNA
3.4 Run Sample on Irys
3.4.1 Loading and Running Irys
4 Notes
References
Chapter 10: Flow Sorting 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, Accumulation of Metaphases, Preparation of Chromosome Suspensions, and Chromosome Sorting
2.2.2 Reagents and Solutions for FISH
2.2.3 Reagents and Solutions for FISHIS
2.3 Instruments and Other Utilities
3 Methods
3.1 Seed Germination (See Note 1)
3.2 Cell Cycle Synchronization Using HU
3.3 Metaphase Accumulation (See Note 3)
3.4 Preparation of Liquid Chromosome Suspensions (See Note 5)
3.5 Chromosome Labeling Using FISH in Suspension (FISHIS)
3.6 Chromosome Sorting Using Flow Cytometry
3.7 Estimation of Purity in Sorted Fractions Using FISH (See Note 11)
4 Notes
References
Chapter 11: Construction of BAC Libraries from Flow-Sorted Chromosomes
1 Introduction
2 Materials
2.1 Solutions
2.2 Special Reagents
2.3 Supplies
2.4 Equipment
3 Methods
3.1 Preparation of HMW DNA from Flow-­Sorted Chromosomes
3.2 Partial Digestion of HMW DNA
3.3 Size Selection of the Digested DNA Fragments
3.4 Electroelution
3.5 Ligation
3.6 Desalting
3.7 Transformation
4 Notes
References
Chapter 13: Immunolocalization on Whole Anther Chromosome Spreads for Male Meiosis
1 Introduction
2 Materials
2.1 Plant Material
2.2 Equipment
2.3 Self-Made Utensils
2.4 Buffers, Solutions, and Chemicals
2.5 Antibody Solutions
3 Methods
3.1 Plant Material Preparation for Maize
3.2 Initial Sample Treatment
3.3 Chromosome Spread
3.4 Antigen Reactivation/Retrieval
3.5 Immunolocal
3.6 DAPI Counterstaining (See Note 19)
3.7 Image Processing with ImageJ
4 Notes
References
Chapter 14: Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation
1 Introduction
2 Materials
2.1 Reagents
2.1.1 Staging and Collecting Meiotic Flowers
2.1.2 Chromatin Crosslinking
2.1.3 Chromatin Extraction and Sonication
2.1.4 Chromatin Immunoprecipitation
2.1.5 ChIP-seq Library Construction and Quality Control
2.2 Supplies and Equipment
2.2.1 Staging and Collecting Meiotic Flowers
2.2.2 Chromatin Crosslinking
2.2.3 Chromatin Extraction and Sonication
2.2.4 Chromatin Immunoprecipitation
2.2.5 ChIP-seq Library Construction and Quality Control
3 Methods
3.1 Staging and Collecting Maize Meiotic Flowers
3.2 Chromatin Crosslinking
3.3 Chromatin Extraction and Sonication
3.4 Chromatin Immunoprecipitation
3.4.1 Blocking Dynabeads (See Note 15)
3.4.2 Immunoprecipi
3.4.3 Decrosslinking
3.4.4 DNA Recovery
3.5 ChIP-seq Library Construction and Sequencing
3.6 Computational Analyses
3.6.1 Processing and Mapping Illumina Reads to the Genome Scaffold
4 Notes
References
Chapter 15: Chromatin Immunoprecipitation to Study  The Plant Epigenome
1 Introduction
2 Materials
3 Methods
3.1 Chromatin Preparation
3.2 Isolation of Specific Chromatin–DNA Complex
3.3 DNA Isolation
4 Notes
References
Index