MOLECULAR PLANT TAXONOMY : methods and protocols.

Preface
Contents
Contributors
List of Abbreviations
Chapter 1: Plant Taxonomy: A Historical Perspective, Current Challenges, and Perspectives
	1 Introduction
	2 Taxonomy and Taxon: Terminology and Fluctuating Meanings
	3 A Historical Perspective to Plant Taxonomy
		3.1 One of the Earliest Scientific Disciplines
		3.2 Toward a Scientific Classification of Plants
		3.3 Naming Plant Names: Major Advances by Linnaeus
		3.4 The Advent of the Theory of Evolution and Its Decisive Impact on Taxonomy
		3.5 New Methods and New Sources of Characters for a Modern Taxonomy
	4 Plant Taxonomy Today: Current Challenges, Methods, and Perspectives
		4.1 How Many Plant Species Are There?
		4.2 Current Threats on Plant Diversity, the Taxonomic Impediment, and Some Initiatives to Overcome It
		4.3 Molecular Taxonomy and the Need for an Accelerated Pace of Species Discovery
			4.3.1 Strengths and Limitations of Molecular Taxonomy
			4.3.2 The Definitive Need for an Integrative Taxonomy
	5 Notes
	References
Chapter 2: Guidelines for the Choice of Sequences for Molecular Plant Taxonomy
	1 The Plant Genome and Regions Targeted for Molecular Plant Taxonomy
		1.1 Repeated Nuclear DNA Sequences
		1.2 Low Copy Nuclear Genes and  SNPs
		1.3 Anonymous Sequences
		1.4 Organellar  DNA
	2 Evolutionary Considerations
	3 Choice of Sequences for Molecular Taxonomy
	4 Genetic Considerations
	5 Analyzing Results
	6 A Temporal Landscape of the most Commonly Used Methods for Molecular Plant Taxonomy
	7 Further Exploration: Chromosomal Organization
	References
Chapter 3: Isolation and Purification of DNA from Complicated Biological Samples
	1 Introduction
	2 Materials
		2.1 DNA Extraction
		2.2 Gel Electrophoresis
		2.3 Equipment
	3 Methods
		3.1 DNA Extraction Protocol
			3.1.1 Tissue Grinding
			3.1.2 Extraction of DNA from Ground Tissue
		3.2 DNA Analysis
	4 Notes
	References
Chapter 4: Herbarium Specimens: A Treasure for DNA Extraction, an Update
	1 Introduction
		1.1 Main Isolation Difficulties
	2 Materials
		2.1 Key to Choice of Protocols
		2.2 General Equipment for all Protocols
		2.3 Method 1: DNeasy Plant Mini Kit (QIAgen) for Plants with Leaves Containing Sclerenchyma Strands
		2.4 Method 2: DNeasy Plant Mini Kit (QIAgen) Modified for AFLP
		2.5 Method 3: DNA Extraction from Seeds
		2.6 Method 4: The STE/CTAB Method for Micro-Scale DNA Extraction from Polysaccharide-Rich Plants
		2.7 Method 5: Modified CTAB Adapted Method for Mucilaginous Tissues
		2.8 Method 6: Modified CTAB Method for Fungi and Lichen Forming Fungi
		2.9 Method 7: CTAB/HNO3 Method for Algae
		2.10 Method 8: DNA Extraction from Dried Mushrooms Using Enzymatic Digestion and Glass-Fiber Filtration (EDGF)
		2.11 Method 9: NucleoSpin Plant II Kit (Macherey-Nagel) Used for Microsatellites
		2.12 Method 10: Extraction of Ultrashort DNA Molecules for ``Next-Generation´´ Sequencing
	3 Methods
		3.1 DNeasy Plant Mini Kit (QIAgen)
			3.1.1 Method 1: DNeasy Plant Mini Kit (QIAgen) for Plants with Leaves Containing Sclerenchyma Strands
			3.1.2 Method 2: DNeasy Plant Mini Kit (QIAgen) Modified for AFLP
			3.1.3 Method 3: DNA Extraction from Seeds
		3.2 CTAB Modified Methods
			3.2.1 Method 4: The STE/CTAB Method for Micro-Scale DNA Extraction from Polysaccharide-Rich Plants
			3.2.2 Method 5: Modified CTAB Adapted Method for Mucilaginous Tissues
			3.2.3 Method 6: Modified CTAB Method for Fungi and Lichen Forming Fungi
			3.2.4 Method 7: CTAB/HNO3 Method for Algae
		3.3 Method 8: DNA Extraction from Dried Mushrooms Using Enzymatic Digestion and Glass-Fiber Filtration (EDGF)
		3.4 Method 9: NucleoSpin Plant II Kit (Macherey-Nagel) Used for Microsatellites
		3.5 Method 10: Extraction of Ultrashort DNA Molecules for ``Next-Generation´´ Sequencing: Modification of DNeasy Plant Mini Ki...
	4 Notes
	References
Chapter 5: Sequencing of Complete Chloroplast Genomes
	1 Introduction
	2 Review of Methods
		2.1 Database of Research Articles
		2.2 Plant Material and DNA Extraction
		2.3 Construction of Sequencing Libraries
		2.4 Sequencing Platforms and Modes
		2.5 Raw Data (Read) Processing
		2.6 Selection of Chloroplast Reads and Assembly
		2.7 Improving the Assembly
		2.8 Description of the Assembly
		2.9 Phylogenetic and General Comparison of Chloroplast Genomes
	3 Recommendations for Reporting Chloroplast Sequences
	4 Conclusions
	References
Chapter 6: Utility of the Mitochondrial Genome in Plant Taxonomic Studies
	1 Mitochondrial Genomes
		1.1 Origin of Mitochondrial Genomes
		1.2 Mitochondrial Structure and Genome  Size
		1.3 Gene Arrangement and the Importance of Homologous Recombination
		1.4 Molecular Evolutionary Rates of the mtDNA
		1.5 Mode of Inheritance of the Mitochondrial Genome
	2 Mitochondrial Molecular Markers in Phylogenetics and Taxonomy
		2.1 Genomic Resources: Complete Mitochondrial Genome
		2.2 Use of mtDNA in Phylogeography and Phylogenetics
	3 Tips for Sequencing Protocol Based on  NGS
		3.1 DNA Purification
		3.2 Construction of Libraries and Sequencing
	4 Recommendations for Bioinformatics Analyses
	References
Chapter 7: Nuclear Ribosomal RNA Genes: ITS Region
	1 Introduction
	2 Materials
		2.1 PCR
		2.2 Electrophoresis
	3 Methods
		3.1 PCR Reaction
		3.2 PCR Program
		3.3 PCR Quality Verification on Agarose Gel
		3.4 Sequencing
	4 Notes
	References
Chapter 8: Plant DNA Barcoding Principles and Limits: A Case Study in the Genus Vanilla
	1 Introduction
		1.1 Barcoding History in Plants
		1.2 Limits of DNA Barcoding
		1.3 A Case Study in the Genus Vanilla
	2 Testing Barcoding Sequences for Vanilla
	3 Which Locus Shows the Greatest Sequence Diversity?
	4 Is there a Barcoding Gap in Vanilla?
	5 Which Locus Shows the Greatest Level of Species Discrimination?
	6 What Is the Discrimination Gain Obtained by Combining Multiple Loci?
	7 Conclusion: Which DNA Barcode for Vanilla and What Limits?
		7.1 Closely Related Species
		7.2 Non-monophyletic Species
		7.3 Hybrid Species
		7.4 Conclusion
	References
Chapter 9: High-Throughput Genotyping Technologies in Plant Taxonomy
	1 Introduction
		1.1 What Are SNPs?
		1.2 Gene Presence and Absence Variation
		1.3 Molecular Marker Applications in Plant Taxonomy
		1.4 Pangenomes as the Future of Molecular Markers and Gene Variance Identification
		1.5 Tools for Plant Genotyping and Taxonomy
			1.5.1 Identification of SNPs In Silico
			1.5.2 Distance Estimation Methods in Genotyping
	2 Materials
		2.1 Bioinformatics Requirements for Mash Analyses
		2.2 Bioinformatics Requirements for SNP Identification Using BCFtools
	3 Methods
		3.1 Mash Analyses
			3.1.1 Mash for Distance Estimation
			3.1.2 Investigate Sample Contamination with Mash
		3.2 SNP Identification Using BCFtools
	4 Notes
	References
Chapter 10: Genotyping-by-Sequencing Technology in Plant Taxonomy and Phylogeny
	1 Introduction
		1.1 Genotyping-by-Sequencing
		1.2 Some Limitations to the GBS Method
	2 Materials
		2.1 DNA Extraction and Quantification
		2.2 GBS Library Construction
		2.3 Illumina Workflow
		2.4 Data Analysis Equipment and Softwares
	3 Methods
		3.1 DNA Extraction and Quantification
		3.2 GBS Library Construction
		3.3 Illumina Sequencing Workflow
		3.4 GBS Data Processing
			3.4.1 Filtering Raw Sequence  Data
			3.4.2 Mapping
			3.4.3 Analysis
	4 Notes
	References
Chapter 11: Development of Microsatellite Markers Using Next-Generation Sequencing
	1 Introduction
	2 Materials
		2.1 Library DNA Construction
			2.1.1 DNA Fragmentation
			2.1.2 Fragmented DNA Purification
			2.1.3 DNA Amplification
			2.1.4 DNA Purification
		2.2 Library Verification
		2.3 Illumina Sequencing
		2.4 Selection and SSR Screening
		2.5 PCR Amplification
		2.6 Sequencer Revelation and Scoring Analysis
	3 Methods
		3.1 DNA Fragmentation
		3.2 DNA Purification with the Zymo Clean-Up  Kit
		3.3 PCR Amplification with Adding Indexes
		3.4 Amplified DNA Purification
		3.5 Library Verification
		3.6 Illumina Sequencing
		3.7 Bioinformatic Analysis and Primer Design
		3.8 Selection and SSR Screening
			3.8.1 Selection of Markers and Primers
			3.8.2 PCR Amplification
			3.8.3 Sequencer Revelation and Scoring Analysis
	4 Notes
	References
Chapter 12: Amplified Fragment Length Polymorphism: Applications and Recent Developments
	1 Introduction
		1.1 Principle of  AFLP
		1.2 Basic Steps Involved in AFLP Analysis
		1.3 AFLP Advantages and Applications
			1.3.1 Genetic Diversity Studies Using AFLP Markers
			1.3.2 Variety/Cultivar Fingerprinting, Kinship, and Genetic Fidelity
			1.3.3 QTL Mapping
			1.3.4 Other Specific Applications of AFLP Marker Systems
		1.4 AFLP Versus Other Popular DNA Markers
		1.5 Disadvantages of AFLP Technique
		1.6 Modifications of  AFLP
			1.6.1 SAMPL
			1.6.2 M-AFLP
			1.6.3 SSAP
			1.6.4 AIMS
			1.6.5 MSAP
			1.6.6 AFLP-RGA
			1.6.7 TE-AFLP
			1.6.8 SDAFLP
			1.6.9 MITE-AFLP
			1.6.10 RNA Fingerprinting Using cDNA-AFLP
			1.6.11 Nonradioactive DD-AFLP
		1.7 Patents and IPR Protection
		1.8 Conclusions
	2 Materials
		2.1 DNA Template Preparation
		2.2 Restriction-Ligation (RL)
		2.3 Pre-Selective PCR Amplification (See Note 1)
		2.4 Selective PCR Amplification
		2.5 Separation and Visualization of Fragments (See Note 3)
	3 Methods
		3.1 DNA Template Preparation
		3.2 RL (Restriction-Ligation)
		3.3 Pre-selective PCR Amplification
		3.4 Selective PCR Amplification (See Note 5)
		3.5 Separation and Visualization of Fragments (See Note 6)
	4 Notes
	References
Chapter 13: Random Amplified Polymorphic DNA (RAPD) and Derived Techniques
	1 Introduction
		1.1 RAPD Technique
		1.2 Recent Applications of RAPD and Its Derived Techniques
			1.2.1 Cultivar Identification
			1.2.2 Genetic Mapping and Tagging
			1.2.3 Assessment of Outcrossing Rates
			1.2.4 Genetic Fidelity Testing
			1.2.5 Inter and Intraspecies Variations and Genetic Diversity
			1.2.6 Others
		1.3 Disadvantages of RAPD Technique and Solutions
	2 Materials
		2.1 Genomic DNA Isolation and Quantification
		2.2 Reagents Used for RAPD-PCR
		2.3 Sequence Characterized Amplified Region (SCAR)
			2.3.1 Genomic DNA Isolation and Quantification
			2.3.2 Reagents for PCR
			2.3.3 Gel Extraction
			2.3.4 Cloning of PCR Amplified Gene
		2.4 Arbitrarily Primed Polymerase Chain Reaction (AP-PCR)
			2.4.1 Genomic DNA Isolation and Quantification
			2.4.2 Reagents for PCR
			2.4.3 Electrophoresis
		2.5 DNA Amplification Fingerprinting (DAF)
			2.5.1 Genomic DNA Isolation and Quantification
			2.5.2 Reagents for PCR
			2.5.3 PAGE Reagents
			2.5.4 Silver Staining Reagents
		2.6 The Sequence-Related Amplified Polymorphism (SRAP) Technique
			2.6.1 Genomic DNA Isolation and Quantification
			2.6.2 Reagents for PCR Conditions
			2.6.3 PAGE Electrophoresis
		2.7 Random Amplified Microsatellite Polymorphism (RAMPO)
			2.7.1 Genomic DNA Isolation and Quantification
			2.7.2 Reagents Used for RAPD and Microsatellite-Primed PCR (MP-PCR)
			2.7.3 Hybridization with Microsatellite-Complementary Probes
		2.8 Random Amplified Hybridization Microsatellites (RAHM)
			2.8.1 Genomic DNA Isolation and Quantification
			2.8.2 Reagents Used for RAPD-PCR
			2.8.3 Hybridization with Microsatellite-Complementary Probes
		2.9 Cleaved Amplified Polymorphic Sequences (CAPS)
			2.9.1 Genomic DNA Isolation and Quantification
			2.9.2 Reagents for PCR Conditions
			2.9.3 Restriction Enzyme Digestion
			2.9.4 PAGE Reagents
			2.9.5 Silver Staining Reagents
	3 Methods
		3.1 Isolation of Genomic DNA (Modified Doyle and Doyle, 1990)
		3.2 DNA Quantification
			3.2.1 By Gel Electrophoresis
			3.2.2 Using UV Spectrophotometer
		3.3 RAPD
			3.3.1 PCR Amplification of Genomic DNA with Primers
			3.3.2 Gel Electrophoresis
			3.3.3 Scoring and Interpretation of RAPD Banding Patterns (See Note 3)
		3.4 Sequence Characterized Amplified Region (SCAR)
			3.4.1 Amplification
			3.4.2 RAPD Fragment Selection and Cloning
		3.5 Arbitrarily Primed Polymerase Chain Reaction (AP-PCR)
			3.5.1 Amplification
			3.5.2 Electrophoresis
		3.6 DNA Amplification Fingerprinting (DAF)
			3.6.1 Amplification
			3.6.2 Silver Staining for DNA Visualization
			3.6.3 Gel Interpretation
		3.7 Sequence-Related Amplified Polymorphism (SRAP) (See Note 9)
			3.7.1 Amplification
			3.7.2 Sequencing of SRAP Marker Bands
		3.8 Random Amplified Microsatellite Polymorphisms (RAMPO)
			3.8.1 Genomic DNA Isolation
			3.8.2 Amplification of Genomic DNA with RAPD Primers/Microsatellite Primers
			3.8.3 Hybridization with Microsatellite-Complementary Probes
		3.9 Random Amplified Hybridization Microsatellites (RAHM)
		3.10 Cleaved Amplified Polymorphic Sequences (CAPS)
	4 Notes
	References
Chapter 14: Inter-Simple Sequence Repeats (ISSR), Microsatellite-Primed Genomic Profiling Using Universal Primers
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 DNA Extraction
		3.2 PCR
		3.3 Gel Electrophoresis
		3.4 Scoring
	4 Notes
	References
Chapter 15: Retrotransposable Elements: DNA Fingerprinting and the Assessment of Genetic Diversity
	1 Introduction
		1.1 LTR Retrotransposons
		1.2 Retrotransposons as DNA Markers
		1.3 Inter-Retrotransposon Amplified Polymorphism (IRAP) and Retrotransposon Microsatellite Amplified Polymorphism (REMAP)
		1.4 Inter-PBS (iPBS) Amplification: A Universal Method for Isolating and Displaying Retrotransposon Polymorphisms
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 DNA Template
		2.4 Primer Design
	3 Methods
		3.1 PCR Protocol for IRAP, REMAP, and iPBS
		3.2 Sample Preparation and Loading
		3.3 Casting the Agarose Gel
		3.4 Gel Electrophoresis
		3.5 DNA Visualization
	4 Notes
	References
Chapter 16: Introduction to Population Genomics Methods
	1 Introduction
	2 Materials
	3 Methods
		3.1 From Raw DNA Data to Genetic Variants
		3.2 Case Study 1: Individual-Based Genotyping
			3.2.1 African Rice
			3.2.2 Variant Discovery from Publicly Available Data
			3.2.3 Population Structure
			3.2.4 Diversity
			3.2.5 Inferring Population Size History
			3.2.6 Deleterious Mutation Load
			3.2.7 FST and Genome Scans for Selection
		3.3 Case Study 2: Sessile Oak Populations
			3.3.1 Pool-seq as a Cost-Efficient Method
			3.3.2 Population Genomics in Wild Sessile Oaks
			3.3.3 From Raw Sequencing Data to Allele Counts
			3.3.4 Inferring the History of a Set of Populations
			3.3.5 FST Fixation Indices
			3.3.6 Genome Scans of Selection
			3.3.7 Genotype-Environment Association (GEA)
	4 Notes
	References
Chapter 17: The Application of Flow Cytometry for Estimating Genome Size, Ploidy Level Endopolyploidy, and Reproductive Modes ...
	1 Introduction
		1.1 Terminology Used for Genome Size Studies
		1.2 The Importance of Cytological Data for Genome Size Studies
	2 Materials
		2.1 Plant Tissue and Reference Standards
		2.2 Equipment
		2.3 Reagents
			2.3.1 Fluorochromes
			2.3.2 Isolation Buffers (See Notes 3 and 4)
	3 Methods
		3.1 Isolation of Plant Nuclei
			3.1.1 Isolation of Plant Nuclei Using the One-Step Protocol
			3.1.2 Isolation of Plant Nuclei Using the Two-Step Protocol
			3.1.3 Isolation of Plant Nuclei Using a Simplified Two-Step Protocol
		3.2 Analysis of the Nuclear DNA Content and DNA Ploidy Level
			3.2.1 Measurement of the Relative Nuclear DNA Fluorescence of a Sample
			3.2.2 Measurement of the Absolute Nuclear DNA Content of a Sample Using a Reference Standard
			3.2.3 Measurement of the Relative Nuclear DNA Content of a Sample Using a Reference Standard to Determine DNA Ploidy Level
			3.2.4 Measurement of the Relative Nuclear DNA Content of a Sample to Determine the Extent of Endopolyploidy
			3.2.5 Using Seeds to Determine Reproductive Pathways Based on the Ratio between the Relative Nuclear DNA Content of the Embryo...
	4 Notes
	References
Chapter 18: Molecular Cytogenetics (Fluorescence In Situ Hybridization - FISH and Fluorochrome Banding): Resolving Species Rel...
	1 Introduction
	2 Materials
		2.1 Pretreatments and Root Tip Fixations
		2.2 Buffers
		2.3 Enzyme Mixture
		2.4 Fluorochrome Banding
		2.5 FISH (Fluorescence In Situ Hybridization)
	3 Methods
		3.1 Pretreatment and Fixation of Root Tips
		3.2 Preparation of Protoplasts
		3.3 Cover Slip Removal
		3.4 Chromomycin Banding
		3.5 Hoechst Banding
		3.6 Destaining Slides After Fluorochrome Bandings
		3.7 FISH
			3.7.1 Day  One
			3.7.2 Day  Two
		3.8 Modified FISH Protocol
			3.8.1 Day  One
			3.8.2 Day  Two
		3.9 Destaining Slides After FISH
	4 Notes
	References
Chapter 19: GISH: Resolving Interspecific and Intergeneric Hybrids
	1 Introduction
		1.1 Genomic In Situ Hybridization (GISH)
		1.2 Example of Application in Sugarcane and Sugarcane Hybrids
			1.2.1 Interspecific Hybrid Between S. officinarum and S. spontaneum
			1.2.2 Intergeneric Hybrid Between S. officinarum and E. arundinaceus
			1.2.3 Revealing the Interspecific and Intergeneric Status of Intergeneric Hybrid Between Saccharum and E. arundinaceus
	2 Materials
		2.1 Equipment
		2.2 Stock Solutions Stored at Room Temperature or on Ice for Immediate Use
		2.3 Stock Solutions Stored at 4 C
		2.4 Stock Solutions Stored at -20 C
	3 Methods
		3.1 Root Pretreatment and Slide Preparation
			3.1.1 Root Treatment
			3.1.2 Slide Preparation (See Note 5)
		3.2 RNase A Treatment
		3.3 GISH Experiment
			3.3.1 Probe Labeling by Random Priming
			3.3.2 Slide Denaturation
			3.3.3 In Situ Hybridization
	4 Notes
	References
Index