Applied Biosystems Agriculture Resource Guide

Contents

• Overview of agricultural applications

• Plant and animal de novo genome sequencing

• Plant and animal genotyping

• QTL mapping and marker-assisted selection

• Plant and animal gene expression analysis

• Synthetic biology and genetic engineering

• Plant cell and tissue analysis

• GMO testing and detection

• Sample preparation and nucleic acid isolation

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Overview of agricultural applications

This application resource guide will help you select Thermo Fisher Scientific's best products for some of the most common agricultural science applications.

Key Technologies: Genetic analyzers, QuantStudio family of real-time PCR systems, Ion Torrent sequencers, TaqMan Assays.

Core Applications:

• GMO testing and detection
• QTL mapping & marker-assisted selection
• Synthetic biology and genetic engineering
• Plant and animal de novo genome sequencing
• Plant and animal genotyping
• Plant and animal gene expression analysis

Objectives: Higher yields, Pest and environmental stress tolerance, Healthier diets, More efficient fertilizer use, Higher quality meat, Higher accuracy breeding values, Higher feed conversion.

Thermo Fisher Scientific offers a range of instruments and reagents including SimpliAmp Thermal Cycler, GeneArt Precision TALs, Phire Direct PCR kits and master mixes, Invitrogen custom DNA oligos, GeneArt Gene Synthesis, GeneArt DNA assembly kits, Competent cells, PCR enzymes, Gateway cloning, Qubit Fluorometer with NanoDrop Spectrophotometer, E-Gel agarose gels, and GeneArt CRISPR products and services.

Plant and animal de novo genome sequencing

Genome sequencing allows researchers to uncover the genetic makeup of plants and animals. Sequencing plant and animal genomes is becoming easier and more cost-effective, especially for de novo sequencing projects aimed at obtaining the primary genetic sequence of a species. Thermo Fisher Scientific offers an extensive portfolio of sequencing instruments, reagents, and analysis software to accelerate research with greater accuracy and reliability.

De novo sequencing solutions: The Ion S5™ and Ion S5™ XL systems provide a DNA-to-data workflow for targeted sequencing with industry-leading speed and affordability, allowing researchers to focus on critical questions. These advancements are transforming agricultural genomics research.

Genome sequencing for all: Fast and affordable genome, exome, and transcriptome sequencing of plants and animals is accessible with the semiconductor sequencing technology in the Ion S5 systems. The system offers:

• Chips with 60–80 million reads, ideal for exomes, transcriptomes, and genotyping by sequencing.
• High accuracy.
• 2–4 hour sequencing run time.
• Single-day sequencing of plant and animal genomes.

Find out more at thermofisher.com/ions5.

Ion Chef System templating made easy: The Ion Chef™ System makes next-generation sequencing more accessible globally, offering:

• Automated template preparation and chip loading.
• Cartridge-based reagent system.
• Sample- and reagent-tracking capability.
• Intuitive user interface.
• Application flexibility with 35–400 bp sequencing.
• Onboard diagnostics.

Find out more at thermofisher.com/ionchef.

Image of the Ion S5 System.

Image of the Ion Chef System.

Find out more and download case studies and technical information at thermofisher.com/plantdenovo.

Plant and animal genotyping

The discovery of novel single-nucleotide polymorphisms (SNPs) is critical for plant and animal genotyping, serving as candidate markers for marker-assisted selection (MAS) programs. Improvements in real-time PCR and next-generation sequencing have simplified SNP discovery and reduced costs. Thermo Fisher Scientific offers solutions to streamline resequencing investigations and achieve experimental and production goals.

Why are SNPs important? SNPs are small DNA variations associated with desired traits. They are used with quantitative trait locus (QTL) mapping to develop assays for MAS programs, aiming to improve characteristics like yield, stress tolerance, or pest resistance.

Targeted resequencing and SNP analysis solutions: Ion Torrent™ sequencers enable discovery of new SNPs in crop genomes. Confirm discoveries with Applied Biosystems™ TaqMan™ Assays or Sanger sequencing on Applied Biosystems™ genetic analyzers.

Genotyping Solutions Comparison:

Find out more at thermofisher.com/gbs.

Plant and animal genotyping by sequencing

Scalability, simplicity, and speed

Genotyping by sequencing: GBS using NGS technologies is a cost-effective tool for association studies and genomics-assisted breeding, especially for species lacking a reference sequence. Ion Torrent sequencers are ideal for GBS due to their scalability, simplicity, and speed.

Advantages of genotyping by sequencing with Ion S5 and Ion S5 XL systems:

• Allow SNP marker confirmation and de novo SNP marker discovery, even without a reference genome.
• Provide attractive, low-cost options for large sample numbers.
• Utilize highly multiplexed sequencing for millions of data points per day.
• Enable 2–4 hour sequencing runs for efficient planning.
• Offer high SNP calling accuracy.
• Provide versatility for applications including de novo sequencing, targeted resequencing, and transcriptome sequencing.

Two strategies for genotyping by sequencing:

• Ion AmpliSeq™ products for GBS: Uses PCR primers to amplify targets of interest. The Ion AmpliSeq™ Designer is a free, online tool for custom panel design using preloaded genomes or uploaded FASTA files.
• Restriction enzyme (RE) digestion: Reduces genome complexity by digesting DNA with restriction enzymes before adapter ligation. This method is suitable for situations with limited genome sequence or no prior SNP variants identified, aiding in the discovery of new markers.

Workflow Overview:

• Day 1: 1. DNA preparation; 2. RE digestion/multiplex PCR; 3. Ligate barcoded adapters; 4. Amplify library fragments.
• Day 2: 5. Prepare template; 6. Sequence; 7. Analyze data.

Thermo Fisher Scientific offers an extensive portfolio of DNA isolation kits.

Image of ripe tomatoes on a vine.

Design for any genome: Design and order custom panels with the Ion AmpliSeq Designer, a web-based tool. Select targets or enter genomic content for custom panel design. Panels can be based on preloaded genomes (Rice, Cow, Tomato, Sheep, Chicken, Pig, Maize, Soybean) or custom reference sequences.

Visit ampliseq.com to start creating designs.

Plant and animal genotyping by qPCR

Genotyping by qPCR: Real-time PCR is a well-established approach for SNP confirmation or screening panels of markers. Thermo Fisher Scientific provides real-time PCR instruments, TaqMan Assays, and reagents for reliable validation and screening.

Applied Biosystems TaqMan Sample-to-SNP Kit: Provides PCR-ready DNA from plant tissues in approximately 5 minutes, along with TaqMan™ GTXpress™ Master Mix for robust PCR amplification.

Applied Biosystems QuantStudio 12K Flex Real-Time PCR System: Offers flexibility with interchangeable blocks and high throughput (110,000 genotypes per workday without robotics).

Custom TaqMan SNP Genotyping Assays: Flexible technology for polymorphism detection. Assays can be created using the Applied Biosystems™ Custom TaqMan™ Assay Design Tool.

Custom Applied Biosystems TaqMan Assay manufacturing and plating service: Tailored services for zygosity analysis, special manufacturing modifications, and plating formats.

Overview of workflow for genotyping by real-time PCR:

• DNA isolation: TaqMan Sample-to-SNP Kit provides PCR-ready DNA in 5 minutes.
• Reaction preparation: Assays available in various formats (single tube, 96/384-well plates, Low Density Array, OpenArray™ plate). Mix DNA with Custom TaqMan GTXpress Master Mix and TaqMan SNP Genotyping Assay.
• Amplification: Perform PCR using QuantStudio systems or Veriti™ Thermal Cycler.
• Endpoint reading: Read amplification with the QuantStudio™ 12K Flex Real-Time PCR System.
• Analysis: Use Applied Biosystems™ TaqMan™ Genotyper Software for fast and accurate genotype calling. Download at thermofisher.com/taqmangenotyper.

Image of Applied Biosystems QuantStudio 12K Flex Real-Time PCR System.

Contact your sales representative to discuss your needs. Learn more at thermofisher.com/agbio.

Results from the Delta Genomics bovine Ion AmpliSeq panel: Delta Genomics used the Ion AmpliSeq panel to target 121 markers, achieving high call rates (>95%) and genotype concordance (>98%).

Results from the International Rice Research Institute (IRRI) rice Ion AmpliSeq panel: IRRI used the Ion AmpliSeq panel to target 500 rice genetic markers, observing high call rates (>94%) and genotype concordance (>98.9%).

QTL mapping and marker-assisted selection

High-throughput genotyping methods for marker-assisted selection (MAS) enhance understanding of breeding crops for desired traits like higher yields or disease resistance. Thermo Fisher Scientific offers technologies for breeding and functional genomic studies, associating traits with genetic variants (SNPs, SSRs), constructing QTL maps, and identifying MAS markers.

What are QTL mapping and marker-assisted selection? QTL analysis links genetic markers with DNA variations (SNPs, microsatellites/SSRs) to QTLs of interest for breeding. It's used in linkage mapping and MAS programs to screen plants for traits like increased yield, disease/stress resistance, or enhanced nutritional profiles.

What is the best approach if I am using SNP markers for breeding? The choice of approach depends on project needs, specifically the number of samples and markers. The chart below provides a high-level representation of technologies offered.

Technology Comparison Chart (Number of SNPs/markers vs. Number of samples):

• GBS on Ion Proton and Ion S5 systems: Low cost, fast/easy workflow, flexibility, affordability.
• TaqMan Assays, high-resolution melt (HRM) analysis, SSRs: Fast/easy workflow, gold standard with high call rate/accuracy (TaqMan Assays), very low-cost HRM analysis.
• TaqMan Assays on QuantStudio OpenArray platform: Low cost, proven TaqMan chemistry, streamlined workflow, reduced consumables.
• TaqMan Assays on Douglas Scientific Array Tape™ strips: High throughput, automated workflow, low cost, proven TaqMan chemistry.

Technology Comparison Table:

Image of a cow.

Image of a Jersey cow.

Image of wheat stalks.

Download application notes and scientific articles at thermofisher.com/qtl.

Abstract image of green cellular structures.

Plant and animal gene expression analysis

Whole-transcriptome analysis is crucial for understanding how altered expression of genetic variants contributes to plant and animal phenotypes. Thermo Fisher Scientific offers a comprehensive product portfolio for gene expression analysis, including transcriptome sequencing, TaqMan™ real-time PCR solutions, and epigenetics research products, facilitating fast, reliable, and accurate gene expression analysis.

What are the different approaches for gene expression analysis? Agricultural researchers use various approaches. Whole-transcriptome profiling is preferred over targeted RNA sequencing for some studies. Epigenetics tools, miRNA, small RNA, and RNAi studies help decipher phenotype causes. Thermo Fisher Scientific provides instruments, reagents, and solutions for all gene expression studies.

Transcriptome sequencing: Unlike traditional microarrays, NGS technologies enable gene expression profiling in any species, offering a higher dynamic range at a lower cost. Transcriptome sequencing is simplified with the Ion S5 or Ion Proton System for profiling entire transcriptomes or reducing complexity.

Targeted gene expression analysis: For experiments focused on a few genes, real-time PCR assays with Applied Biosystems™ TaqMan™ probes are ideal. Applied Biosystems™ instruments and software, along with predesigned and custom assays, ensure success. Predesigned assays for soybean, corn, rice, cattle, and pig save time on assay design.

Find out more at thermofisher.com/taqmangex.

Epigenetics, miRNA, and noncoding RNA research: Gene regulation involves epigenetic changes (DNA methylation, chromatin structure) and noncoding RNAs. Thermo Fisher Scientific offers technologies for epigenetics research, covering sample preparation, sequencing, qPCR, functional analysis, and bioinformatics. View solutions at thermofisher.com/epigenetics.

Image of the Ion S5 System.

Image of laboratory equipment for gene expression analysis.

Browse all solutions for gene expression at thermofisher.com/plantge.

Synthetic biology and genetic engineering

Generating transgenic plants is key to introducing new crop traits and conducting gene studies. Developing genetically modified plants often requires complex DNA element design for optimal expression. Thermo Fisher Scientific offers solutions for genome editing, cloning, DNA assembly, amplification, analysis, and Agrobacterium tumefaciens LBA4404 for plant-cell transformation.

Manipulate, synthesize, and clone plant DNA easily: Construction of T-DNA vectors or vectors for particle bombardment/electroporation is simplified by Thermo Fisher Scientific's DNA cloning and assembly products. These solutions allow testing regulatory sequences or stacking traits on expression vectors.

• Invitrogen™ GeneArt™ Seamless PLUS Cloning and Assembly kits and GeneArt™ Gene Synthesis services: Enable simultaneous cloning of complex DNA elements and design of synthetic genes for enhanced heterologous gene expression. Learn more at thermofisher.com/geneart.
• Invitrogen™ Gateway™ cloning: An expression platform for plant systems with a wide range of vectors. Gateway™ vectors are adapted for Agrobacterium-mediated transformation and silencing studies. Learn more at thermofisher.com/gateway.
• Basic cloning reagents: Offer competent cells, DNA oligos, and Invitrogen™ TOPO™ cloning kits. Browse offerings at thermofisher.com/cloning.

Precise, efficient gene sequence targeting and modification technologies: Thermo Fisher Scientific provides solutions for genome editing, gene modification, and high-efficiency delivery systems. Technologies like Invitrogen™ GeneArt™ CRISPRs and TALs enable reliable genomic sequence modification and phenotypic analysis. Their cell engineering portfolio is built on 20 years of innovation.

GeneArt CRISPRs: A complete genome editing solution designed to expedite research. Solutions are optimized and validated for accessibility. The portfolio spans cell culture reagents, delivery reagents, sample preparation, genome modification, and detection/analysis. Features include an online CRISPR search and design tool, CRISPR-Cas9 formats, an all-in-one expression vector, Cas9 mRNA, Cas9 protein, and CRISPR Libraries services.

Image of Invitrogen GeneArt reagents and kits.

GeneArt Precision TALs: Custom DNA-binding proteins based on Xanthomonas effector proteins that accurately target genomic loci. TAL technology is effective in plants, algae, and fungi. Explore precision TALs at thermofisher.com/tal.

Plant transformation: Agrobacterium-mediated transformation is a widely used technique in plant biotechnology. Thermo Fisher Scientific offers A. tumefaciens LBA4404 cells and antibiotics for research. Browse products for plant genetic engineering at thermofisher.com/plantengineering.

Image of ripe corn cobs.

Plant cell and tissue analysis

Studying plant cell morphology, gene regulation, and expression requires specialized techniques and tools. Immunohistochemistry is routinely used to label plant tissues and organelles. Thermo Fisher Scientific offers a portfolio of fluorescent reagents, kits, and instruments for plant cell and tissue analysis.

Invitrogen fluorescent dyes and probes: Reagents including Invitrogen™ Alexa Fluor™ dyes, Qdot™ nanocrystals, Click-iT™ detection assays, and ProLong™ Gold Antifade Reagents are used for various plant research applications, including:

• Antibody labeling
• Immunohistochemistry
• Fluorescence in situ hybridization
• Organelle and cytoskeletal stains
• Endocytosis studies
• Transport studies
• Glucose metabolism
• Viability staining
• Glutathione detection
• Calcium and other ion-imaging applications
• Tyramide signal amplification (TSA)

Rubisco localization in maize leaf section: Visualized using Invitrogen™ Alexa Fluor™ 488 goat anti-rabbit IgG antibody (Cat. No. A11034). The image illustrates immunolocalization of rubisco in chloroplasts.

Pectin associated with plasmodesmatal pit fields of kiwifruit cells: Tagged with an anti-pectin monoclonal antibody, JIM 5, detected and visualized with Invitrogen™ Alexa Fluor™ 488 goat anti-rat IgG (Cat. No. A11006).

Microscopic image showing Rubisco localization in maize leaf section.

Microscopic image showing pectin associated with plasmodesmatal pit fields of kiwifruit cells.

Flow cytometry: An ideal means for analyzing cells and subcellular particles, providing statistically exploitable data and facilitating subpopulation detection. It is essential for understanding plant growth, development, and function, with applications in ploidy levels, genome mapping, and sex-specific chromosome localization.

Find out more at thermofisher.com/plantimaging.

Cell Imaging: The Invitrogen™ EVOS™ line of cell imaging systems makes cell imaging accessible for various applications, from cell culture to complex protein analysis and fluorescence imaging.

Cell Counting: The Invitrogen™ Countess™ II FL Automated Cell Counter is a benchtop platform for rapid cell assessment in suspension, offering brightfield and fluorescence channels for counting, monitoring protein expression, evaluating apoptosis, and measuring cell viability.

Image of the Attune NxT Flow Cytometer.

Flow cytometry plots showing nuclear holoploid genome sizing analysis.

GMO testing and detection

Thermo Fisher Scientific offers real-time and digital PCR solutions for genetically modified organism (GMO) testing, allergen detection, and quantification of mutant/rare alleles in plant genomes, addressing throughput, accuracy, and sensitivity needs.

Products and technologies for GMO testing: Testing for GMOs is increasingly important for quality control. Thermo Fisher Scientific offers kits based on TaqMan chemistry for detecting GMO-specific DNA sequences in seed, grain, and processed foods.

Digital PCR solutions for GMO detection: Digital PCR is an advanced approach for accurately quantifying low amounts of plant transgenes, offering high tolerance for PCR inhibitors found in plant samples.

Find out more at thermofisher.com/digitalpcr.

Explore GMO testing and detection solutions at thermofisher.com/gmo.

Applied Biosystems™ QuantStudio™ 3D Digital PCR System: Enables high accuracy, sensitivity, and absolute quantification.

Diagram illustrating the Applied Biosystems QuantStudio 3D Digital PCR System workflow, showing sample partitioning into reactions and positive/negative results for absolute quantification.

Case study: Soybean (Glycine max): Dr. Wing Cheung (DNA LandMarks, BASF) partnered with Thermo Fisher Scientific to design TaqMan Assays for GMO soybean DNA detection and validate assay specificity using digital PCR. The study confirmed the measured ratio for GMO and wild-type targets was comparable to the estimated ratio.

Download the study at thermofisher.com/gmo.

Image of soybean plants.

Sample preparation and nucleic acid isolation

Plant DNA isolation presents challenges due to carbohydrates, phenolics, and other compounds in plant tissues. Thermo Fisher Scientific offers an expansive portfolio of kits, reagents, and devices for isolating, purifying, and quantifying plant genomic DNA, total RNA, mRNA, and microRNA/transcriptome RNA.

Which DNA isolation kit is right for you?

Which RNA isolation kit is right for you?

Explore solutions at thermofisher.com/plantnap.

Technology platforms and applications:

For a full selection of Thermo Fisher Scientific's products for agricultural biotechnology research, including resources, live events, recorded webinars, and more, visit thermofisher.com/agbio.