The ICAR-IARI, New Delhi, has made significant contributions in the development of crop protection and production technologies for all major crops in India. Genomics of Agriculturally Important Insects (18-28 September 2019) Venue for Lectures: Virology Auditorium, ICAR, IARI, New Delhi.
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Glossary of terminologies used in Insect Genetics, Genomics and Molecular Biology
In a narrow sense: the ratio between the additive genetic variance and the total phenotypic variance (VA/VP). DNA synthesis occurs, resulting in a doubling of the number of DNA molecules defined by the primers.
Regulatory gene A gene that codes for a protein involved in regulating the expression of other genes. The twisting of the DNA molecule in a direction opposite to the twists of the strands in the double helix is called negative supercoiling.
DNA A structural form of DNA in which the two strands are wound into a left-handed helix rather than a right-handed form
X chromosome A sex chromosome that is usually present in two copies in female insects (XX) and in one (unpaired) copy in males (XO or XY). Y chromosome A sex chromosome that is characteristic of males in species in which the male usually has two different sex chromosomes (XY).
Extraction of DNA from Insect tissue using CTAB method
Dissolve the pellet in 500 μl Lysis buffer with gentle vortexing for 30 seconds and incubate at 37°C overnight. Mix the mixture with vigorous shaking for 2 minutes and then centrifuge the tubes at 13,000 rpm for 10 minutes at room temperature.
RNA isolation and cDNA synthesis for gene expression analysis in insects
First, the dissolved RNA was kept at 4˚C for 10-15 minutes and after 30 minutes the OD was measured in a nanospectrophotometer. First, the dissolved RNA was kept at 4˚C for 10-15 minutes and after 30 minutes the OD was measured in a nanospectrophotometer and used for cDNA synthesis.
Polymerase Chain Reaction and Agarose Gel Electrophoresis
Page | 46. A gel is prepared with an agarose concentration appropriate to the size of the DNA fragments to be separated; (2) the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a period of time that will achieve optimal separation; and (3) the gel is stained or, if ethidium bromide is included in the gel and electrophoresis buffer, immediately visualized after illumination with UV light. Turn OFF the power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
PCR Primer’s Characteristics, Designing and Resuspending
The stability of the primer-template DNA duplex can be measured by the melting temperature (Tm). Tm of the primer is the melting temperature of the less stable primer-template pair Tm of the product is the melting temperature of the PCR product.
Gene expression analysis through qPCR in insects
Real Time PCR has become one of the most reliable tools in gene expression analysis studies. Analysis of relative gene expression data using real-time quantitative PCR and the 22DDCT method.
DNA Barcodes for Insects
The ethanol was slowly drained off and the pellet dried under vacuum in a desiccator for 5 to 10 minutes. The gel tray was removed and the gel was observed under the UV transilluminator and documented using the Gel Doc system.
Molecular gut analysis of the predators
Optimization of the primers for their specificity and cross-specificity against other prey on which the predator feeds should be performed. Primer optimization using single and multiplex PCR for specificity and cross-specificity against other prey on which a predator feeds.
Restriction Fragment Length Polymorphism (RFLP)
When fractionated by agarose or acrylamide gel electrophoresis, RE-digested PCR products will yield readily distinguishable patterns. Separately digest the amplicons with one or more restriction enzymes on the PCR-amplified DNA fragments from the target genotypes.
Genotyping of phosphine resistance in Red flour beetle Tribolium castaneum- A Practical Approach
PCR is performed using 2μl genomic DNA template with gene-specific primers for the rph2 gene in T.castaneum. The amplified 368-bp PCR product should be digested with MboI RE at 37 °C overnight followed by heating at 65 °C for 20 min with the following reaction volume.
Insect Preparation for Genomics
- Subramanian
- Important considerations for insect genomics
- DNA EXTRACTION PROTOCOL FOR INSECTS Protocol 1: CTAB /Mercaptoethanol Method
- Rapid, Cost efficient genomic DNA isolation protocol
- Chelex Technique
- Chelax method for 96 well plates Material needed (for a 96-well PCR plate)
But some of the above strategies require relatively small amounts of DNA (although the DNA still should not be degraded). Pick up the leg(s) and place them at the bottom of the first well of a 96-well PCR plate.
Insect Metagenomics: Principles and Practices
The quality of the sequence bases is checked and their composition and GC content determined. The read sequences at the paired end contain part of the V3 region, the spacer and the conserved region.
Terminologies and concept of sequence analysis
Paralogous Gene
TBLASTN : Compares a protein query sequence to a nucleotide sequence database dynamically translated in all six reading frames (both strands). Fast pairwise alignment: calculate distance matrix …> directory tree…> Progressive alignment to directory tree.
Unrooted dendrogram
The bootstrap is a method to determine the statistical significance of the positions of branches in a phylogenetic tree. For each aligned pair, it takes scores from random positions in the alignment, and adds the scores.
Use an online service such as VecScreen (www.ncbi.nlm.nih.gov/tools/vecscreen/) for this purpose. If it is a circular molecule, then find the origin of the sequence (eg - in the case of begomoviruses it is TAATATT↓ACC).
Evaluation of RNAi constructs by feeding assay against insects
Prepare different concentrations, namely 5.0 µg dsRNA/g diet, using a stock solution suitably diluted to a volume of 0.1 ml nuclease-free water, to mix evenly in 10 g diet. Wash the collected larvae with 70% alcohol followed by nuclease-free water and store them in RNAse-free tube at -80oC in RNAse later™ for further use of expression analysis by Real Time PCR.
Estimation of detoxification enzymes associated with insecticide resistance in insects
Plate-read cytochrome P450 (general oxidase) activity was expressed as cytochrome P450 equivalent units (EU) per milligram of protein using a cytochrome C standard curve. Cytochrome P450-Based Mechanism of Resistance and Pyrethroid Resistance in the Field Anopheles Resistance Management albimanus trial, Pestič.
Gene Silencing and Genome Editing in Insect Pest Management
Asokan
Other components of the RNAi and miRNA silencing pathways have been shown to be closely related, notably Argonaute, which is a key component of the RNA-induced Silencing Complex (RISC) and miRNAs function in a silencing complex that is similar, if not identical not. , to RISC to regulate expression of target genes, either through splicing of mRNA or translational repression: if the miRNA shows perfect complementarity to its target mRNA, the mRNA is cleaved (typically in plants), if there is only partial complementarity, find place translational repression (typically in animals). Most of the studies on RNAi in entomology have been on determining the function of genes involved in various metabolic processes.
RNAi (dsgst) in the Management of B. tabaci
In terms of its application in insect research, CRISPR/Cas9 has lagged behind its growing popularity in mammalian studies. However, CRISPR/Cas9 applications in insects have been increasingly reported, mainly in Drosophila melanogaster, Bombyx mori and Aedes aegypti.
Enzyme kinetics – an indispensable tool for understanding metabolic pathways
Most competitive inhibitors have structural similarity to the substrate and bind only to the free enzyme. They increase the Km value but do not modify the Vmax of the enzyme.
DNA Barcoding and Its Application in Identification of Species
Venkatesan
DNA barcoding is being done for several organisms including insects and other arthropods under various initiatives like barcode of life. Recently, the multilocus DNA barcoding approach (ITS region and 18S RNA) is progressively emerging and is now widely accepted (especially in cases where COI is not species-specific).
Computational Tools for Gene Annotation
R. Rao
This is multiplied by the transition probability from the first state to the second, which is 1. The rest of the alignment are the columns that will correspond to the main states in the model.
Genome Sequencing: An Overview
The turning point in genome sequencing was in the latter part of the last decade when next-generation sequencers were released by 454 and Solexa (Fig 1 & 2). NGS technology changed everything and whole genome sequencing (WGS) became a routine feature where only a few micrograms of genomic DNA was enough to obtain a snapshot of the genomic assembly.
Metagenomics: An overview
Annapurna, Rajeev Kaushik and V. Govindasamy
The most commonly used methods of microbial identification using high throughput metagenome sequencing data are
The study of the proteome expressed in the microbial community is known as Meta-proteomics. New cellulases with improved enzymatic properties have been identified. d) The use of metagenomics, metatranscriptomics and metaproteomics approaches improve enzyme discovery and can be used to efficiently screen for highly active enzymes. e) Recent studies have shown that metagenomics can be used as a bioremediation tool; compared to other bioremediation approaches, metagenomics yielded better degradation rates.
Molecular markers for Entomological Research
Mohankumar
The use of molecular markers is a very reliable method for diagnosing the integration of both genes in plants. Ten species in one: DNA barcoding reveals cryptic species in the Neotropical skipper butterfly Astraptesfulgerator.
Quantitative PCR-Principles and Practices
Ramakrishnan
The higher the initial number of target sequences in the sample, the faster the fluorescence intensity will increase during the PCR reaction. The standard templates (DNA or RNA) should be quantified using the standard methods (UV spectrophotometry or nucleic acid binding dyes) in replicates, without template controls (NTC).
RNAi in Insect Pest Management
Feeding WCR with this modified plant resulted in larval stunting and premature death of the insect. When the stability of dsRNA in the insect body is the main issue, polymer- or liposome- or liposome- and bacteria-nanoparticles can be used.
The Genomic Basis of Nematode Parasitology: as a Host & a Pathogen
The most convincing success of in planta delivery of dsRNA to feeding nematodes, resulting in the knockdown of several key nematode genes, comes from root-knot nematodes. Although RNAi-based transgenics is still in the preliminary stage, but it offers new management strategy for the future.
Plant-Parasitic Nematode Genomics: An Update
A list of all plant parasitic nematodes sequenced, the technologies used for sequencing and their genome statistics are given in Table 1. Recent advances in PPN genomics and transcriptomics have made it possible to identify promising molecular targets and metabolic bottlenecks that can be exploited for their management through transgenic approaches or target-based drug discovery. Although new drugs or chemicals designed using genomic information are still awaited, approaches based on transgenic crops, such as host-delivered RNAi, have been used with great success for nematode management. It is expected that the increased use of genomic information would provide a major impetus to find smarter options for managers of animal and plant parasitic nematodes.
Transcriptomic Approaches for Elucidating the Genes Network Associated with Source and Sink in Wheat
SBEs show a specificity for the length of the α(1-4) glucan chain that they will use as a substrate. Even the transcriptional regulation of the genes coding for these enzymes has not yet been fully explored.
Real time PCR Technique
Sample Preparation
Alternatively, a one-step qPCR reaction can be performed today, where cDNA synthesis and subsequent amplification can be performed in one reaction.
Data Analysis
Real-time PCR is based on the detection of the fluorescence produced by a reporter molecule that increases as the reaction proceeds. The real-time PCR test can simultaneously detect and quantify bacterial, fungal and viral pathogens.
Recipe for molecular biology reagents
Lysis buffer for DNA Extraction Components Total volume (10ml)
Preparation of 1 M Tris Cl (pH 8.0)
Preparation of TAE Buffer One liter 50X stock of TAE
Ethidium Bromide (10 mg/ml)
