Plant Genotyping: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Genotyping by Sequencing (GBS) for Genome-Wide SNP Identification in Plants
1 Introduction
2 Materials
2.1 Reagents and Kits
2.2 Consumables
2.3 Equipment
3 Methods
3.1 Adapter Preparation (See Note 1)
3.2 DNA Normalization and Digestion
3.3 Adapter Ligation
3.4 Multiplexing (See Note 5)
3.5 PCR Amplification (See Note 6)
3.6 Size Selection and Library Quantification
4 Notes
References
Chapter 2: Genotyping by Multiplexed Sequencing (GMS) Using SNP Markers
1 Introduction
2 Materials
2.1 Consumables
2.2 Kits
2.3 Reagents
2.4 Equipment
3 Methods
3.1 SNP Primer Design for Primer Pools
3.2 Creating Primer Pools
3.3 GMS PCR-1
3.4 PCR-1 Dilution
3.5 GMS PCR-2
3.6 GMS Library Pool Cleanup
3.7 GMS Library Pool Bead Cleanup
3.8 4% Gel Size Selection
3.9 Gel Purification
3.10 2% Gel Size Selection
3.11 GMS Library Pool Cleanup
3.12 GMS Library Pool Quantification and Length Assays
4 Notes
References
Chapter 3: Computational Protocol for DNA Methylation Profiling in Plants Using Restriction Enzyme-Based Genome Reduction
1 Introduction
2 Materials
2.1 Reagents and Kits
2.2 Equipment
2.3 Software
2.4 Data
3 Methods
3.1 Profiling Methylation by Double-Digestion with Restriction Enzymes
3.2 PCR Amplification
3.3 Next-Generation Sequencing (NGS)
3.4 Computational Workflow
3.5 Software Installation
3.6 Pipeline Execution
3.7 Pipeline Steps
3.8 Quality Control
3.9 Identifying Potential DNA Methylation Sites
3.10 Counting Sequencing Reads
3.11 Identification of DNA Methylation by Differential Counts Analysis
3.12 Biological Inference Using Detected DNA Methylation
4 Notes
References
Chapter 4: Double Digest Restriction-Site Associated DNA Sequencing (ddRADseq) Technology
1 Introduction
2 Materials
2.1 Plasticware and Consumables
2.2 Enzymes and Kits
2.3 Equipment
3 Methods
3.1 General Description of ddRADseq Method
3.1.1 Double Digestion
3.1.2 Adapters Ligation
3.1.3 Sample Pooling
3.1.4 Size Selection
3.1.5 PCR Amplification
3.1.6 Next Generation Sequencing
3.2 Preparing of Genomic DNA
3.3 DNA Digestion
3.4 First Round of Purification with Magnetic Beads
3.5 Ligation
3.6 Pooling Libraries
3.7 Second Round of Purification with Magnetic Beads
3.8 Automated Size Selection
3.9 Third Round of Purification with Magnetic Beads
3.10 PCR Enrichment of Libraries
3.11 Fourth Round of Purification with Magnetic Beads
3.12 Library Validation
3.13 Preparing Libraries for Sequencing
4 Notes
References
Chapter 5: Whole Genome Wide SSR Markers Identification Based on ddRADseq Data
1 Introduction
2 Materials
2.1 Reagents Used
2.2 Software Used
3 Methods
3.1 Library Preparation and Sequencing
3.2 Data Pre-processing and Construction of Consensus Sequences
3.3 Microsatellite Identification
3.4 Primer Design
3.5 Whole Genome SSR Markers Identification with Example of ddRADseq Data
3.6 Utility of ddRAD Derived SSR Markers
4 Notes
References
Chapter 6: High-Throughput Association Mapping in Brassica napus L.: Methods and Applications
1 Introduction
2 Materials and Methods
2.1 QTL Analysis
2.1.1 Plant Material
2.1.2 Phenotyping
2.1.3 Genotyping
2.1.4 QTL Mapping
2.1.5 Identification of Candidate Genes in the Target Region
2.2 Genome Wide Association Analysis
2.2.1 Plant Material
2.2.2 Phenotypic Data Analysis
2.2.3 Brassica 60K SNP Array Analysis
2.2.4 DNA Resequencing
2.2.5 mRNA Sequencing
2.2.6 Calling of SNPs
2.2.7 Filtering of SNPs
2.2.8 Association of SNPs with Phenotypic Traits
2.2.9 Identification of Candidate Genes
3 Application of SNPs in Association Mapping Targeting Agronomic Traits in Brassica napus
3.1 Root Architecture-Related Traits
3.2 Plant Architecture-Related Traits
3.3 Flowering Time-Related Traits
3.4 Reproduction and Silique-Related Traits
3.5 Seed Quality-Related Traits
4 Conclusion and Future Perspectives
References
Chapter 7: Polyploid SNP Genotyping Using the MassARRAY System
1 Introduction
2 Materials
2.1 Plasticware and Consumables
2.2 Kits and Reagents
2.3 Equipment and Software
3 Methods
3.1 Workflow Overview
3.2 Designing Genotyping Assays
3.3 Amplifying DNA for Genotyping in Multiplex (Capture Reaction)
3.4 Neutralizing Unincorporated dNTPs (SAP Reaction)
3.5 Creating the iPLEX Gold Reaction (Extension Reaction)
3.6 iPLEX Reaction Cleanup with Resin
3.7 Dispensing onto SpectroCHIP Arrays
3.8 Designing the Plate Input File and Acquiring the Spectrum Profile
3.9 Exporting Results on the MassARRAY TYPER 4.0
3.10 Dosage Estimation Using SuperMASSA Software
4 A Practical Example: Ploidy and Dosage Estimation in the Hexaploid Urochloa humidicola
5 Notes
References
Chapter 8: qPCR Genotyping of Polyploid Species
1 Introduction
2 Materials
2.1 Genomic DNA Extraction
2.2 gDNA Quantitative PCR Amplification
3 Methods
3.1 Genomic DNA Extraction
3.2 Reference Sequence and Allele-Specific Primer Design
3.3 Quantitative PCR Amplification
3.4 Analyzing qPCR Genotyping Date
4 Notes
References
Chapter 9: Genome-Wide Association Studies (GWAS)
1 Introduction
2 Simple Association Test Approach
3 General Linear Model Approach
4 Mixed Linear Model Approach
5 Multi-Locus Multi-Allele Model (RTM-GWAS) Approach
5.1 Constructing SNPLDB Marker for Multiple Alleles Detection
5.2 Detecting QTL-Allele System Using Efficient Multi-Locus Model
5.3 Potential Applications of RTM-GWAS
6 Example: QTL-Allele System of Seed Protein Content in Northeast China Soybeans
6.1 Plant Materials and Field Experiment
6.2 SNP Genotyping
6.3 SNPLDB Marker Construction
6.4 Genetic Similarity Matrix Calculation
6.5 Multi-Locus Multi-Allele Model GWAS
6.6 SPC QTL-Allele System of the NECSGP
6.7 SPC QTL-Allele Changes in the Evolution from Late to Early Maturity Groups
6.8 Prediction of Recombination Potential for Optimal Cross Design
6.9 Annotation of Candidate Gene System of SPC
References
Chapter 10: Transcriptomic Approach for Global Distribution of SNP/Indel and Plant Genotyping
1 Introduction
2 Materials
2.1 Consumables
2.2 Equipment
3 Methods
3.1 RNA Extraction (Modified ``Hot Borate´´ Method)
3.2 Library Synthesis and Sequencing
3.2.1 RNA Purification and Fragmentation
3.2.2 RNA First Strand Synthesis
3.2.3 RNA Second Strand Synthesis
3.2.4 RNA Purification
3.2.5 RNA End Repair
3.2.6 Adenylate 3′-Ends
3.2.7 Ligation of Adaptors
3.2.8 Enrichment of DNA Sequences with Ligated Adaptors
3.2.9 Final Steps for Library Sequencing
3.3 SNP and InDel Calling and Annotation
3.4 Primer Design
4 Notes
References
Chapter 11: Specific-Locus Amplified Fragment Sequencing (SLAF-Seq)
1 Introduction
2 Application of SLAF-Seq in Ornamental Plants
3 SLAF Method
3.1 Experimental Scheme Design Based on Bioinformatics Information
3.2 Construct SLAF Library According to the Scheme of Preliminary Experiment
3.3 High-Throughput Sequencing
3.4 Data Processing and Analysis
3.5 Conclusion
4 Notes
References
Chapter 12: Modifications of Kompetitive Allele-Specific PCR (KASP) Genotyping for Detection of Rare Alleles
1 Introduction
1.1 General Description of Assay
1.2 Primer Design
1.3 Template DNA
1.4 PCR Conditions
1.5 Standard Data Interpretation: Homozygous or Heterozygous Alleles
1.6 Determining Allele Ratios
1.7 Angle of Amplification (θ) Method of KASP Data Analysis
1.8 Delta Method of KASP Data Analysis
2 Materials
3 Methods
3.1 Preparation of Reaction Mixture for Rare Allele Detection
3.2 Plate Preparation
3.3 Running KASP Assay
3.4 Data Interpretation
3.4.1 Arctan Transformation
3.4.2 Delta Transformation
4 Notes
References
Chapter 13: Amplifluor-Based SNP Genotyping
1 Introduction
2 Materials
2.1 Primer Design
2.2 Amplifluor Assay
3 Methods
3.1 Design of SNP Specific Primers
3.2 Genotyping
4 Notes
References
Chapter 14: SNP Genotyping with Amplifluor-Like Method
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 15: Semi-Thermal Asymmetric Reverse PCR (STARP) Genotyping
1 Introduction
2 Materials
2.1 Primer Design
2.2 STARP Assay
3 Methods
3.1 Primer Design
3.2 STARP Assay
4 Notes
References
Chapter 16: Modified Allele-Specific qPCR (ASQ) Genotyping
1 Introduction
2 Materials
3 Methods
3.1 Primer Design and Tail-Tag Attachment
3.2 Molecular Probes Setup for Ordering
3.3 DNA Requirement, Master-Mix, and Reaction Setup
3.4 Microplate Preparation and Loading
3.5 qPCR Instrument Setup, Fluorescence Amplification, SNP Genotyping, and Allele Discrimination
3.6 Examples
3.6.1 Example of Plant Genotyping Using Variant A (Short-Tag) ASQ Method
3.6.2 Example of Plant Genotyping Using Variant B (Long-Tag) ASQ Method
4 Notes
References
Chapter 17: Allele-Specific Mutation Genotyping with Mismatches in Primer Design
1 Introduction
2 Example A. Detection of SNPs Specific for the Red Flesh Trait in Sweet Cherry Cultivars
2.1 Materials
2.1.1 PCR Amplification
2.1.2 Gel Electrophoresis
2.2 Methods
2.2.1 Primer Design
2.2.2 Standard PCR Amplification
2.2.3 Direct PCR
2.2.4 Gel Electrophoresis
3 Example B. Detection of Sex Trait-Specific SNP in Figs
3.1 Materials
3.1.1 PCR Amplification
3.1.2 Gel Electrophoresis
3.2 Methods
3.2.1 Primer Design
3.2.2 PCR Amplification
3.2.3 Gel Electrophoresis
4 Example C. Detection of a Self-Incompatible-Specific InDel in Sweet Cherry
4.1 Materials
4.1.1 PCR Amplification
4.1.2 Gel Electrophoresis
4.2 Methods
4.2.1 Primer Design
4.2.2 PCR Amplification
4.2.3 Gel Electrophoresis
5 Notes
References
Chapter 18: PCR Allele Competitive Extension (PACE)
1 Introduction
2 Materials
2.1 Primer Design
2.2 PACE Assay
3 Methods
3.1 Primer Design
3.2 PACE Assay
4 Notes
References
Chapter 19: Molecular Beacon Probe (MBP)-Based Real-Time PCR
1 Introduction
2 Materials
2.1 Plant Material
2.2 Genomic DNA Isolation (See Note 2)
2.3 Purification of Genomic DNA
2.4 Molecular Beacon Probe Based Real-Time PCR
2.5 Equipment
3 Methods
3.1 Plant Material and Tissue Collection
3.2 Genomic DNA Isolation Using the CTAB Method
3.3 DNA Quality and Quantity Assessment
3.4 Purification of DNA by RNAse Treatment
3.5 Design of Molecular Beacon Probes (MBPs) and Primers
3.6 Validation of Molecular Beacon Probe Based on Real-Time PCR Assay
4 Notes
References
Chapter 20: Molecular Beacons - Loop-Mediated Amplification (MB-LAMP)
1 Introduction
2 Materials
2.1 Requirements for Molecular Beacons
2.2 Reagents for MB-LAMP
2.3 Consumables and Specialist Equipment
2.4 Consumables for Genome Extraction from Plant Material
3 Methods
3.1 Preparation of LAMP Primers and Molecular Beacon
3.2 Preparation of Plant Samples
3.3 Preparation of the LAMP-MB Mix
3.4 LAMP-MB Assay
4 Notes
References
Chapter 21: TaqMan Probes for Plant Species Identification and Quantification in Food and Feed Traceability
1 Introduction
2 Materials
2.1 Equipment and General Laboratory Supplies
2.2 Reagents
2.3 Software
3 Methods
3.1 Design of Specific Primers and TaqMan Probes
3.2 Evaluation of Assay Specificity
3.3 Evaluation of Sensitivity and Linearity of TaqMan Assay
3.4 Absolute DNA Quantification Methodology
3.4.1 Construction of DNA Calibrator Plasmids
3.4.2 Quantification of the Target gDNA Amount
4 Notes
References
Chapter 22: Tetra-Primer Amplification Refractory Mutation System (T-ARMS)
1 Introduction
2 Materials
2.1 Equipment
2.2 Chemicals and Reagents
2.3 Plasticware
2.4 Buffers and Stock Solutions
3 Methods
3.1 Designing of Primers
3.2 Genomic DNA Isolation
3.3 PCR and Gel Electrophoresis
4 Notes
5 Conclusion and Future Perspective
References
Chapter 23: Penta-Primer Amplification Refractory Mutation System (PARMS) with Direct PCR-Based SNP Marker-Assisted Selection ...
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Consumables and Equipment
3 Methods
3.1 Plants and Sampling
3.2 DNA Extraction
3.3 SNP Genotyping Assay
4 Notes
References
Chapter 24: High-Resolution Melting (HRM) Genotyping
1 Introduction
2 Materials
2.1 Consumables and Components of the Real Time-PCR Thermocycler
2.2 HRM PCR Reagent
2.3 Software
3 Methods
3.1 Primer Design
3.2 Preparation of Reaction Samples for HRM
3.3 Real-Time PCR Running Program Parameters and Instructions
3.4 HRM Data Analysis
4 Notes
References
Chapter 25: Modified High-Resolution Melting (HRM) Marker Systems Increasing Discriminability Between Homozygous Alleles
1 Introduction
1.1 Plant Genotyping and the Role of the HRM Method
1.2 The Principal Behind of NNNs-HRM Markers
2 Methods
2.1 Suitable NNNs Selection
2.2 The Design of Primers for NNNs-HRM Markers
2.3 PCR Conditions
2.4 Examples of HRM Pattern
2.5 Conclusion
3 Notes
References
Chapter 26: A New SNP Genotyping Technology by Target SNP-Seq
1 Introduction
2 Materials
2.1 Plasticware and Consumables
2.2 Enzymes and Kits
2.3 Equipment
3 Methods
3.1 Discovery of Genome-Wide Perfect SNPs for Target SNP-seq
3.2 DNA Extraction and Quality Testing
3.3 Construction of Target SNP-seq Library
3.4 Target SNP Genotype Calling
4 Notes
References
Chapter 27: Derived Polymorphic Amplified Cleaved Sequence (dPACS) Assay
1 Introduction
2 Materials
2.1 Template DNA in Tris-EDTA (TE) Buffer or Sterile Water
2.2 Polymerase Chain Reaction
2.3 Restriction Digestion
2.4 MetaPhor Gel Electrophoresis
3 Methods
3.1 Enzyme Selection and Primer Design with the dPACS 1.0 Program
3.1.1 Inputs into the dPACS Program
3.1.2 Outputs from the dPACS Program
3.1.3 Choice of Restriction Enzyme
3.1.4 Primer Design
3.2 DNA Template Preparation
3.3 Polymerase Chain Reaction (PCR)
3.4 Restriction Digestion of PCR Products
3.5 Horizontal MetaPhor Gel Electrophoresis
3.5.1 Preparing the MetaPhor Gel
3.5.2 Loading Samples and Running Electrophoresis
3.5.3 Staining and Visualization of the Gel
3.6 dPACS Results for S264G Mutation Analysis
4 Notes
References
28: Tubulin-Based Polymorphism (TBP) in Plant Genotyping
1 Introduction
2 Materials
2.1 gDNA Qualitative, Quantitative Evaluation, and Dilution
2.2 TBP Amplification Protocol
2.3 Agarose Gel Electrophoresis and Sample Dilution
2.4 Sample Preparation and Capillary Electrophoresis Separation
2.5 Data Analysis
3 Methods
3.1 gDNA Qualitative, Quantitative Evaluation, and Dilution
3.2 TBP Amplification Protocol
3.3 Agarose Gel Electrophoresis and TBP Amplicons Dilution
3.4 Capillary Electrophoresis Separation
3.5 Data Analysis
4 Notes
References
Chapter 29: Multiplexed ISSR Genotyping by Sequencing (MIG-Seq)
1 Introduction
2 Materials
2.1 Reagents and Kits
2.2 Equipment
3 Methods
3.1 DNA Extraction
3.2 PCR Amplification
3.3 Purification and Size Selection of the PCR Products
3.4 Estimation of DNA Concentration and Preparation of the Size-Selected Library
3.5 Detection of SNPs
4 Notes
References
Chapter 30: Application of SolCAP Genotyping in Potato (Solanum tuberosum L.) Association Mapping
1 Introduction
1.1 Evolution in Breeding and Development of SolCAP Array
1.2 SolCAP Array: Recent Outcomes and Applications in Potatoes
2 Materials
2.1 Plant Material and DNA Extraction
2.2 Genotyping Platforms and Software
3 Methods
3.1 SolCAP 8 K SNP Array
3.1.1 Plant Material and DNA Extraction
3.1.2 Cluster Development and SNP Genotyping
3.1.3 Data Analysis
3.1.4 Managing the Results
3.2 SolCAP 12 K SNP Array
3.2.1 Plant Material and DNA Extraction
3.2.2 SNP Genotyping
3.2.3 Data Analysis
3.2.4 Population Structure
3.2.5 Managing the Results
3.3 SolCAP 20 K SNP Array
3.3.1 Development of SolSTW
3.3.2 Managing the Results
4 Notes
References
Chapter 31: Fluorescence In Situ Hybridization (FISH) for the Genotyping of Triticeae Tribe Species and Hybrids
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Consumable, Instruments, and Equipment
3 Methods
3.1 Preparation of Slides with Mitotic Metaphase Chromosomes
3.2 DNA Probes for FISH Genotyping
3.3 Plasmid DNA and Genomic Plant DNA Isolation
3.4 Probe Labeling
3.5 In Situ Hybridization Procedure
3.5.1 Denaturing of Slides
3.5.2 Preparation of the Hybridization Mixture and Hybridization
3.5.3 Posthybridization Washes
3.6 Detection and Amplification of Hybridization Signals
4 Notes
References
Chapter 32: Innovative Advances in Plant Genotyping
1 Introduction
2 SNP-Based Genotyping
2.1 SNP Arrays
2.2 Genotyping by Sequencing
2.3 Specific-Locus Amplified Fragment Sequencing
2.4 Whole-Genome Resequencing
2.5 Kompetitive Allele-Specific PCR
3 Genotyping Structural Variants
4 A Pangenome Approach to Genotyping
5 Genotype-Phenotype Prediction Using Machine Learning
6 Conclusion
References
Index